“recombinant cellulase production in...

“Recombinant cellulase production in plants”

Von der Fakultät für Mathematik, Informatik und Naturwissenschaften der

RWTH Aachen University zur Erlangung des akademischen Grades eines

Doktors der Naturwissenschaften genehmigte Dissertation

vorgelegt von

Holger Klose, M.Sc.

aus Lüdenscheid

Berichter: Universitätsprofessor Dr. Rainer Fischer

Universitätsprofessor Dr. Björn Usadel

Tag der mündlichen Prüfung: 12. Mai 2014

Diese Dissertation ist auf den Internetseiten der Hochschulbibliothek online verfügbar.

Meinen Eltern

Table of contents

i

Table of contents

Table of contents .......................................................................................................................................i

Abstract .................................................................................................................................................... iii

Kurzzusammenfassung ........................................................................................................................... iv

Chapter 1: Introduction ............................................................................................................................ 1

1. Bioenergy from biomass .................................................................................................................. 1

2. The next generation of advanced biofuels ....................................................................................... 2

3. Processing lignocellulosic biomass ................................................................................................. 4

3.1 Biomass recalcitrance – the structure and composition of plant cell walls ................................ 4

3.2 Pretreatment technologies for lignocellulosic biomass .............................................................. 7

3.3 Enzymes for cellulose conversion .............................................................................................. 9

3.4 Advanced feed stocks for fuels ................................................................................................ 11

4. Expression of recombinant glycosylhydrolases in plants .............................................................. 12

Objectives of this study ...................................................................................................................... 16

Chapter 2: Differential targeting of a mesophilic endoglucanase .......................................................... 17

Results ............................................................................................................................................... 17

Analyzing the transient expression of TrCel5A in tobacco plants .................................................. 17

Analyzing the localization of TrCel5A by transient expression in tobacco plants .......................... 19

Generation of transgenic tobacco plants expressing TrCel5A ....................................................... 21

Biochemical characteristics of TrCel5A produced in planta ........................................................... 23

Subcellular localization of TrCel5A in tobacco plants .................................................................... 24

Growth characteristics of the transgenic tobacco plants ................................................................ 25

Chemical analysis of tobacco cell walls ......................................................................................... 27

Hydrolysis of leafy biomass from transgenic tobacco .................................................................... 29

Discussion .......................................................................................................................................... 30

Chapter 3: Ethanol-inducible endoglucanase expression in tobacco.................................................... 35

Results ............................................................................................................................................... 35

Generation of transgenic tobacco plants expressing inducible TrCel5A ....................................... 35

Inducible TrCel5A expression in tobacco plants ............................................................................ 37

Growth characteristics and histology ............................................................................................. 38

Chemical analysis of tobacco cell walls in alcR::TrCel5AAP plants ................................................ 40

Discussion .......................................................................................................................................... 41

Table of contents

ii

Chapter 4: Expression of a hyperthermophilic endoglucanase in tobacco ........................................... 43

Results ............................................................................................................................................... 44

Expression of SSO1354 in tobacco plants and downstream purification ...................................... 44

Biochemical properties and catalytic activity of plant-derived SSO1354 ....................................... 47

SSO1354 activity in ionic liquids .................................................................................................... 49

Discussion .......................................................................................................................................... 50

Chapter 5: Summary, Conclusion and Outlook ..................................................................................... 53

Chapter 6: Materials and experimental procedures .............................................................................. 57

Materials ............................................................................................................................................. 57

Synthetic oligonucleotides .............................................................................................................. 57

Plasmids ......................................................................................................................................... 58

Bacterial strains .............................................................................................................................. 58

Plant material ................................................................................................................................. 59

Experimental procedures ................................................................................................................... 59

Cloning ........................................................................................................................................... 59

Protein production in tobacco plants .............................................................................................. 60

Protein chemical methods .............................................................................................................. 62

Enzymatic assays ........................................................................................................................... 62

Chemical analysis of plant cell wall polymers ................................................................................ 64

Histological methods ...................................................................................................................... 65

Conversion of transgenic tobacco leaf material ............................................................................. 65

Statistical methods ......................................................................................................................... 66

References ............................................................................................................................................ 67

Appendix ................................................................................................................................................ 80

Abbreviations ..................................................................................................................................... 80

Register of equipment ........................................................................................................................ 82

Table of figures .................................................................................................................................. 83

Scientific publications ......................................................................................................................... 84

Acknowledgement .............................................................................................................................. 86

Declaration ......................................................................................................................................... 87

Curriculum vitae ................................................................................................................................. 88

Abstract

iii

Abstract

Enzyme-catalyzed lignocellulose conversion is a promising pathway to increase the

feasibility of second generation biofuel production. The development of viable

processes for enzymatic lignocellulose utilization must address different issues to

become economically feasible. One important issue is the production of appropriate

and cost-effective enzymes to deconstruct the complex and heterogeneous structure

of the lignocellulose. Plant based expression of cell wall degrading enzymes is a

promising technique, but it currently faces a number of challenges, such as enzyme

expression levels and auto-hydrolysis of the plant cell walls.

This thesis deals with establishing and evaluating cellulase expression strategies in

tobacco plants, including differential protein targeting, inducible protein expression

and the implementation of enzymes with different biochemical properties, such as

thermophile. Different approaches of in planta produced cellulases are evaluated by

the analysis and their properties as well as by detailed phenotypical analyses, such

as biochemical characterization of plant cell walls. Constitutive high level expression

of a mesophilic cellulase led to detrimental changes in the plant phenotype, including

growth and development. The retention of the enzyme inside the endoplasmic

reticulum could not completely avoid this impact on the plant development. The

application of an alcohol inducible promoter for cellulase production led to enzyme

expression rate without any significant phenotypical effects on the plant.

Hyperthermophilic cellulases are inactive under normal plant growth conditions,

allowing their expression in plants without negative effects on growth and

development. Subsequent heat-inducible activation enables lignocellulose

degradation. The expression rate of these hyperthermophilic enzymes is at lower

levels than that of mesophilic enzymes. However, this enzyme was shown to survive

harsh pretreatment conditions. Therefore it is a promising candidate for an advanced

enzymatic conversion of lignocellulosic biomass.

This study established advanced basic knowledge for plant based production of

cellulolytic enzymes and subsequently the developed and evaluated new production

strategies for cellulases would provide an advanced enzymatic conversion processes

of plant biomass.

Kurzzusammenfassung

iv

Kurzzusammenfassung

Die Umwandlung von Lignocellulose zu fermentierbaren Zuckern durch Enzyme ist

ein vielversprechender Ansatz zur nachhaltigen Herstellung von Biokraftstoffen. Die

Entwicklung realisierbarer Prozesse für die Enzym-katalysierte Nutzung von

Lignocellulose muss verschiedene Gesichtspunkte berücksichtigen, um ökonomisch

rentable und nachhaltige Verfahren zu erstellen. Die kostengünstige Produktion

geeigneter Enzyme zum Abbau der komplexen Struktur lignocellulosehaltiger

Biomasse ist dabei ein wesentlicher Kernpunkt. Pflanzen bieten eine kostengünstige

und vielversprechende Alternative zur Produktion solcher Enzyme. Zurzeit müssen

solche pflanzlichen Systeme jedoch noch hinsichtlich der Enzym-Ausbeute und der

potentiellen Schädigung der Pflanze durch das Enzym optimiert werden.

Die vorliegende Studie diente der Entwicklung und Evaluation von Strategien zur

Expression von Cellulasen in Tabakpflanzen. Hierbei wurden unterschiedliches

Protein-Targeting, induzierbare Expression sowie die Expression Temperatur-

aktivierbarer Enzyme untersucht. Die verschiedenen Ansätze wurden hinsichtlich der

biochemischen Eigenschaften der produzierten Cellulasen analysiert. Zudem wurden

die erstellten transgenen Tabakpflanzen auf Veränderungen des Phänotyps,

insbesondere der biochemischen Zusammensetzung der pflanzlichen Zellwände hin

untersucht. Die konstitutive Expression einer mesophilen Cellulase führte zwar zu

hohen Expressionsraten, jedoch wurden auch signifikant Änderungen im Wachstum

und der Entwicklung der transgenen Pflanzen festgestellt. Die Retention der

Cellulase im Endoplasmatischen Reticulum konnte diesen Einfluss auf die

Entwicklung der Pflanze nicht vollständig verhindern. Die Verwendung eines Alkohol-

induzierbaren Promotorsystems zur Produktion der Cellulase führte hingegen nicht

zu signifikanten Änderungen im Phänotyp der Pflanzen. Vergleichbare Ergebnisse

wurden mit der Verwendung einer hyperthermophilen Cellulase erzielt, die unter den

Wachstumsbedingungen der Pflanze inaktiv vorliegt und erst durch hohe

Temperaturen aktiviert werden kann. Zwar wurde für dieses Enzym eine geringere

Ausbeute erzielt als für das mesophile Enzym, jedoch zeigt es sehr hohe Toleranz

gegenüber den harschen Bedingungen während bei konventioneller

Vorbehandlungsmethoden zur Verzuckerung von Biomasse und ist ein

vielversprechender Kandidat für neue verbesserte Prozesse zur enzymatischen

Biomassekonversion.

Kurzzusammenfassung

v

Die innerhalb dieser Arbeit erzielten Daten ermöglichen erweiterte Erkenntnisse zur

Expression von Cellulasen in Pflanzen. Die daraus erfolgte Entwicklung und

Evaluierung neuer Produktionsstrategien kann in Zukunft einen Beitrag zu einer

verbesserten und nachhaltigen Nutzung pflanzlicher Biomasse liefern.

Chapter 1: Introduction

1


1. Bioenergy from biomass

One of the most important challenges in 21st century is the rapid depletion of fossil

fuel resources. Energy from the sun is the most sustainable alternative to oil, gas and

coal. The surface of the earth receives more than 9000 times more energy than is

consumed by humans (Lewis and Nocera, 2006). This energy can be utilized directly

to produce electricity, e.g. wind turbines and photovoltaic cells. However in contrast

to these utilization forms which are highly fluctuating, plant biomass can also be used

to create liquid or solid fuels, and this process is currently considered the most

effective route to convert the sun’s energy into power (Carroll and Somerville, 2009).

Bioethanol has a comparably long history as a transportation fuel and fuel additive. In

Brazil it has been mandatory to supplement transportation fuels with bioethanol

derived from sugarcane since 1929 (Graham-Rowe, 2011). However, it was the

worldwide oil crisis in 1973 and the launching of the “ProAlcool” program by the

Brazilian government that transformed bioethanol production into an economically

relevant industrial sector (Amorim et al., 2011). In 2008, sugarcane-based fuel

surpassed petroleum-based gasoline in Brazil (Chaddad, 2010) and 95% of all

vehicles sold in Brazil use “flex-fuel”, i.e. any blend of ethanol and gasoline (Amorim

et al., 2011).

First-generation biofuels, e.g. ethanol from starch or sugar based, are

disadvantageous because they compete with food and feed crops. Recent concerns

about energy security and the massive increase in energy consumption has

increased the use of edible crops for the production of fuels, e.g. one third of all

harvested corn was used for bioethanol generation in 2007, resulting in a 73% price

rise by the end of 2010 (Graham-Rowe, 2011). The World Bank and the UN Food

and Agriculture Organization (FAO) reported this as the highest price in the last 20

years, contributing to food riots in Mexico and other developing countries (Fairley,

2011). This underlines the need and the great challenge to provide the growing world

population with sustainable supplies of food and energy.


2

Figure 1: Global demographic development. Between today and 2050, the world population is

predicted to rise above 9 billion people. Each person will also consume more calories per day and use

more energy to power their lives. Food and fuel supplies will need to increase massively to meet this

demand. Reproduced from (Graham-Rowe, 2011).

2. The next generation of advanced biofuels

Instead of using the edible parts of the plant, fuels can be sourced from

lignocellulosic residues to avoid competition between food and fuel. Lignocellulose

(from plant cell wall residues) is a completely different starting material for fuel

production, which contains much more energy than grain at the cost of more difficult

and intensive processing (Carroll and Somerville, 2009). The production costs (up to

US$123 per barrel or €0.6 per liter) are much higher than first-generation biofuels

and this may be the most important drawback to ethanol production from

lignocellulosic sources (Tyner, 2008). However, these production costs are estimates

based mostly on laboratory or pilot scale plants (Sainz, 2011), because there are only

a few large scale facilities worldwide.


3

Nevertheless world leading energy suppliers have started to focus on production

facilities for lignocellulosic biofuels. The joint venture “project liberty” between POET

and Royal DSM will be one of the first commercial-scale facilities for cellulosic

ethanol. The plant is under construction in Emmetsburg, Iowa, and will produce 20-

25 million gallons of ethanol per year from corn cobs, leaf husks and stalks starting in

late 2013 (http://www.poetdsm.com/). Likewise, DuPont is constructing a commercial-

scale cellulosic ethanol facility in Nevada, Iowa, which will become operational in

mid-2014 and will produce 30 million gallons per year

(http://biofuels.dupont.com/cellulosic-ethanol/nevada-site-ce-facility/). Both projects

benefit from the US Department of Energy thanks to the Energy Independence and

Security Act 2007.

Oil and automobile companies have also funded cellulosic ethanol research. Dutch

Royal Shell and Volkswagen have invested in, among others, the Canadian company

Iogen to further develop cellulosic ethanol at a large pilot scale facility in Ottawa,

Canada (http://www.iogen.ca).

European examples include the Spanish company Abengoa Bioenergy and the

Danish company Inbicon. Abengoa runs 14 bioethanol facilities worldwide and a

demonstration plant in Salamanca, Spain, which uses wheat and barley straw for the

production of cellulosic ethanol. Inbicon is a subsidiary of Denmark´s largest energy

supplier Dong Energy. Its demonstration plant in Kalundborg, Denmark, produces 1.5

million gallons of cellulosic ethanol per year (www.inbicon.com). In Germany, the

Swiss company Clariant launched a pilot scale plant for cellulosic ethanol in

Straubing in 2012, supported by German governmental research initiative

BioEconomy 2030 (www.clariant.com). This increased investment is encouraged by

the potential of energy and transportation fuels derived from lignocellulosic biomass.

Up to 30% of global gasoline demand could be met from agricultural crop residues

(Michael et al., 2007). Funding and investment has recently accelerated, focusing on

all steps of the process chain (Sainz, 2011).


4

3. Processing lignocellulosic biomass

Plants have evolved to resist microbial attack (Scheller and Ulvskov, 2010). Plant cell

walls play an important role in this process, by providing a barrier against potentially

pathogenic organisms. A greater understanding of plant cell wall architecture and

adaptive strategies for the deconstruction of plant biomass are therefore key

requirements in the development of novel and sustainable processes for

lignocellulose utilization.

3.1 Biomass recalcitrance – the structure and composition of plant cell walls

Many plant cell walls are basically composed of lignocellulose, which comprises three

carbon-based polymers: lignin, hemicellulose and cellulose.

Figure 2: The primary cell wall of a growing cell as found in most flowering plants. The cellulose

chains were organized in microfibrils which are embedded in a matrix of heterogenous

polysaccharides. This matrix-phase consists mainly of pectic polysaccharides like polygalacturonic

acid and rhamnogalacturonan, the latter is often substituted with small polymeric side groups of

arabinan, galactan and arabinogalactan. Xyloglucans have only a single face and therefore can attach

other glucan chains via hydrogen bonds. Highly-esterified PGA is an important component of the

young cell wall and the junction zones are enriched in the middle lamella. Abbreviations: PGA,

polygalacturonic acid; RG I, rhamnogalacturonan I. Figure reproduced from (Carpita and Gibeaut,

1993).


5

There are primary and secondary cell walls. The primary cell wall is formed when

cells develop and typically does not contain any lignin (Carpita and Gibeaut, 1993;

Cosgrove, 1997).

Secondary cell walls were formed after differentiation as part of structural and

functional specialization (Boerjan et al., 2003; Bonawitz and Chapple, 2010). Lignin

contributes up to 30% of the total weight of some secondary cell walls (Scheller and

Ulvskov, 2010). It is a heterogeneous complex polymer comprising diverse

phenylpropanoid subunits (Boerjan et al., 2003). It provides strength, rigidity and

hydrophobicity, although the amount varies between different species and tissues

(Bonawitz and Chapple, 2010).

Cell wall polysaccharides form two different subgroups; the stereoregular homo-

polymer cellulose forms the microfibrilar phase in all cell walls, whereas the stereo-

irregular heteropolysaccharides are associated with these microfibrils. The latter

consist of structurally and compositionally distinct polysaccharides, often described

as hemicellulose and pectin (Pauly et al., 2013). These polymers are located in the

matrix phase between the fibers and are responsible for covering the fibrils and

connecting them to the lignin polymer in the secondary cell wall (Popper, 2008).

Hemicelluloses are categorized by the components of their backbone, mainly

mannans, glucans and xylans (Pauly et al., 2013; Scheller and Ulvskov, 2010). Pectic

polysaccharides contain large amounts of galacturonic acid and their structure and

composition is even more complex and diverse (Atmodjo et al., 2013). The

biosynthesis of these heterogenous polysaccharides takes place in the cisterna of

the Golgi apparatus by plant glycosyltransferases (GTs) that generate the sugar

backbone and subsequently decorate it with side chain sugars (Scheller and Ulvskov,

2010). These precursors can be trimmed by glycosylhydrolases (GHs) before transfer

to the apoplast and incorporation into the cell wall (Günl and Pauly, 2011; Minic,

2008).

Cellulose is usually the most abundant component of plant cell walls (Endler and

Persson, 2011). It consists of long unbranched chains of β-glucopyranose which are

linked via β 1,4 glycosidic bonds. The sugar chains have a ribbon-like conformation

which allows them to be packed into microfibrils stabilized by intermolecular

hydrogen bonds and van der Waals interactions (Harris and Stone, 2009). Although

cellulose has been described as crystalline this is not true in the classical sense.


6

Native cellulose in living plants varies in structure from amorphous to crystalline

depending on species and tissue (Atalla et al., 2009).

Figure 3: Schematic representation of hemicellulose polysaccharides. Symbols representing

different monosaccharides were adopted from the Nomenclature Committee Consortium for Functional

Glycomics (Varki et al. 2009). Glycosidic linkages are presented in their anomeric configurations (α

and β) and their position, e.g. β4 indicates a β 1,4 linkage. Mannans are abundant and are present in

variable amounts in the cell wall. Glucomannes are connected to the secondary cell wall. Xyloglucans

(XyGs) vary in structure and composition between dicots (where predominantly fucogalactoXyG

appears) and monocots and solanales (where arabinogalactoXyG is present). Xylan is found as

Glucoronoxylan (GX) in dicots but more often as glucoronoarabinoxylan (GAX) in grasses.

Reproduced from (Pauly et al., 2013).


7

Cellulose is synthesized by globular protein complexes located in the plasma

membrane. These so-called cellulose synthase complexes probably use a steryl

glycoside primer and therefore first accept UDP-glucose before forming the long

β 1,4-glucan chains. GHs are also required for cellulose biosynthesis (Minic, 2008).

For example, the endoglucanase KORRIGAN may contribute to the assembly of the

microfibrils and/or chain termination (Molhoj et al., 2001).

The heterologous composition of lignocellulose and the variety of linkages within and

between the different polymers explain the recalcitrance of biomass, and this must be

overcome to develop an economically feasible process for lignocellulose conversion

(Chundawat et al., 2011).

3.2 Pretreatment technologies for lignocellulosic biomass

The first step in the conversion of lignocellulose to single sugars is the pretreatment

of the biomass, which had been practiced for more than 100 years but has seen a

resurgence of interest in the last decade (Saville, 2011). The aim of pretreatment is to

improve the subsequent enzymatic hydrolysis and fermentation. The recovery of

soluble and therefore fermentable sugars should be maximized, whereas energy

costs, chemical inputs and waste stream disposal should be minimized (Galbe and

Zacchi, 2007). Currently, pretreatment represents 18% of overall production costs,

which is the second most expensive step (Mosier et al., 2005; Yang and Wyman,

2008) . The greatest hurdle in the pretreatment step is the resistance of plant cell

walls to deconstruction. This recalcitrance of lignocellulosic biomass reflects the

highly crystalline structure of cellulose, which is embedded into a complex matrix of

hemicellulose and lignin (Himmel et al., 2007). Pretreatment can involve physical,

chemical or biological processes, and based on the heterogeneous nature of

lignocellulosic substrates it is difficult to develop a universal process (Alvira et al.,

2010).

Physical pretreatment technologies include mechanical comminution or extrusion in

order to reduce the size of particles and increase the surface area. Milling, chipping

and grinding produce particles with a size between 30 and 0.2 mm (Sun and Chen,

2008) and are often combined with other pretreatment technologies.

Chemical pretreatment methods can be classified based on the chemical agent.

Alkaline pretreatment uses sodium or ammonium hydroxide to de-esterify the lignin

polymer (Cheng et al., 2010) and to cause swelling and the partial decrystallization of


8

cellulose and hemicellulose (McIntosh and Vancov, 2010). Acid pretreatment uses

diluted or concentrated acids for the solubilization of hemicellulose and lignin with

minimal degradation (Carrasco et al., 1994). However extensive washing and

detoxification is required in both cases before subsequent hydrolysis and

fermentation (Saha et al., 2005), particularly in the case of acid pretreatment which

can generate potential fermentation inhibitors such as furfurals (Marzialetti et al.,

2008).

Over the last decade, so-called green solvents have emerged as alternatives for the

processing of lignocellulosic biomass, including the extensively studied ionic liquids

(ILs). These are salts comprising a small anion and a large organic cation, with a low

vapor pressure that typically means they remain liquid at room temperature (Vancov

et al., 2012). Ionic liquids can dissolve diverse biomaterials including crystalline

cellulose, which is most efficiently dissolved by ILs based on imidazolium (Vancov et

al., 2012; Zavrel et al., 2009). Cellulose regenerated after treatment with ILs was

more amorphous and porous than before (Kilpeläinen et al., 2007), and underwent

more efficient saccharification (Bodirlau et al., 2010). However, ILs are toxic and tend

to inhibit or inactivate enzymes even at low concentrations, thus increasing the costs

of regeneration (Turner et al., 2003). Therefore needed regeneration or separation

steps included in the process increased the overall cost. The development of

improved biorefinery concepts such as consolidated bioprocessing hence requires

the evaluation of solvents that support the activity of enzymes and microorganisms

(Brodeur et al., 2011).

Biological pretreatment involves the use of microorganisms such as brown, white or

soft-rot fungi to break down lignin and hemicellulose (Sánchez, 2009). The fungi

secrete peroxidases and laccases that oxidize lignin (Kumar et al., 2009) and may

also produce additional enzymes and proteins to enhance subsequent

saccharification, such as swollenins (Jäger et al., 2011). The low capital cost of these

systems and their minimal energy and chemical inputs offer important advantages,

but they still offer low conversion rates compared to other systems (Sun and Cheng,

2002). To establish an economically-feasible approach including biological

pretreatment the conversion rates must be improved substantially.

Other technologies for biomass conversion have been developed that combine those

described above, including ammonia fiber expulsion (AFEX) and organosolv, which


9

combine high temperature and pressure with alkaline treatment and organic solvents,

respectively.

3.3 Enzymes for cellulose conversion

The glycosylhydrolase (GH) superfamily includes many diverse polysaccharide-

degrading enzymes (http://www.cazy.org/). Cellulases are less efficient than other

sugar-hydrolyzing enzymes mainly due to the crystalline nature of the substrate.

Figure 4: Current overview on fungal enzymatic degradation of cellulose. EGs cleave

β-glycosidic bonds at internal amorphous sites, generating oligosaccharides of variable length down to

cellobiose and creating new chain ends (Lynd et al., 2002; Zhang and Lynd, 2004). CBHs release

cellobiose from the ends of cellulose chains acting in a processive manner (Lynd et al., 2002). CBH1

and CBH2 hydrolyze from the reducing and non-reducing ends, respectively. BGLs finally convert

cellobiose to glucose. Non-hydrolytic or expansin-related proteins like swollelin (Swo) can transform

crystalline areas into amorphous areas by loosening the hydrogen bonds between the chains (Jäger et

al., 2011). Recently discovered PMOs such as the CBM33 and GH61 families create new ends by

oxidizing glucopyranose units within the cellulose chains (Horn et al., 2012). Most cellulolytic systems

comprise multiple EGs and CBHs, which vary in their specificity for the substrate, acting on different

crystal faces or parts differing in crystallinity and accessibility. The PMO depicted here represents a

GH61 class enzyme, which acts on C4 atoms to generate non-oxidized ends for CBH1 (oxidized

sugars represented by white circles). CDHs could provide PMOs with electrons by oxidizing

cellobiose. Also other non-enzymatic reductants (electron donors) can induce oxidative activity, e.g.

reduced glutathione, ascorbic acid and gallic acid (Žifčáková and Baldrian, 2012). Abbreviations: BGL,

beta-glucosidase; CBH, cellobiohydrolase; CDH, cellobiose-dehydrogenase; EG, endoglucanase;

PMO, polysaccharide mono-oxigenase; Swo, Swollenin. Adapted from Horn et al. (2012) and Jäger et

al. (2012).


10

The complete conversion of cellulose to glucose requires the activity of three different

classes of enzymes: endoglucanases, exoglucanases and β-glucosidases (Jäger and

Büchs, 2012; Sainz, 2011).

Exo-β 1,4-D-glucanases or cellobiohydrolases (CBH; EC3.2.1.91) act on the ends of

the cellulose chains and processively cleave off cellobiose units. There are two

subgroups, depending on whether they act from the reducing or non-reducing end of

the sugar chain. Endo-β 1,4-D-glucanases (EG; EC3.2.1.4) hydrolyse β–glycosidic

bonds within sugar chains and are more active on amorphous rather than crystalline

cellulose.Finally, β-1,4-D-glucosidases (BGL; EC3.2.1.21) cleave cellobiose units into

glucose. The whole enzyme set acts synergistically to convert cellulose into its

subunits. For example, EGs generate new ends for CBHs, and BGLs remove the

cellobiose which inhibits both EGs and CBHs (Kumar et al., 2008). Many GHs feature

additional domains responsible for adsorption onto the substrate. Such carbohydrate-

binding modules (CBMs) were initially found to bind the surface of crystalline

cellulose (Gilkes et al., 1988; Tomme et al., 1988), but more recent studies have

shown that some CBMs also have disruptive functions (Boraston et al., 2004;

Forsberg et al., 2011).

The mesophilic fungus Trichoderma reesei is one of the best-known sources of

cellulolytic enzymes and is the most widely studied cellulolytic system, secreting a set

of five endoglucanases and two exoglucanases in different ratios (Peterson and

Nevalainen, 2012; Rosgaard et al., 2007). It is a key source of commercial cellulase

preparations, but bacterial and archaeal systems have also been investigated

because these microorganisms and their enzymes demonstrate thermotolerance and

survive under other harsh conditions. The incorporation such enzymes into industrial

processes therefore seems a promising approach (Barnard et al., 2010; Blumer-

Schuette et al., 2008). One example is the hyperthermophilic archaean Sulfolobus

solfataricus, which produces several enzymes involved in carbohydrate degradation

(She et al., 2001) that have been evaluated for biomass degradation activity (Cannio

et al., 2004; Cobucci-Ponzano et al., 2010; Girfoglio et al., 2012; Huang et al., 2005;

Limauro et al., 2001).

Metagenomic libraries can also be screened for novel carbohydrate-degrading

enzymes with advanced features for biomass conversion (Ilmberger et al., 2011;

Pottkämper et al., 2009; Voget et al., 2006). Known enzymes can also be re-


11

engineered to enhance their stability and activity (Lehmann et al., 2012; Liang et al.,

2011; Percival Zhang et al., 2006).

Nevertheless, enzymes for cellulose hydrolysis still represent a major component of

overall process costs (Carroll and Somerville, 2009). Up to 25 kg of enzyme is

required per ton of biomass (Taylor et al., 2008). Despite progress towards the

economical production of enzymes by commercial suppliers such as Novozyme and

Danisco-Genencor this remains a major bottleneck in the production of biofuels from

biomass.

3.4 Advanced feed stocks for fuels

Ethanol is produced from starch and sugarcane by established mature industries

(Geddes et al., 2011) but they provide only marginal societal benefits over fossil fuels

(Hill et al., 2006). Environmental life-cycle analysis has revealed the negative impact

caused by the movement of agrichemicals such as nitrogen/phosphorous fertilizers

and pesticides (Vitousek et al., 1997). The competition between food and fuel crops

also remains a major concern (Geddes et al., 2011). Lignocellulosic feed stocks can

address these concerns. The net energy balance (NEB) is the difference between

energy inputs and outputs throughout the production chain, and NEB models for

lignocellulosic biomass estimate ratios of >4.0 which is a major improvement over

corn grain ethanol (1.25) and soybean diesel (1.93) (Hill et al., 2006). Mixtures of

grasses and fast-growing woody plants are promising candidates for the sustainable

production of biofuels from lignocellolosic biomass (Tilman et al., 2006).

The recalcitrance of the plant cell wall could be overcome by genetic engineering,

which is widely used to modify the properties of crops (Taylor et al., 2008). Strategies

to improve plants for biofuel production include increasing the accessibility of the cell

wall, and modification of the lignin biosynthesis pathway (Vanholme et al., 2008), e.g.

by modulating genes that control the ratio of guaiacyl and syringyl subunits (Chen

and Dixon, 2007) or the overall content of lignin (Jung et al., 2013a). Modification can

lead to unwanted side effects such as dwarf phenotypes or reduced water

conductivity and elasticity (Abramson et al., 2013). However, the modification of plant

polysaccharides by the expression of bacterial CBMs and plant GHs has already

achieved increases in yield, growth rate and porosity even though their exact role

and impact on cell wall biosynthesis is yet not fully understood (Shoseyov et al.,

2006) (Abramson et al., 2013).


12

4. Expression of recombinant glycosylhydrolases in plants

The expression of GHs in plants would reduce the costs of production and improve

the economic feasibility of biomass conversion (Sticklen, 2006; Taylor et al., 2008).

This concept was initially thought unfeasible because the enzymes would disrupt

vegetative growth. Indeed the impact of GHs on cell wall biosynthesis depends on

the species, tissue and the enzyme itself (Abramson et al., 2013; Rudsander et al.,

2008; Takahashi et al., 2009), resulting in both beneficial and deleterious effects

(Maloney and Mansfield, 2010; Park et al., 2003). Different strategies to produce GHs

in plants have therefore been tested in model species such as Arabidopsis and

tobacco (Sainz, 2011; Taylor et al., 2008), and in economically-relevant crops such

as rice and maize (Chou et al., 2011; Harrison et al., 2011; Hood et al., 2007).

However, the use of these crops for biofuel production competes with food and feed

resources, hence the recent focus on grasses and woody plants as alternatives (Jung

et al., 2013b; Vogel and Jung, 2001). Several endoglucanases and xylanases have

been successfully expressed in plants using regulatory elements that target different

tissues and subcellular compartments (Sainz, 2011; Taylor et al., 2008). Complex

enzymes with additional CBMs are often expressed in a truncated form which still

confers full activity on soluble substrates (Harrison et al., 2011; Sun et al., 2007). In

this context, endoglucanase Cel5A from Thermotoga maritime fused to CBM6 from

Clostridium stercorarium xylanase was stably produced in tobacco and showed an

improved efficiency for autohydrolysis (Mahadevan et al., 2011).

The stable accumulation of plant-derived GHs has been achieved by targeting

different subcellular compartments and tissues (Sainz, 2011; Taylor et al., 2008).

Subcellular targeting and tissue-specific expression are also promising for the

sequestration of potentially harmful enzymes during plant growth and development

(Taylor et al., 2008). Inducible expression systems can produce enzymes at similar

levels to constitutive systems while avoiding unwanted deleterious effects, and can

also regulate the amount of enzyme by adaptive induction (Lebel et al., 2008).


13

Table 1: Examples of recent progress in the heterologous expression of GHs in plants. Table

adapted and updated from Taylor et al. (2008)

Enzyme Source Host Targeting Properties

and comments

References

AcE1 endoglucanase Acidothermus

cellulolyticus

Tobacco Chloroplast Transplastomic,

modified CD leads to

high expression levels

(Ziegelhoffer et

al., 2009)

PttCel9A1 endoglucanase Populus tremula Arabidopsis Apoplast/

Cell wall

Modified cell wall

sugar contents

(Takahashi et

al., 2009)

AtCel1 endoglucanase

AaXEG xyloglucanase

HvXylI Xylanase

Tv6GAL galactase

RsAraf1

arabinofuranosidase

Arabidopsis

thaliana,

Aspergillus sp.,

Hordeum vulgare,

Raphanus sativus

Poplar Apoplast/

Cell wall

Altered fiber structure

and composition,

accelerated hydrolysis

of plant material

(Kaida et al.,

2009)

BglB β-glucosidase Thermotoga

maritima

Tobacco Cytoplasm,

chloroplasts

Thermostable

enzyme, preserved

activity after

lyophilization

(Jung et al.,

2010)

TmCel5A endoglucanase Thermotoga

maritima

Tobacco Chloroplast Optimized targeting

with RuBisCO small

subunit signal peptide

(Kim et al.,

2010)

CsXynB Xylanase Clostridium

stercorarium

BY2-cells,

Tobacco,

Rice

Secretion Bacterial signal

peptide is partially

functional in plants

(Kimura et al.,

2010)

endoglucanases, cutinase,

exoglucanase, lipase

pectate lyases, swollenin,

xylanase, acetyl xylan

esterase, β-glucosidase

Different bacteria

and fungi

Tobacco Chloroplast Transplastomic plants,

enzyme cocktails

derived from plat

crude extract

hydrolyzed filter paper,

pine wood or citrus

peel

(Verma et al.,

2010)

AtKOR endoglucanase Arabidopsis

thaliana

Poplar Apoplast/

Cell wall

Led to severe defects

in plant growth,

significantly impacts

the ultrastructure of

cellulose

(Maloney and

Mansfield,

2010)

DtXYnB Dictyoglomus

thermophilum

Maize Apoplast Intein modification

enables heat

activation

(Shen et al.,

2012)

Bsx xylanase

CsXynB xylanase

Bacillus sp.

Clostridium

stercorarium

Maize Apoplast Stunted growth

phenotype

(Gray et al.,

2011b)


cellulolyticus

Rice Apoplast Accelerated

Saccharification of the

transgenic plant

material

(Chou et al.,

2011)

TrMan1 mannase Trichoderma reesei Tobacco Chloroplast Transplastomic plant (Agrawal et al.,

2011)


14


cellulolyticus

Tobacco,

Maize

Accelerated

Saccharification of the

transgenic plant

material

(Brunecky et

al., 2011)

TmCel5A endoglucanase Thermotoga

maritima

Tobacco Chloroplast Engineered CBM for

enhanced hydrolysis

rate

(Mahadevan et

al., 2011)

TfBglC β-glucosidase

TfE3 exoglucanase

TfE4 endoglucanase

TfXeg74 xylanase

Thermobifida fusca Tobacco Chloroplast Transplastomic plants,

homoplasmic plants

showed severe effects

on plant health and

fertility

(Petersen and

Bock, 2011)

DtXynA endoxylanase

TlXynB endoxylanase

NtEGA endoglucanase

AcEGB endoglucanase

Dictyoglomus

thermophilum,

Thermomyces

lanuginosus,

Nasutitermes

takasagoensis,

Acidothermus

cellulolyticus

Maize not specified Transgenic stover

showed enhanced

properties when

utilized in SSF

process for ethanol

generation

(Zhang et al.,

2011)


cellulolyticus

Maize Seed embryo Cross breeding for

high enzyme level

accumulation

(Hood et al.,

2012)

TrXyn2 xylanase Trichoderma reesei Poplar Chloroplast Seed embryo due to

sequestration

(Kim et al.,

2012)

Cutinase

Swollenin

Fusarium solani

Trichoderma reesei

Tobacco Chloroplast (Verma et al.,

2013)

Thermophilic GHs with little activity below 50°C can be expressed functionally in

plants without adverse effects because they are inactive during the growth phase and

can be activated post-harvest by a temperature shift (Basu, 2001; Sainz, 2011;

Taylor et al., 2008). The in planta production of thermophilic GHs is also promising

because such enzymes facilitate lignocellulose hydrolysis under extreme conditions

e.g. thus increasing product and reactant solubility, increasing the speed of the

reaction and reducing the amount of enzyme that is needed (Bhalla et al., 2013). The

catalytic domain of endoglucanase E1 from A. cellulolyticus has been produced in

different plant species and showed great potential for biomass processing (Brunecky

et al., 2011; Chou et al., 2011; Hood et al., 2012) but the incorporation in an existing

pretreatment process (AFEX) revealed that there is still room for improvement

(Teymouri et al., 2004).

Plastid transformation could also facilitate the production of GHs in plants, because

proteins accumulate to high levels in chloroplasts without affecting vegetative growth,

there is no gene silencing, and transgene containment is achieved by the maternal


15

inheritance of chloroplast genomes (Scotti et al., 2012). The high-level expression of

individual functional GHs has been achieved (Gray et al., 2011a; Ziegelhoffer et al.,

2009) but these studies have focused predominantly on bacterial enzymes because

the environment of the chloroplast is similar to that of the bacterial cell. However, a

set of GHs from the fungus T. reesei has also been expressed in transplastomic

tobacco, demonstrating the potential of plastid-derived GHs for lignocellulose

hydrolysis (Verma et al., 2010). Remarkably high levels of T. fusca GHs have also

been expressed in transplastomic tobacco, and although there were severe

deleterious effects in homoplasmic plants, the heteroplasmic plants with wild-type as

well as transformed plastids could be grown to maturity (Petersen and Bock, 2011).

Achieving high yields of recombinant enzymes is one of the key challenges when

enzymes are expressed in plants to facilitate biomass conversion in situ.

Conservative estimates suggest that cellulase must represent at least 10% of TSP to

achieve complete hydrolysis, which is far higher than most reported yields (Sticklen,

2006). Approximately 4 kg of cellulase per hectare has been produced in maize

(Hood et al., 2007) but even this is insufficient for the complete conversion of the

stover (Sainz, 2011). Improved cellulase variants are helpful, but further increase in

productivity is also required (Hood et al., 2012).

Recent developments have shown that even low level expression of recombinant

GHs could contribute to an economically-feasible process for biomass conversion.

The impact of GHs on the structure and composition of the plant cell wall could be

used to make the plant biomass less recalcitrant. Maize and tobacco plants

expressing A. cellulolyticus E1cd showed more efficient saccharification and reduced

the severity of pretreatment (Brunecky et al., 2011). Similar approaches include the

expression of recombinant xylanases to enhance saccharification by altering the

structure of hemicellulose (Borkhardt et al., 2010; Shen et al., 2012; Zhang et al.,

2011). Even though the role of GHs in the construction of plant cell walls is not yet

fully understood and the performance of these transgenic plants has not been

evaluated in the field, the in planta production of GHs nevertheless offers hope for

sustainable bioprocessing.


16

Objectives of this study

The objectives of the study were to establish and evaluate different strategies for the

expression of heterologous cellulases in plants, focusing not only on enzyme

properties such as expression level and functionality, but also on their impact on the

plant development. The model system Nicotiana tabacum was used to generate

transgenic lines expressing different endoglucanase genes targeted to different

subcellular compartments, under the control of constitutive and inducible promoters.

The activity and biochemical characteristics of the enzymes were investigated in the

context of their bioconversion potential. The phenotype of the transgenic plants was

studied at the morphological level to determine the impact of cellulases on growth

and development, and also at the cellular and subcellular level to characterize the

impact on the cell wall. This study will provide new insight in the interdependency

between recombinant cellulase expression and the physiological characteristics of

the host plant.

Chapter 2: Differential targeting of a mesophilic endoglucanase

17

Chapter 2: Differential targeting of a mesophilic

endoglucanase

The results of this chapter have been or will be published as follows:

The Analysis of the biochemical characteristics of TrCel5A has been published in:

Klose H, Günl M, Usadel B, Fischer R, Commandeur U. (2013) Ethanol inducible

expression of a mesophilic cellulase avoids adverse effects on plant development.

Biotechnology for Biofuels, 6:53.

The transient localization studies of TrCel5A-mCherry fusion have been published in:

Garvey M, Klinger J, Klose H, Fischer R, Commandeur U (2013) Expression of recombinant

cellulase Cel5A from Trichoderma reesei in tobacco plants. Journal of Visualized

Experiments, in press

The comparison of different localizations of recombinant TrCel5A in transgenic tobacco is prepared to

be published in:

Klose H, Günl M, Usadel B, Fischer R, Commandeur U. Plant cell wall modification by

differential targeting of recombinant endoglucanase from Trichoderma reesei

The Trichoderma reesei genome contains 22 open reading frames (ORFs)

corresponding to known cellulolytic enzymes (Ouyang et al., 2006). This includes

eight genes representing different endoglucanases. TrCel5A (formerly EGII or EGIII)

is the second most abundant endoglucanase in the cell (Jäger et al., 2010; Ouyang

et al., 2006). It comprises a C-terminal catalytic domain from glycoside hydrolase

family 5, a Ser/Pro-rich linker and a family 1 carbohydrate-binding module (CBM)

(Saloheimo et al., 1988). Transient expression studies have already indicated that

this enzyme can be expressed in tobacco plants (Klose, 2009; Werthmann, 2009).

Here, the results were confirmed and analyzed in more detail so that the

endoglucanase could be produced in transgenic tobacco allowing its impact on plant

development to be investigated.

Results

Analyzing the transient expression of TrCel5A in tobacco plants

Constructs containing the TrCel5A sequence had already been prepared, and

included a plant codon-optimized leader peptide derived from the heavy chain of the

murine monoclonal antibody mAb24 for secretion to the apoplast, and a C-terminal

His6 tag for detection and purification. An additional construct was prepared


18

containing a C-terminal KDEL tag to retain the recombinant protein in the

endoplasmic reticulum (ER). Both genes were controlled by the constitutive double

CaMV 35SS promoter. Transient expression was used to confirm the constructs were

functional before using them for the stable transformation of tobacco plants. A further

construct containing only the catalytic domain from codon 92 to 418 of TrCel5A was

cloned for comparison.

Figure 5: Schematic presentation of plant expression cassettes for the differential targeting of

TrCel5A. The CaMV promoter (P35SS) and terminator (pA35S) are indicated in light blue. The 5'-UTR

of chalcone synthase (CHS) and the His6 coding sequence (His6) are indicated in blue. The plant

codon-optimized leader peptide (LPH) derived from the heavy chain of the murine mAb24 is shown in

green. LPH achieves the secretion of the recombinant protein to the apoplast (A). The C-terminal

KDEL signal indicated in red retrieves the protein to the ER (B). Arrows show primer annealing sites to

amplify the CaMV 35SS expression cassette.

Transient expression of the holoenzyme followed by western blots of total soluble

protein (TSP) revealed two major bands with molecular weights of approximately 45

and 35 kDa, as well as additional intermediate degradation products (Figure 6). The

major bands were similar to the molecular weights of the holoenzyme (Ouyang et al.,

2006) and the catalytic domain (Nakazawa et al., 2008). Transient expression of the

catalytic domain TrCel5Acd produced one main product with a molecular weight of

approximately 35 kDa. Freezing and storage without protease inhibitors resulted in

additional small degradation products but these could be avoided by adding PMSF.

Even the addition of several different protease inhibitors did not prevent cleavage into

the two main fragments (Figure 6) although the serine protease-specific inhibitor E-64

prevented the appearance of the intermediate fragments (approximately 40 kDa).


19

Figure 6: Transient expression of TrCel5A in N. tabacum. Western blot of 10 µg TSP per lane,

derived from frozen and stored crude plant extract following the transient expression of TrCel5AAP,

TRCel5AER and the catalytic domain TrCel5Acd (A). Extraction of recombinant TrCel5AAP in liquid

nitrogen with different protease inhibitors (B). NC: 10µg TSP from N. tabacum SR1 wild-type. PC:

purified His6-tagged phenylalanine ammonium lyase (PAL). Recombinant cellulase was

immunostained with the MαHis primary antibody and GAMFCAP as the secondary antibody.

Analyzing the localization of TrCel5A by transient expression in tobacco plants

The subcellular localization of TrCel5A was investigated by joining the C-terminus in-

frame with the mCherry fluorescent marker protein lacking an additional KDEL tag.

Transient expression was confirmed by the appearance of red fluorescence in the

infiltrated leaf areas and also a 70-kDa band on the corresponding Western blot. The

fusion proteins generated different degradation products depending on the presence

of a KDEL tag, e.g. full version (with the N-terminal CBM1) has only been obeserved

in the tagged version (Figure 7). Infiltrated leafs (representing each of the constructs)

showing red fluorescence were used to prepare 90-µm sections with a Leica

Vibrating Blade Microtome and the subcellular localization of the recombinant

proteins was determined by fluorescence microscopy. Fluorescence for TrCel5A-

mCherryAP is clearly located in the apoplastic space (Figure 8 A-C), whereas for

TrCel5A-mCherryER only a diffuse fluorescence signal was observed (Figure 8 D-E).


20

Figure 7: Transient expression of TrCel5A-mCherry fusion in tobacco. Western blots show the

expression of TrCel5AmCherryAP (A) and TrCel5AmCherryER (B and C). Lanes 1, 3 and 5 were loaded

with 20 µg of total soluble protein from transient expression experiments. Lanes 2, 4 and 6 contain

purified and TCA-precipitated recombinant protein from 0.5 g of leaf material. NC: 20 µg of total

soluble protein from wild-type tobacco cv. SR1. The positive control (PC) in (A) and (B) is His6-tagged

PAL, and in (C) it is KDEL-tagged DsRed. The antibody system used in (A) and (B) is

MαHis5/GAMFcAP, and in (C) it is MαKDEL/GAMFc

AP. The presence of the 70-kDa fusion protein was

confirmed by detecting either the His6 tag or (in the case of TrCel5AmCherryER) the KDEL tag.

Figure 8: Localization of transiently-expressed TrCel5A-mCherry fusion in 90-µm sections of

tobacco leaf tissue. The LPH signal peptide promotes the secretion of TrCel5A-mCherry as indicated

by the strong red fluorescence in the apoplast (A-C). A C-terminal KDEL tag retrieves the recombinant

enzyme to the interior of the cell, as indicated by the strong but diffuse red fluorescence.


21

Generation of transgenic tobacco plants expressing TrCel5A

The constructs encoding the holoenzyme with and without the KDEL tag were

introduced into tobacco (Nicotiana tabacum SR1) leaf discs by Agrobacterium-

mediated transformation (Horsch, 1985). Each generation of plants was screened for

the presence of the enzyme by Western blot. The enzymatic activity was tested by

the conversion of azo-dyed carboxymethylcellulose (azoCMC) and

4-methylumbelliferyl-β-D-cellobioside (4MUC). Transgene integration was confirmed

by genomic PCR and activity assays (data not shown). Homozygous lines revealing a

3:1 segregation ratio consistent with a single locus insertion were used to produce

subsequent generations of plants (Table 2).

Table 2: Expression of TrCel5A in transgenic tobacco plants. The table shows transgenic events

and fertile plants transformed with vectors designed to express T. reesei endoglucanase. The mean

expression level was tested by 4MUC conversion for of all plants transformed with each construct.

*Values for the second generation represents the mean of three homozygous lines (T2) based on 10

samples each.

Construct Gene

[GeneBank]

Subcellular

localization

Total

plants

Number of

events

Expression

level T1

[mU/mg TSP]

Expression

level T2*

[mU/mg TSP]

TrCel5AER

AAA34213.1 ER 50 39 15.9±11.8 40.8±5.2

TrCel5AAP

AAA34213.1 Apoplast 35 13 9.4±7.5 29.9±6.4

Western blots showed that homozygous transgenic plants expressing the secreted

version of TrCel5A yielded a recombinant protein with a molecular weight of ~35 kDa,

similar to that of the native catalytic domain (Figure 9C). In contrast, both western

blots and substrate SDS-PAGE activity staining of extracts from homozygous lines

expressing TrCel5A with a KDEL tag revealed the same two major bands produced

in the transient expression experiments.


22

Figure 9: Analysis of transgenic tobacco expressing TrCel5A. Amplification of genomic DNA

prepared from wild-type and transgenic plants, using primers pSS-5 and pSS-3 which anneal within

the CaMV 35SS expression cassette (A) and primers spanning a highly conserved region in the

chloroplast genome as a positive control (Demesure et al., 1995) (B). Lanes: M, GeneRuler™ 1-kb

ladder; 1,2 TrCel5AAP; 3,4 TrCel5AER; 5,6 SR1 wild-type; 7 H2O; 8 control plasmid DNA pTRAkc-

TrCel5A-ER. Western blot of recombinant TrCel5A from different TrCel5AAP lines (lanes 1-3) and

TrCel5AER lines (lanes 4-6) (C). Lanes were loaded with 10 µg TSP from transgenic plants (1-6), wild-

type plants (7) and IMAC purified PAL-His6. The MαHis5/GAMFcAP antibody system was used in (C).

All transgenic lines produced TrCel5A degradation products, but small amounts of the full enzyme

were detected in the TrCel5AER lines. The expression level of TrCel5A in transgenic plants was

determined by the conversion of 4MUC (D). TrCel5AER plants showed approximately 1.25-fold higher

activity than TrCel5AAP plants.

Recombinant protein expression levels in transgenic tobacco plants were determined

by measuring the activity of total soluble proteins (TSP) against the substrates

azoCMC and 4MUC. T2 plants expressing the secreted version of TrCel5A achieved

a specific enzyme activity of up to 1.5 U mg-1 TSP on azoCMC (data not shown) and

35 nmol 4MU min-1 mg-1 on 4MUC (Figure 9D), whereas the ER-localized version

achieved a specific enzyme activity of up to 2.1 U mg-1 on azoCMC and

46 nmol 4MU min-1 mg-1 on 4MUC (Figure 9D).


23

Biochemical characteristics of TrCel5A produced in planta

Figure 10: Enzymatic activity of TrCel5A at different pH values and temperatures. TrCel5A

activity was determined using the azoCMC assay for pH values between 3.0 and 7.0 (A) and

temperatures in the range 20–70°C (B). The maximum activity measured for each system (at pH 4.8

and 55°C, respectively) was set to 100%. For panel A, buffer systems are used as indicated in the

figure. For panel B, acetate buffer (pH 4.8) was used throughout.

Plant-derived TrCel5A was extracted and purified by IMAC to determine its

biochemical characteristics, such as its activity on azoCMC in different buffer

systems within the pH range 3.0–7.0 and the temperature range 20–70°C (Figure

10). The optimal conditions were pH 4.8 and 55°C. Under these conditions, 50% of

the enzyme remained active for 90 min. There was no significant activity against

highly crystalline substrates such as Avicel, but there was significant activity against

heterogeneous substrates including lichenan and barley-β-glucan (Table 3).

Table 3: Hydrolytic activity of TrCel5A against different polymeric carbohydrates. The quantity

of reducing sugars released in the reaction was determined using a PAHBAH assay. Activity against

carboxymethylcellulose (CMC) was set as 100%. N.D: no detectable activity.

CMC β-Glucan Lichenan PASC Avicel Xylan

Relative

activity [%] 100±2 120±3 136±5 22±2 N.D. N.D.


24

Subcellular localization of TrCel5A in tobacco plants

The localization of TrCel5A in the transgenic tobacco plants was determined by

immunostaining and fluorescence microscopy. Thin sections [10 µm] of the vascular

bundles of tobacco leaves were prepared using a Leica Cryostat Microtome.

Figure 11: Subcellular localization of recombinant TrCel5AER in transgenic tobacco cells. Plants

expressing TrCel5AER were immunostained with the MαKDEL primary antibody and GAMFCFITC as the

secondary antibody. Images were obtained by fluorescence microscopy. The SR1 wild-type control (A)

was compared with the TrCel5AER transgenic plants (B). Intracellular fluorescent signals were

detected in the TrCel5AER transgenic plants in some cells (B), whereas control plants did not show

significant fluorescent staining (A).

When the sections were stained with either RαCell and GARFCFITC or MαKDEL and

GAMFCFITC, strong green fluorescence indicated the presence of the recombinant

enzyme in in some cells of the transgenic plants but not the wild-type control.

Fluorescence was more easily detected in the TrCel5AAP tissue despite the higher

recombinant protein expression levels in the TrCel5AER plants (Figure 12). As

expected, MαKDEL only detected the enzyme carrying the KDEL tag (in the

TrCel5AER tissues) and intracellular localization was confirmed (Figure 11).


25

Figure 12: Subcellular localization of recombinant TrCel5A in transgenic tobacco cells. Plants

expressing variants of TrCel5A targeted to the apoplast and ER were immunostained with the RαCell

primary antibody and GARFCFITC as the secondary antibody. Images were obtained by fluorescence

microscopy. The SR1 wild-type control (A) was compared with TrCel5AER (B) and TrCel5AAP (C)

transgenic plants. Intracellular fluorescent signals were detected in the TrCel5AER tissues, whereas a

peripheral signal in the cell walls was detected in the TrCel5AAP tissues. Control plants did not show

any significant fluorescent staining (A).

Growth characteristics of the transgenic tobacco plants

The constitutive expression of TrCel5A and its localization in the apoplast reduced

the growth of tobacco plants and significantly delayed their development. The stem

length and germination rate were both reduced (Figure 13 and Figure 14). The

expression of TrCel5A with a KDEL peptide for retention in the ER resulted in a much

less significant difference in growth and development (Figure 13 and Figure 14). For

both of the constructs, a few transgenic plants displayed a curly leaf phenotype, and

in the TrCel5AAP plants this was accompanied by necrotic areas on the leaf (Figure

13C).


26

Figure 13: Transgenic tobacco plants expressing TrCel5A. Eight-week-old N. tabacum SR1 wild-

type plants were grown in soil under standard conditions (A & D). TrCel5AAP plants showed a

significant reduction in the growth and germination rate (E), whereas TrCel5AER plants showed a near

normal growth and development phenotype (F). Several transgenic plants displayed a curly leaf

phenotype (B & C) which in the case of TrCel5AAP plants included additional necrotic lesions (C).


27

Chemical analysis of tobacco cell walls

The structural carbohydrate composition of plants expressing differentially-targeted

TrCel5A was determined in leaf material from six-week-old tobacco plants.

Destarched alcohol-insoluble residues (AIRs) were hydrolyzed with weak acid (Foster

et al. 2010) and the solubilized carbohydrates were measured as alditol acetate

derivatives by GC/MS (Albersheim et al., 1967). The crystalline cellulose content of

the tobacco leaf material was determined by Updegraff hydrolysis (Updegraff, 1969a)

and an anthrone cellulose assay. Wild-type plants were compared with transgenic

lines expressing TrCel5A in the apoplast and ER.

Figure 14: Phenotypic analysis of transgenic tobacco plants. The crystalline cellulose content and

stem length was analyzed in six-week-old wild-type and TrCel5A transgenic tobacco plants grown in

soil. Asterisks indicate a significant decrease (P ≤ 0.01) in the crystalline cellulose content for

TrCel5AAP compared to wild-type plants whereas TrCel5AER plants show no significant difference (A).

The comparison of stem length between TrCel5A transgenic and wild-type plants showed that

TrCel5AAP lines with the lowest cellulose levels also has the shortest stems (B).

Transgenic lines accumulating TrCel5A in the apoplast showed dramatic changes in

the content of crystalline cellulose, which was significantly lower than in wild-type

plants, whereas transgenic plants accumulating TrCel5A in the ER did not show such

differences (Figure 14A). Analysis of the matrix phase polysaccharides also revealed

dramatic differences in the composition of simple sugars (Table 4). The TrCel5AAP

lines contained less glucose and more xylose in the matrix phase polysaccharides,

whereas the TrCel5AER lines showed only a marginal difference from the wild type


28

levels. The amount of galactose increased in all the transgenic lines. Arabinose and

rhamnose levels were lower in the TrCel5AAP lines, but there was no change in the

level of mannose.

Table 4: Compositional analysis of matrix-phase polysaccharides in different transgenic

tobacco lines. Relative values [%] for sugar distribution in matrix polysaccharide fraction. Data

analyzed by two-sided Student’s t-test; * indicates a significant P < 0.01; + indicates a significant P <

0.05

Line Arabinose Glucose Galactose Mannose Rhamnose Xylose

Wild-type 20.41

±2.61

23.60

±8.42

16,85

±2.14

6.12

±1.10

17.90

±2.28

13.96

±1.81

TrCel5AAP-1 14.95*

±1.36

10.81 *

±1.27

32.71*

±6.71

5.31

±1.69

14.42+

±3.68

21.79*

±3.44

TrCel5AAP-2 16.63+

±1.86

13.90 *

±4.68

34.08*

±4.68

5.01

±1.18

13.43*

±1.69

16.94*

±1.93

TrCel5AER-1 20.72

±1.65

17.93+

±2.46

27.70*

±2.93

4.99+

±0.74

16.92

±1.55

13.91

±1.35

TrCel5AER-2 16.11*

±1.09

19. 74+

±2.59

33.11*

±2.37

4.73+

±1.56

13.36*

±1.13

11.04*

±1.10

TrCel5AER-3 18.96

±1.93

20.54+

±3.44

27.22*

±3.51

4.50*

±1.42

16.21

±1.85

11.97*

±1.19

Two of three TrCel5AER lines contained lower levels of xylose, and all three lines

contained slightly lower levels of mannose compared to wild-type plants. There were

no significant changes in the levels of arabinose and rhamnose in two of the

TrCel5AER but there was a significant reduction for both sugars in the remaining line.

The biochemical changes in the cell wall sugars of TrCel5AAP lines were associated

with the growth reduction phenotype, but as discussed above there was no

significant visible change in the TrCel5AER lines despite the modulation of sugar

levels.


29

Hydrolysis of leafy biomass from transgenic tobacco

To determine the rate at which transgenic leaf material was hydrolyzed by

commercially-available cellulase preparations, tobacco plants of both varieties were

selected, with similar levels of TSP and TrCel5A activity (based on the release of

4MU) and compared to wild-type controls. Following enzymatic hydrolysis, the

crystalline cellulose remaining in the insoluble material was quantified.

Figure 15: Enzymatic hydrolysis of transgenic tobacco leaves. Leaf tissue from transgenic

tobacco lines TrCel5AAP and TrCel5AER, both expressing ~30 mU/mg TSP (based on 4MUC

conversion) were hydrolyzed with the appropriate amount of the commercial cellulose preparation

CTec2 (Zhang et al., 2011). Digestion was carried out at 55°C with constant shaking at 1000 rpm. The

TrCel5AAP leaves underwent more rapid hydrolysis than the TrCel5AER and wild-type leaves, but

eventually reached the same level of total cellulose hydrolysis.

The TrCel5AER transgenic leaves did not show any difference in the rate of hydrolysis

compared to wild-type leaves, regardless of the TrCel5A expression level. In

contrast, the TrCel5AP transgenic leaves were hydrolyzed more rapidly, although

ultimately the relative amount of solubilized crystalline cellulose was the same as that

in TrCel5AER and wild-type leaves (Figure 15).


30

Discussion

Current research in the area of biofuels aims to achieve the economically feasible

production of cellulolytic enzymes for the conversion of lignocellulosic biomass into

soluble sugars. Plants offer a relatively inexpensive production platform for industrial

proteins (Fischer et al., 2004; Streatfield, 2007) and therefore plants have been used

to express a number of biomass-degrading enzymes (Table 1 and reviewed in Sainz,

2011; Taylor et al., 2008).

Several studies have demonstrated the potential of in planta recombinant cellulases

by achieving the accumulation of functional enzymes at high levels (Brunecky et al.,

2011; Hood et al., 2012; Petersen and Bock, 2011). However, several drawbacks are

associated with constitutive cellulase expression, including growth inhibition, changes

in leaf morphology, loss of cell wall integrity and reduced stress tolerance (Abdeev et

al., 2004; Petersen and Bock, 2011; Taylor et al., 2008).

Plants express endogenous GHs that play an important yet barely understood role in

cell wall synthesis (Lopez-Casado et al., 2008). Both the overexpression and

silencing of these GHs can have a negative impact on growth and cell wall

development (Maloney and Mansfield, 2010; Takahashi et al., 2009).

These issues have been addressed by targeting cell wall-degrading enzymes to

different subcellular compartments, which can increase the overall enzyme yields

(Buanafina et al., 2010; Dai et al., 2005; Mahadevan et al., 2011) as well as keeping

potentially harmful enzymes away from the cell wall (Kim et al., 2010; Ziegelhoffer et

al., 2009). We therefore compared transgenic plants in which the T. reesei mesophilic

endoglucanase TrCel5A was either secreted to the apoplast or retained in the ER.

Already in the transient studies the localization of TrCel5A has been monitored with

mCherry fusions of the enzyme, proving the correct targeting for TrCel5A-mCherryAP

in the apoplastic space and TrCel5A-mCherryER inside the cells. For transgenic

plants TrCel5A localization was confirmed by immunostaining of tissue thin sections,

which approved the result of the transient study, though the staining did not reflect

the expression level of the enzyme. Further studies using embedding techniques

before sectioning might lead to improved results.

Transient expression revealed that TrCel5A underwent proteolytic cleavage within

the linker region, probably caused by papain-like proteases in the apoplast (Beyene

et al., 2006) that are known to degrade T. reesei cellulases (van Tilbeurgh et al.,

1986). Adding a specific inhibitor of this protease class (E-64) during extraction


31

reduced the amount of degradation, albeit not completely. However, the truncated

enzyme remained active against soluble substrates such as azoCMC and other

β-glucans with a β1,4-linked glucose backbone, whereas carbohydrates with different

linkages remained unaffected (Nakazawa et al., 2008). The apoplast targeting

strategy yielded the truncated and active form of TrCel5A alone, whereas retention in

the ER yielded a mixture of the truncated and full-length enzyme, although the latter

was a minority product. TrCel5A activity in the transgenic plants was determined by

measuring the conversion of 4MUC into 4MU, resulting in a maximum of

33 nmol 4MU min-1 mg-1 in the TrCel5AAP plants and 46 nmol 4MU min-1 mg-1 in the

TrCel5AER plants. This probably reflects the relative paucity in the number and

quantity of proteases in the ER lumen compared to the apoplast (Beyene et al.,

2006).

The optimal reaction conditions for the recombinant TrCel5A were found to be 55°C

and pH 4.8, highlighting the mesophilic origin of the enzyme. However, the enzyme

also showed remarkable residual activity under physiological conditions typical for

tobacco plants (20–30°C, pH 5.0) suggesting it potentially interferes with the

synthesis of cell wall cellulose during normal plant growth and development. Thus it

was not surprising that the TrCel5AAP transgenic plants grew more slowly and

germinated less frequently, whereas there was no significant difference between the

TrCel5AER and wild-type plants. Despite the differences between the transgenic

plants accumulating TrCel5A in different compartments, there was no significant

change in the overall amount of crystalline cellulose for TrCel5AER plants compared

to the wild-type lines. Cell wall cellulose is synthesized by cellulose synthase

complexes in the plasma membrane. After polymerization, the glucan chains are

extruded into the extracellular space (Endler and Persson, 2011) and therefore

endoglucanase expression targeted to the apoplast has the potential to interfere with

endogenous cellulose synthesis. This corresponds to our observation that the

content of crystalline cellulose is reduced in TrCel5AAP plants but not in TrCel5AER

plants. However the role of endoglucanases during the synthesis of cell wall cellulose

appears far more intricate than previously reported. Plant endoglucanases such as

KORRIGAN are necessary for the accumulation of sufficient cellulose and its

absence leads to severe deficit (Molhoj et al., 2001; Takahashi et al., 2009). Recent

studies have shown that the overexpression of KORRIGAN and other recombinant


32

endoglucanases can also reduce the crystallinity of cellulose (Brunecky et al., 2011;

Maloney and Mansfield).

The analysis of matrix polysaccharide composition of transgenic and wild-type

tobacco plants revealed a significant reduction in the relative values of glucose,

arabinose and rhamnose in the TrCel5AAP lines, but much higher levels of galactose

and xylose. Plant GHs have been shown to remodel cell wall polysaccharides in the

apoplastic space and during the transport of polysaccharide precursors through the

Golgi apparatus (Minic, 2008). TrCel5A is a strict endoglucanase and is only known

to cleave β1,4-linkages between glucopyranose units, while showing no activity

against complex mixed glucans (Nakazawa et al., 2008). The degradation is

therefore likely to take place at an earlier stage of XyG synthesis, when the glucan

chains are still undecorated. The higher levels of galactose and xylose would directly

reflect the loss of glucose.

The relative level of glucose was also lower in the TrCel5AER lines, albeit not to the

extent seen in the TrCel5AAP lines. Galactose levels were also higher, as in the

TrCel5AAP plants, but xylose levels were lower in two of three tested lines. All

TrCel5AAP plants had lower levels of mannose and one line also showed lower levels

of arabinose and rhamnose. These changes in the matrix polysaccharide

composition for TrCel5AER lines were not associated with a visible phenotype in

contrast to the observed changes in the TrCel5AAP plants.

ER localization was used to prevent the enzyme from entering the Golgi apparatus

where most hemicellulose and pectin synthesis takes place (Moore and Staehelin,

1988; Staehelin and Moore, 1995). However, recent studies have shown that ER-

localized enzymes can still affect matrix polymer synthesis. The deferuloylation of

arabinoxylans in tall fescue can be achieved using a fungal ferulic acid esterase A

(FAEA). Even if the detected values for Golgi apparatus localization - the place of the

synthesis - were higher, significant cleavage was observed for ER retardation of the

enzyme (Buanafina et al., 2010). This may reflect the promiscuous behavior of

KDEL-tagged proteins, which can in some cases enter the Golgi network from where

they are later retrieved (Pelham, 1990).

XyG is the most abundant hemicellulose in the cell walls of dicotyledonous plants,

representing up to 30% by weight (Scheller and Ulvskov, 2010). In most dicots the

main form is fucogalacto-XyG, but arabinogalacto-XyG is more abundant in

solanaceous plants and monocots (Pauly et al., 2013; Ring and Selvendran, 1981).


33

In fucogalacto-XyG, galactose is transferred to the xylosyl residue and then

substituted with fucose, whereas in arabinogalacto-XyG galactose or arabinose can

be transferred to the xylosyl residue (Pauly et al., 2013). The scarcity of glucose

could then lead to the reduced levels of xylose in the matrix polysaccharides of

TrCel5AER plants. In contrast, xylose levels were normal in the TrCel5AAP lines.

Interestingly, galactose levels were elevated in all the plants. However, despite these

differences in biochemistry, there was no significant morphological or developmental

change in the TrCel5AER plants, which is consistent with previous observations of

mutant plants with changes in the XyG side chain composition (Jensen et al.). All

biochemical ana-lyses were carried out with plants grown in the greenhouse without

the addition of fertilizer. Therefore it is possible that the addition could reduce the

metabolic stress in the transgenic plants and diminish the observed changes.

However, if the same lines will be tested in field experiments, this stress is more likely

increased.

The remaining amount of crystalline cellulose was measured in the tobacco leaves

after digestion with a commercial cellulase preparation, revealing a two-fold greater

reduction in TrCel5AAP plants after 8 h compared to wild-type and TrCel5AER plants.

After 65 h however, all three lines contained the same relative amounts of residual

cellulose, showing that degradation was accelerated but not enhanced overall in the

TrCel5AAP plants. Quantitative effects were ruled out by carefully selecting transgenic

plants of both lines with similar overall enzyme expression levels, and in any case we

found no overall difference between the transgenic and wild-type lines.

Endoglucanases produced in planta have previously been shown to make the plant

cell wall less recalcitrant to hydrolysis (Brunecky et al., 2011). Accordingly, our

results confirmed a significant reduction in the levels of crystalline cellulose in

TrCel5AAP plants followed by accelerated hydrolysis.

The altered matrix polysaccharide fraction of the TrCel5AER plants appeared to have

little impact on the enzymatic hydrolysis of leaf material, which was similar to the

profile in wild-type leaves. This may reflect the relatively simple assay substrate,

which does not consider the impact of lignin due to its low abundance in leaves

compared to stems and roots (Van Dyk and Pletschke, 2012). The modification of cell

wall hemicellulose using hydrolytic enzymes can promote lignocellulose degradation

by reducing the amount of crosslinking between polymers (Buanafina et al., 2010;

Pogorelko et al., 2011; Zhang et al., 2011).


34

In summary, the heterologous expression of the mesophilic endoglucanase TrCel5A

had a significant impact on the composition of plant cell wall polysaccharides,

although the precise changes were dependent on the subcellular localization of the

enzyme. Secretion to the apoplast reduced the levels of crystalline cellulose in the

cell wall and had a significant impact on plant growth and development, whereas

targeting to the ER resulted in compositional changes among the matrix-phase

polysaccharides but did not cause any adverse morphological or developmental

phenotypes.

Chapter 3: Ethanol-inducible endoglucanase expression in tobacco

35

Chapter 3: Ethanol-inducible endoglucanase expression in

tobacco

The comparison of inducible and constitutive expression of TrCel5A in transgenic tobacco has been

published in:




The high-level constitutive expression of GHs can affect the structure and

composition of plant cell walls by altering the quality and/or quantity of cellulose

(Chapter 2). The regulation of endoglucanase expression using inducible promoters

is therefore a promising approach to avoid the drawbacks caused by cellulase

accumulation during vegetative growth. Many different inducible expression systems

have been developed for plants (Bortesi et al., 2012; Bruce et al., 2000; Okuzaki et

al., 2011) and the Aspergillus nidulans alc platform has been widely used (Caddick et

al., 1998; Roslan et al., 2001; Tomsett et al., 2004). This platform comprises two

elements, the coding sequence for the transcription factor AlcR (controlled by the

CaMV 35S promoter), and the alcAmin 35S promoter, in which two AlcR binding sites

are joined to the CaMV 35S minimal promoter (Caddick et al., 1998). This promoter

shows negligible basal expression but strong induction ratio in most plants in

response to ethanol (Caddick et al., 1998; Roslan et al., 2001; Salter et al., 1998).

We used the alc platform to express the secreted form the TrCel5A endoglucanase

from T. reesei resulting in its accumulation in the apoplast of tobacco cells.

Results

Generation of transgenic tobacco plants expressing inducible TrCel5A

The cDNA encoding TrCel5A (lacking the native signal peptide sequence) was

placed under the control of the ethanol-inducible alcAmin35S promoter (Caddick et

al., 1998). The construct included a plant-codon-optimized signal peptide derived

from the heavy chain of the murine monoclonal antibody mAb24 for secretion to the

apoplast, and a C-terminal His6 tag for detection and purification. This construct was

then introduced into N. tabacum SR1 leaf discs by Agrobacterium-mediated


36

transformation (Horsch, 1985). Integration of the transgene was confirmed by

genomic PCR (Figure 16). After induction, each generation of plants was screened

for the presence and activity of the enzyme by Western blot and activity staining on

azoCMC and 4MUC gels. Positive T1 lines showing 3:1 transgene segregation

consistent with a single locus were selfed to produce the subsequent homozygous

generations.

Figure 16: Generation and analysis of transgenic tobacco expressing TrCel5A under the

control of an alcohol-inducible promoter. Schematic presentation of the plant inducible expression

cassette (A). The chimeric promoter (alcAmin35S) comprising the minimal CaMV35S promoter (±31 to

+5) fused to the upstream promoter sequences of alcA (Caddick et al., 1998) is shown in dark blue.

The terminator signal (pA35S) is shown in light blue. The chalcone synthase (CHS) gene 5'-UTR and

His6 tag sequence are indicated in navy blue. A plant-codon-optimized leader peptide (LPH) derived

from the heavy chain of the murine mAb24 is shown in green. Genomic DNA prepared from wild-type

and transgenic plants was tested by PCR using primers binding within the trcel5a gene (cel5a-seq)

and the pA35S terminator sequence (pss-3) (B) and primers binding in a highly-conserved region in

the chloroplast genome (C) (Demesure et al., 1995). Lanes: M, GeneRuler™ 1kb; 1-4,

alcR::TrCel5AAP lines; 5,6 SR1 wild-type; 7 water; 8 control plasmid pTRAkc-alcR-TrCel5A-AP.


37

Inducible TrCel5A expression in tobacco plants

The impact of alcohol induction on plant growth and TrCel5A expression was tested

on T2 alcR::TrCel5A transgenic plants and wild-type controls. The induction of

TrCel5A expression with 2% (v/v) ethanol led after 24 h to a maximum activity of

0.4 U mg–1 TSP on azoCMC and 5 nmol 4MU min-1 mg-1 (Figure 17). Further analysis

showed that significant expression could be achieved by inducing with 0.1% (v/v)

ethanol, but optimal expression levels required 2% (v/v) ethanol (Figure 17B).

TrCel5A expression did not increase when plants were induced with 5% (v/v) ethanol,

and higher concentrations were detrimental to both wild-type and transgenic plants,

resulting in physiological stress symptoms such as leaf yellowing. Significant basal

expression in the absence of ethanol was not detected.

Figure 17: Inducible TrCel5A expression in tobacco plants. Time course of ethanol induction in

six-week-old soil-grown T2 plants irrigated with ethanol solutions. Plants were induced at t0 by

irrigating with 2% (v/v) ethanol, and cellulase activity was monitored over a 96-h time course (A).

Transgenic lines were monitored for cellulase activity 24 h after watering with different ethanol

solutions. Values represent the mean of three plants per independent transgenic line (B). The effect of

sequential ethanol induction (C). Six-week-old soil-grown plants from the homozygous line

alcR::TrCel5A-3 were induced with 2% (v/v) ethanol at t0 and t48 (asterisks). Cellulase activity was

monitored over a six-day time course. Each data point represents the mean of three replicates. Wild-

type plants were monitored in parallel, and no activity was observed throughout the experiment.


38

To determine the time course of induction, TrCel5A activity was monitored for 4 days

following induction with 2% (v/v) ethanol (Figure 17A). Peak activity was observed

48 h after induction (7 nmol 4MU min-1 mg-1) followed by a significant decline.

Continuous or sequential induction has been shown to boost transgene expression

levels (Roslan et al., 2001), so a second induction step was introduced 48 h after the

first induction. This increased the enzyme activity significantly, to

27 nmol 4MU min-1 mg-1 (Figure 17C). This was lower than the enzyme activity

achieved in constitutive TrCel5A plants, but the two-step induction nevertheless

achieved an expression level within the same order of magnitude as the constitutive

promoter.

Growth characteristics and histology

The inducible expression of the secreted form of TrCel5A had no impact on the

growth or development of the transgenic tobacco plants compared to wild-type

controls (Figure 18). Plants with constitutive expression and secretion of TrCel5A as

described in chapter A, exhibited a significantly reduced growth and a delayed

development. The mature stems of the transgenic lines were 21–36% shorter than

controls and delayed flowering. Cross-sections of stems stained with calcofluor-white

showed no significant differences in cell structure between alcR::TrCel5A and wild-

type plants, whereas there was a slight increase in the number of small vessels in

TrCel5AAP plants (Figure 18).


39

Figure 18: Phenotype of transgenic tobacco plants. Cross sections of wild-type (A), TrCel5AAP (B)

and alcR::TrCel5AAP (C) stems (10x magnification) stained with calcofluor white and visualized under

UV light. There were no significant differences other than a marginal increase in the number of small

vessels (white arrow) in TrCel5AAP plants. Scale bars = 50 µm. Morphological phenotype of wild-type

(D), TrCel5AAP (E) and alcR::TrCel5AAP (F) plants grown under photoautotrophic conditions in soil.

TrCel5AAP plants were significantly shorter and had a lower germination rate compared to wild-type

and alcR::TrCel5AAP plants.


40

Chemical analysis of tobacco cell walls in alcR::TrCel5AAP plants

The impact of inducible TrCel5A expression on the structure of tobacco cell walls was

determined by measuring the amount of cellulose, alcohol-insoluble residues (AIRs)

and crystalline cellulose as described in Chapter 2.

Figure 19: Crystalline cellulose content in transgenic tobacco plants expressing TrCel5A. The

cellulose content was measured in six-week-old soil-grown transgenic tobacco plants transformed with

constitutive and inducible expression constructs, compared to wild-type controls (A). Values represent

the mean ± standard error of 10 plants per tobacco line (transgenic or wild-type). Asterisks indicate a

significant difference compared to the wild-type (P ≤ 0.01). Cellulase activity and cellulose content was

also measured in alcR::TrCel5AAP-3 plants after double induction with ethanol (B). A relative value of

100% cellulase activity represents the conversion of 27 nmol 4MU min-1 mg-1, whereas a value of

100% for cellulose content represents 140 µg glucose per mg alcohol insoluble residue (AIR). For all

panels, values represent the mean of three plants per independent transgenic line. Error bars show

standard deviations normalized against the wild-type controls.

Here wild-type plants were compared with three constitutive TrCel5AAP lines and two

inducible alcR::TrCel5AAP lines. As stated earlier, the constitutive transgenic lines

contained significantly lower levels of crystalline cellulose than the wild type plants

and the TrCel5AAP line with the highest enzyme activity also showed the greatest

reduction in crystalline cellulose (Figure 19A). In contrast, there was no difference in

crystalline cellulose levels between the inducible transgenic plants and wild-type

controls, even up to 120 h after the induction of cellulase activity (Figure 19B).


41

Discussion

As discussed in Chapter 2, the precise role of GHs in the development of plant cell

walls is not yet understood. The depletion and overexpression of GHs can each

affect plant growth and the formation of cell walls, and this represents a major

challenge when recombinant GHs are expressed in plants for biomass degradation

(Maloney and Mansfield, 2010; Park et al., 2003; Takahashi et al., 2009). In several

studies, the constitutive expression of biomass-degrading enzymes has been shown

to affect plant health, e.g. inhibiting growth, altering leaf morphology, reducing stress

tolerance and affecting the structure of the cell wall (Abdeev et al., 2003; Brunecky et

al., 2011; Petersen and Bock, 2011; Taylor et al., 2008). Therefore, alternative

strategies are required for the production of GHs such as cellulases in plants, without

affecting normal growth and development. These include sequestration into

subcellular compartments (Chapter 2) or the expression of enzymes that are inactive

under physiological conditions (Dai et al., 2000). However, the inducible expression

of cellulases could allow the expression of functional enzymes just prior to harvest,

thus avoiding detrimental effects during vegetative growth.

Ethanol-inducible expression cassettes are widely used in plant biotechnology (Ait-ali

et al., 2003), and here we combined the alcAmin 35S promoter, a fusion of the AlcR

binding site with the minimal CaMV 35S promoter (Caddick et al., 1998), to achieve

the inducible expression of TrCel5A, and used the doubled CaMV 35SS promoter as

a comparator for constitutive expression. Both promoters were used to express the

secreted form of the enzyme, which was shown to accelerate biomass degradation in

Chapter 2.

The one-step induction procedure with 2% (v/v) ethanol had no significant impact on

the health of wild-type tobacco plants and achieved a significant level of cellulase

activity, but after 24 h this was 4–6 times lower than achieved by constitutive

expression. Different induction times and ethanol concentrations were tested, but 2%

ethanol was found the optimal concentration for induction, in agreement with

previous reports (Roslan et al., 2001). TrCel5A activity peaked at 7 nmol 4MU min-1

mg-1 48 h after induction, compared to 35 nmol 4MU min-1 mg-1 achieved by

constitutive expression. We introduced a second induction step after 48 h because

previous reports have shown this can boost transgene expression (Roslan et al.,

2001). Accordingly, this boosted the maximum enzyme activity to

27 nmol 4MU min-1 mg-1 5 days after the first induction step. Although still lower than


42

the activity achieved by constitutive expression, it was in the same order of

magnitude (and was obtained without a negative impact on plant growth and

development) and was comparable to the levels achieved by other researchers

(Brunecky et al., 2011; Dai et al., 2005).

There was little difference in the crystalline cellulose content of the alcR::TrCel5AAP

transgenic plants before and after induction, suggesting that the endoglucanase only

reduces the crystallinity of the cell wall when cellulose is synthesized (evidently this

occurs during constitutive expression). This theory is also supported by the lower

endoglucanase activity of TrCel5A against more crystalline substrates, such as Avicel

and PASC (Table3). The truncation of the enzyme, which removes the CBM, could

also contribute to the lower affinity for crystalline substrates (Boraston et al., 2004).

Alternatively, the efficient hydrolysis of crystalline cellulose may require more than

one cellulolytic activity (Henrissat et al., 1985). These data suggest that the absence

of the CBM might also be advantageous for systems based on the constitutive

expression of TrCel5A. Potentially, the expression of CBMs alone could disrupt cell

wall architecture and interfere with plant development (Obembe et al., 2007), or act

as a microbe-associated molecular patterns (MAMPs) to trigger a defense

mechanism, such as the accumulation of sesquiterpene cyclase (Brotman et al.,

2008; Gaulin et al., 2006).

In summary, the constitutive expression of the mesophilic endoglucanase TrCel5A

has a negative impact on plant growth and development, but this can be avoided by

using the ethanol-inducible alcAmin35S system. Furthermore, double induction

resulted in the accumulation of more recombinant cellulase, approaching the levels

achieved by constitutive expression. We propose that the inducible expression of

biomass degrading enzymes in plants is beneficial because it allows controlled

applications such as expression during specific developmental stages or defined

expression levels. This system also uses an economical inducer and a comparatively

simple application process suitable for large-scale biomass degradation.

Chapter 4: Expression of a hyperthermophilic endoglucanase in tobacco

43

Chapter 4: Expression of a hyperthermophilic

endoglucanase in tobacco

Results presented in this chapter have been published in:

Klose H, Röder J, Girfoglio M, Fischer R, Commandeur U (2012) Hyperthermophilic

endoglucanase for in planta lignocellulose conversion. Biotechnology for Biofuels 5: 63.

Cellulases have been produced in a number of plant species (Dai et al., 2005;

Ziegelhoffer et al., 1999) and the isolated recombinant enzymes have been shown to

digest cell wall preparations (Kawazu et al., 1996; Montalvo-Rodriguez et al., 2000).

As discussed, the constitutive expression of mesophilic cellulases can inhibit the

growth of tobacco plants and generate abnormal morphological phenotypes reflecting

changes in the composition of the cell walls (Brunecky et al., 2011; Taylor et al.,

2008). Different strategies have been used in an attempt to avoid direct contact

between cellulases and cell wall polysaccharides during vegetative growth, including

targeting to intracellular compartments (chapter 2) and inducible expression (chapter

3). The degradation of plant cell walls during normal growth can also be avoided by

the expression of thermophilic or hyperthermophilic cellulases with extreme

temperature optima beyond the physiological range for mesophilic plantgrowth

(Montalvo-Rodriguez et al., 2000; Xue et al., 2003).

Extremophiles represent an ideal source for enzymes that can tolerate high

temperatures, extreme pH environments and high salt concentrations (Turner et al.,

2007; Unsworth et al., 2007). They may therefore provide thermophilic cellulases that

are compatible with standard pretreatment technologies for the conversion of

lignocellulose into fermentable sugars. For example, A. cellulolyticus endoglucanase

E1 has been expressed in transgenic tobacco plants that were subsequently

processed by AFEX pretreatment, and approximately 30% of the enzyme activity was

retained after treatment (Teymouri et al., 2004). Similarly, ionic liquids are efficient

lignocellulose solvents that can be combined with enzymatic hydrolysis, but the

enzymes must retain their activity in highly concentrated ionic solutions with a water

content of only 10–20% (Zavrel et al., 2009).

The genome of the hyperthermophilic archaeon Sulfolobus solfataricus encodes

three different endoglucanases named SSO1354, SSO1949 and SSO2534 (She et

al., 2001) that have already been shown to perform efficiently under harsh physical


44

and chemical conditions (Girfoglio et al., 2012; Huang et al., 2005; Limauro et al.,

2001; Maurelli et al., 2008). The transient expression of SSO1354 has already been

described in an earlier study (Röder, 2010). Here, stable SSO1354 expression in

N. tabacum was achieved and the ability of the enzyme to convert plant biomass into

fermentable sugars under different pretreatment conditions was determined, offering

a promising new approach for the bioconversion of lignocellulose in planta.

Results

Expression and downstream purification of SSO1354 in tobacco plants

The sso1354 gene was amplified from S. solfataricus genomic DNA without the

putative leader peptide as described elsewhere (Girfoglio et al., 2012). Ten potential

N-glycosylation sites (matching the consensus Asn X Ser/Thr) were identified using

the NetNGlyc 1.0 Server (http://www.cbs.dtu.dk/services/NetNGlyc). The amplified

sequences were flanked by an N-terminal His6 tag and an optional C-terminal KDEL

sequence for ER retention, depending on the primers. The products were transferred

to the pTRAkc expression vector, which included an expression cassette containing

the Cauliflower mosaic virus (CaMV) double 35S promoter (P35SS), the 5'-UTR

translational enhancer from the Petroselinum hortense chalcone synthase gene and

a plant-codon-optimized LPH signal peptide for secretion to the apoplast (Figure 20).

Figure 20: Schematic representation of the SSO1354 expression cassettes including an

N-terminal His6 tag. The double CaMV promoter (P35SS) and terminator (pA35S) are shown in light

blue, the chalcone synthase 5'-UTR (CHS) in dark blue and the codon-optimized leader peptide from

the murine antibody mAb24 (LPH) is shown in green. The His6 and KDEL coding sequences are

indicated in red. LPH facilitates the secretion of the recombinant protein to the apoplast (A). An

additional C-terminal KDEL signal retains the protein in the ER lumen (B).


45

The expression vectors were introduced into A. tumefaciens strain GV3101 and

infiltrated into tobacco leaves. The infiltrated leaves were harvested 4 days later and

the TSP was extracted for analysis by SDS-PAGE, activity staining and Western blot.

Previous transient expression tests showed that enzyme variants containing a

C-terminal His6 tag could not be detected in the infiltrated plants (Röder, 2010).

However constructs carrying an N-terminal His6 tag either with or without the KDEL

sequence could be detected by means of Western blot. The ER-localized enzyme

showed the highest activity against azoCMC per gram leaf material and was selected

for expression in transgenic plants.

Figure 21: Substrate SDS-PAGE in the presence of 0.15% (w/v) CMC and extracts from different

SSO1354 transgenic plants. Lanes 1-12, T0 transgenic plants; lane 13, wild-type tobacco plants; lane

14, culture supernatant from K. lactis expressing SSO1354. Samples were incubated in 50 mM

sodium acetate buffer (pH 4.8) at 90°C for 15 min.

T0 transgenic plants expressing the ER-tagged enzyme were transferred to soil and

maintained in the greenhouse. The leaves were screened for enzyme activity using

the substrate SDS-PAGE method (Figure 21), and T1 seeds from positive candidates

were used to produce the next generation of plants. The leaves from the T1 plants

were tested using the substrate 4MUC to determine the activity of the recombinant

SSO1354 protein, which was comparably high, ranging from 92 to 488 nmol 4MU

min-1 mg-1 in the different transgenic plants (Figure 22). There was no difference in

morphology or growth behavior between the transgenic plants and wild-type controls

(Figure 22) and no differences in the biochemical composition of matrix-phase

polysaccharides or the content of crystalline cellulose (data not shown).


46

Figure 22: Tobacco plants expressing active SSO1354. SSO1354 activity in transgenic tobacco

lines (F1 to F6) based on the conversion of 4MUC (A). Values represent the mean of three in different

transgenic plants. Phenotypes of transgenic tobacco plant expressing SSO1354 (B) compared to

wild-type controls (C). Both lines were grown in soil under greenhouse conditions, and pictures were

taken six weeks after seeding. Monitoring different purification steps following SSO1354 expression in

tobacco with Western blot (D) and zymography (E) in the presence of 0.15 % (w/v) CMC. Lanes

contain 1 µg of purified SSO1354. Antibody system used in (D) is MαHis5 /GAMFcAP. The multiple

bands may represent alternative forms of N-glycosylation.

The recombinant SSO1354 was partially purified from crude plant extracts by thermal

precipitation. Heating to 85°C for 10 min caused 95% of the host cell proteins to

precipitate while SSO1354 remained soluble and active. Reducing sugars in the

extract, which could potentially interfere with subsequent enzyme activity assays,

were removed by passing the remaining extract through a PD10 desalting column.


47

Biochemical properties and catalytic activity of plant-derived SSO1354

The substrate profile of SSO1354 was determined by testing the enzyme against a

panel of β-glucans. The p-hydroxybenzoic acid hydrazide (PAHBAH) assay was used

to measure the release of reducing sugars, showing that SSO1354 was active

against CMC (arbitrary value set at 100% activity) and also barley β-glucan (136%

relative activity) and lichenan (130% relative activity), but not against pachyman

(Figure 23D).

Figure 23: Biochemical characterization of plant-derived SSO1354. The influence of temperature

on SSO1354 activity was measured using the azoCMC assay at 60–99°C (A). The maximum activity

measured at 90°C was set at 100%. The influence of buffer and pH on SSO1354 activity was

measured using the azoCMC assay at pH values between 2.0 and 7.0 (B). The maximum activity

measured at pH 4.5 was set at 100%. The influence of solvents and different salt concentrations on

SSO1354 activity (C) was measured using the azoCMC assay at optimal conditions (90°C, pH 4.5) by

adding different concentrations of salts and solvents. SSO1354 activity in the absence of salts and

solvents was set at 100%. The activity of SSO1354 was measured on different substrates (D). The

PAHBAH assay was used to determine the release of reducing sugars, and the amount reduced from

CMC was set at 100%.


48

The enzyme showed no significant activity against Avicel or oat xylan. This

suggested that SSO1354 has a β1,4-hydrolytic mode of action and is only active on

soluble substrates.

Further analysis using the azoCMC assay showed that SSO1354 has an optimum

temperature at 90°C, although it retains 79% residual activity at 99°C. The activity

was reduced substantially at lower temperatures, falling to ~30% activity at 70°C

(Figure 23A). The optimal pH was 4.5, although the enzyme retained 58% residual

activity at pH 2.7 and 62% residual activity at pH 6.5 (Figure 23B). There was only

minimal activity below pH 2.2 and above pH 7.0. Under optimal conditions (90°C, pH

4.5), the enzyme retained 50% of its activity after incubation for 100 min. SSO1354

also tolerated high salt concentrations, with 49.5% residual activity in the presence of

2.4 M NaCl, and 46.5% residual activity in the presence of 2.4 M CaCl2 (Figure 23C).

SSO1354 activity was inhibited by 15% (v/v) solutions of both protic solvents

(capable of donating protons) such as methanol and ethanol, and aprotic solvents

such as acetone and dimethylsulfoxide (DMSO). The enzyme retained 79% residual

activity in the presence of methanol, 75% in the presence of DMSO, 51% in the

presence of acetone and 48% in the presence of ethanol. Increasing the solvent

concentration to 30% (v/v) exacerbated the effect, with methanol and acetone

causing near-complete inhibition, DMSO leaving a residual activity of 14% and

ethanol leaving a residual activity of 16%.


49

SSO1354 activity in ionic liquids

As discussed above, SSO1354 cannot hydrolyze crystalline cellulose or cell wall

polysaccharides efficiently in aqueous solutions, but concentrated solutions (80–90%

v/v) of ionic liquids such as 1-ethyl-3-methylimidazolium acetate (EMIM-Ac) and 1,3

dimethylimidazolium dimethylphosphate (MMIM-DMP) can dissolve crystalline

cellulose substrates such as Avicel and cell wall polysaccharides, making them more

accessible to hydrolytic enzymes.

We therefore tested the activity of SSO1354 on Avicel that had been dissolved in

80% (v/v) EMIM-Ac and MMIM-DMP, by measuring the release of reducing sugars

and subtracting the quantity of sugars released by spontaneous hydrolysis in the

absence of the enzyme. The normalized release rates were 5.5 mg l-1 h-1 in EMIM-Ac

and 3.5 mg l-1 h-1 in MMIM-DMP (Figure 24). We also found that cell wall

polysaccharides dissolved in EMIM-Ac and MMIM-DMP were hydrolyzed by

SSO1354 with release rates of 1.8 mg l-1 h-1 for EMIM-Ac and 1.2 mg l-1 h-1 for

MMIM-DMP (Figure 24).

Figure 24: Activity of SSO1354 in high concentrations of ionic liquids. SSO1354 released

reducing sugars from 0.5% (w/v) Avicel (A) and tobacco cell wall polysaccharides (PCWS) (B)

dissolved in 80% (v/v) EMIM-Ac or MMIM-DMP. Assays were carried out at 90°C. The amount of

reducing sugar produced after ionic liquid precipitation was measured using the PAHBAH assay. The

sugars released by spontaneous hydrolysis in the absence of enzyme were measured in control

reactions and the values were subtracted from the test reactions. The release rate of reducing sugars

from dissolved Avicel was 5.5 mg l-1 h-1 in EMIM-Ac and 3.5 mg l-1 h-1 in MMIM-DMP, whereas the

release rate from dissolved cell wall polysaccharides was 1.8 mg l-1 h-1 for EMIM-Ac and 1.2 mg l-1 h-1

for MMIM-DMP.


50

Discussion

The conversion of lignocellulosic biomass into fermentable sugars is an important

step in the production of second-generation biofuels. A typical approach involves the

enzymatic hydrolysis of cellulose using microbial β-glucanases, but the efficiency of

saccharification is limited by the accessibility of the substrate, which must therefore

be pretreated (Galbe and Zacchi, 2007). Common cellulase preparations are derived

from mesophilic fungi and have usually an optimal activity around 50°C and pH 4–5

(Saddler and Gregg, 1998). In contrast, standard biomass pretreatment methods

involve harsh conditions that would invariably denature these enzymes, which means

they can only be added after pretreatment (Galbe and Zacchi, 2007). The production

of cellulases directly in the target biomass would be more economically and

environmentally sustainable than microbial fermentation, but the common mesophilic

enzymes are incompatible with current pretreatment regimens (Ragauskas et al.,

2006). Constitutively expressed and secreted mesophilic cellulases can also inhibit

normal plant growth, by reducing the integrity of the cell wall and therefore leaving

the plant vulnerable to environmental stress (Brunecky et al., 2011). These

challenges can be addressed by the sequestration of recombinant cellulases into

subcellular compartments with no direct access to cell wall components (Chapter 2)

or inducible expression when the plant is fully developed (Chapter 3).

The expression of cellulases from hyperthermophilic organisms is a promising

alternative because the temperature optima of these enzymes would ensure normal

plant growth and development at physiological temperatures and additionally enzyme

survival and activity under conventional pretreatment conditions. The sso1354 gene

from S. solfataricus encodes an endoglucanase from GH family 12 (She et al., 2001)

and does not contain a CBM. This enzyme is active at high temperatures and low pH

values, and is one of the first enzymes known to retain its activity in high

concentrations of ionic liquids. These features make SSO1354 an ideal candidate for

enzymatic biomass conversion directly in plants.

We demonstrated that SSO1354 can be functionally expressed in transgenic tobacco

plants by targeting the protein either for secretion into the apoplast or for retention in

the ER. The addition of a His6 tag to the N-terminus and an optional C-terminal KDEL

sequence had no adverse effects on enzyme activity. The Western blot revealed

different detectable forms of the protein, with molecular weights between 40 and 50

kDa (data not shown). The predicted molecular weight of the protein was 38 kDa, and


51

the discrepancy may reflect the presence of glycan residues on one or more of the

ten potential N-glycan acceptor sites.

Analysis of the morphological phenotype and the cell wall composition revealed no

significant differences between wild-type tobacco plants and transgenic lines

producing SSO1354, confirming that the enzyme remains inactive under

physiological conditions. The activity of plant-derived SSO1354 reached a maximum

of 0.5 µmol 4MU min-1 mg-1, reflecting its highly specific activity against 4MUC, but

this represents only a modest calculated yield of 1.1% TSP which is lower than other

cellulolytic enzymes in plant expression systems (Brunecky et al., 2011; Dai et al.,

2005; Ziegelhoffer et al., 2009).

Already known features of SSO1354 could be confirmed (Girfoglio et al., 2012;

Maurelli et al., 2008) and additionally, novel characteristics, such as the efficient

hydrolysis of substrates with β 1,4-linked glucose units in ionic liquids were

discovered. There was no hydrolysis of crystalline cellulose or substrates lacking β

1,4 linkages, nor did we observe the hydrolysis of substrates like oat xylan, which

comprises β 1,4-linked xylose, even though such activity has been reported

previously (Maurelli et al., 2008). This confirms that the enzyme is a strict β 1,4-

endoglucanase. The enzyme has a temperature optimum of 90°C, which is the

highest reported temperature for any cellulase expressed in plants and one of the

highest in any heterologous expression system (Bauer et al., 1999; Bronnenmeier et

al., 1995; Huang et al., 2005). The activity of the enzyme drops sharply as the

temperature declines, displaying only 25% activity at 70°C and virtually no activity at

temperatures below 30°C, suggesting there would be no hydrolysis of plant cellulose

under normal plant growth conditions. This is supported by the phenotype of the

transgenic plants which is not different compared to the wildtype and suggests that

post-harvest cellulose hydrolysis could be induced by a temperature shift.

SSO1354 is also advantageous because it tolerates a relatively broad pH range

(2.7–6.5, retaining at least 60% residual activity) whereas SSO1949 from the same

species has a sharp optimum at pH 1.8 (Huang et al., 2005). Under the optimal

conditions (pH 4.5 and 90°C), SSO1354 retains 85% activity after 1 h of incubation.

In addition to its remarkable thermostability and pH tolerance, SSO1354 also

tolerates moderate levels of organic solvents and high levels of salt.

Ionic liquids are very efficient in dissolving crystalline cellulose, however they are still

expensive (Abels et al., 2012; Zavrel et al., 2009). Promising results of their recycling


52

have been published recently (Abels et al., 2012). A unique feature of SSO1354 is its

activity in ionic liquids such as EMIM-Ac and MMIM-DMP. The hydrolysis rate was

3.5 mg l-1 h-1 in MMIM-DMP, showing this is a less suitable ionic liquid for SSO1354-

catalyzed biomass hydrolysis. SSO1354 was able to produce 5.5 mg l-1 h-1 of

reducing sugars from Avicel dissolved in EMIM-Ac, which is low compared to water

soluble substrates but still very promising for future optimization.

Although testing SSO1354 on artificial substrates confirmed its activity under a range

of conditions, plant biomass is a complex substrate with different components such

as cellulose, hemicelluloses and lignin, the latter acting as a significant inhibitor of

cellulases (hence the need for pretreatment) (Rahikainen et al., 2011). To investigate

the activity of SSO1354 against such complex substrates, we exposed the enzyme to

cell wall polysaccharides from tobacco leaves. This substrate is more heterogeneous

than crystalline cellulose but it has approximately 0.8% (dry weight) lignin content

and is therefore less complex than material derived from tobacco roots or stems

(Lagrimini et al., 1997). SSO1354 showed no significant activity against cell wall

polysaccharides in aqueous suspension, as determined by the release of reducing

sugars, but when the substrate was dissolved in ionic liquids the results were more

impressive. The enzyme released 1.8 mg l-1 h-1 of reducing sugars in EMIM-Ac and

1.2 mg l-1 h-1 in MMIM-DMP. These data indicate the possibility of combined

pretreatment and enzymatic hydrolysis.

The use of plants as an enzyme production system and subsequent utilization

requires two important criteria to be satisfied. The enzyme should not interfere with

cell wall synthesis or integrity and it must tolerate the extreme conditions found in

standard pretreatment processes. Hyperthermophilic cellulases like the

endoglucanase SSO1354 from S. solfataricus combine both features. This study

demonstrates the successful production of inactive SSO1354 at normal growth

conditions followed by the induction of enzyme activity at higher temperatures. To our

knowledge, SSO1354 is the first enzyme expressed in planta that remains active in

ionic liquids such as EMIM-Ac and MMIM-DMP, which are used for biomass

pretreatment. Plants have already been developed as a powerful platform for the

expression of biomass-processing enzymes, but the use of enzymes that function

optimally under harsh pretreatment conditions opens the way to develop combined

pretreatment and enzymatic hydrolysis strategies for the efficient conversion of

lignocellulosic plant biomass into fermentable sugars.

Chapter 5: Conclusion, Summary and Outlook

53

Chapter 5: Summary, Conclusion and Outlook

Lignocellulose is the major component of plant cell walls and is therefore an

abundant resource and a promising alternative to fossil fuel commodities. But plants

have evolved resistance to microbial and enzymatic attack, which makes them

recalcitrant to lignocellulosic conversion. Based on the complex structure and

composition of the lignocellulosic biomass, this recalcitrance is one the main barriers

preventing the large-scale industrial engineering of lignocellulose to produce fuels

and basic materials (Himmel et al., 2007).

Conversion strategies typically consist of three main steps: pretreatment (i) should

attenuate the recalcitrance of the substrate and therefore allow better access for the

enzymes used in the subsequent hydrolysis and saccharification steps (ii). The

resulting sugars are subsequently used for fermentation (iii) into fuels and

commodities. A significant reduction in enzyme costs, which still represent up to one

third of overall costs, is one of the most important goals for lignocellulose conversion

(Carroll and Somerville, 2009; Jørgensen et al., 2007). In planta production of

appropriate enzymes, e.g. GHs for lignocellulose conversion, is therefore a promising

option to improve the economic feasibility of such conversion processes (Sainz,

2011; Taylor et al., 2008).

Differential subcellular targeting plays an important role for in planta expression of

cellulases especially when functional enzymes are produced (Dai et al., 2005;

Ziegelhoffer et al., 2001). In chapter 2, the mesophilic endoglucanase TrCel5A from

the fungus T. reesei was produced in transgenic tobacco plants by targeting two

different subcellular compartments. A functional enzyme that was able to cleave

artificial substrates such as CMC and 4MU was produced by targeting either the

apoplast or the ER. However, the expression of TrCel5A was demonstrated to result

in an abnormal morphological phenotype and dramatic changes to the composition of

plant cell wall polysaccharides. The secreted enzyme reduced the content of

crystalline cellulose and inhibited both growth and germination, whereas keeping the

enzyme in the ER only altered the matrix phase polysaccharide composition without

visible effects on plant development, suggesting a more important role of the

crystalline cellulose for growth and development. Alternative strategies are therefore

required to produce cellulases in planta without a negative impact on growth and

development.


54

Inducible expression could be a promising alternative because potentially harmful

enzymes can be produced in fully-developed plants to avoid damage during

vegetative growth. In Chapter 3, the ethanol-inducible promoter system alcAmin35S

was used to produce TrCel5A endoglucanase. A single-step induction was inefficient

compared to constitutive expression but the optimization of the induction procedure

led yields of recombinant cellulase similar to the constitutive expression strategy.

Measurement of the composition of plant cell wall sugars in transgenic plants

carrying the induced expression cassette and phenotypical analysis were used to

characterize the impact of inducible TrCel5A expression on transgenic tobacco

plants. No significant difference was observed between transgenic lines with high

enzyme expression levels and wild-type controls. This underlines the advantage of

an induced expression strategy allowing a controlled application under different

expression conditions such as a specific developmental stage or defined expression

level without deleteriously impacting the plant development.

A high-level expression of functional cellulases is clearly beneficial for the

development of economically feasible in planta biomass conversion platforms, but the

enzymes must also survive the harsh conditions in the pretreatment step. Previous

studies already have shown that most enzymes do not survive common pretreatment

procedures like AFEX (Ragauskas et al., 2006; Teymouri et al., 2004).

Hyperthermophilic cellulases are more likely to remain active after or even during

pretreatment (Barnard et al., 2010; Blumer-Schuette et al., 2008). Accordingly, in

chapter 4, the successful production of S. solfataricus endoglucanase SSO1354 in

tobacco plants was described. Due to the enzyme´s inactivity under normal growth

conditions, SSO1354 was expressed without any morphological changes of the

plants or alterations in the plant cell wall sugar composition. However, the enzyme

can be activated when shifted to its optimal temperature, thus allowing full activity

during biomass conversion without the negative impact on growth and development

seen with mesophilic cellulases. Uniquely, it has been shown that SSO1354 can also

convert cellulose into smaller oligomers in the presence of ionic liquids, and can also

act on prepared cell walls in this environment. Plants therefore represent a powerful

platform for the expression of biomass-processing enzymes, including those that

function optimally under harsh pretreatment conditions. This offers new ways to

develop combined pretreatment and enzymatic hydrolysis strategies for the more

efficient conversion of lignocellulosic plant biomass into fermentable sugars.


55

The principal goal of in planta cellulase production is a high enzyme yield. Although

the achieved expression levels for TrCel5A in both strategies, constitutive and

inducible, are comparable to previous reports (Brunecky et al., 2011; Dai et al., 2005;

Ziegelhoffer et al., 2001), the expression level for SSO1354 is considerably lower.

However all obtained values are significantly lower than the amounts necessary for

complete self-conversion. Rough calculations, which ignore the requirement for

different activities and therefore different enzymes, estimate that ~15 kg of cellulase

would be required to convert 1 ton of biomass (Sainz, 2011). Even the best-

performing transgenic plants do not yet reach this goal (Hood et al., 2012). Potential

options for boosting in planta enzymes levels include plastid transformation, which

can produce impressive yields although homotransplastomic plants do often suffer

from sever damages that makes them unable to grow or flower in soil (Kim et al.,

2011; Petersen and Bock, 2011). However, there are approaches to significantly

increase the expression levels in transgenic plants like special breeding procedures

(Hood et al., 2012). Still the complete replacement of microbial enzymes by plant-

based production platforms seems unlikely in the short term, but in planta enzymes

could at least reduce the demand for microbial enzymes by contributing to the

biomass-degradation process thus increasing its economic feasibility. It is further

necessary to consider the loss of in planta produced enzyme during the pretreatment.

Even a comparably thermostable enzyme undergoes a dramatic loss of activity

during conventional AFEX pretreatment (Teymouri et al., 2004). A mesophilic

cellulase like TrCel5A would be completely inactivated. Enzymes from extremophilic

sources offer a more promising strategy to at least partially overcome the challenge

of inactivation by the combination of high temperature, high pressure, extreme pH,

and the presence of organic solvents used as pretreatment agents. Ionic liquids have

emerged as a powerful solvent for lignocellulose, though they are expensive and

highly toxic towards the microbes used for subsequent fermentation steps. The

endoglucanase SSO1354 is a promising enzyme for an application under these

harsh conditions. It is thus far unique in its ability to remain active in the presence of

high concentrations of ionic liquids, although the hydrolysis rate is comparatively low

and therefore unsuitable for economic conversion processes at the moment.

Besides high expression levels, characteristics like a good germination rate, plant

health and stress resistance are important for an industrial application of transgenic

plants producing cellulolytic enzymes. Most certainly the phenotypical alterations as


56

described here for TrCel5AAP plants would prevent an implementation as industrial

crop. The inducible expression of TrCel5A in fully developed plants or the post-

harvest activation of SSO1354 both led to plants with a normal phenotype. Therefore

both strategies seem to be promising for a later application.

Nevertheless cell wall modifications by heterologous expression of an endoglucanase

(E1 from A. cellulolyticus) have also been described as beneficial for subsequent

saccharification (Brunecky et al., 2011). The examined plants so far did not show an

abnormal phenotype as observed for TrCel5A in this study. This might be related to

different biochemical properties of the enzyme, though a detailed analysis of the cell

wall structure and composition is missing. However the effect, enabling an improved

saccharification, of both endoglucanses seems to be at least comparable. Also other

GHs, successfully implemented for in planta expression, have been leading to a

reduction of cell wall recalcitrance and therefore mitigating the pretreatment

conditions or reducing the amount of enzymes used in the saccharification (Shen et

al.; Zhang et al., 2011). Yet, there are no field studies for GHs expressing plants

concerning the impact of environmental stress. For the transgenic grass

Festuca arundinacea (tall fescue) with an altered esterification in the plant cell walls

an increased vulnerability to herbivore attack has been described (Buanafina and

Fescemyer, 2012). Future studies have to elucidate the effect of GHs in transgenic

plants in the field in order to design novel traits for industrial relevant crops.

In this study transgenic plants expressing recombinant cellulases have been

developed and analyzed. Novel insights have been obtained on the impact of such

enzymes on the plant especially the cell wall polysaccharides. Strategies have been

evaluated to avoid adverse effects on plant health. Developed analysis procedures

and the gained knowledge of this study established a promising basis for new

designed energy crops to contribute to novel sustainable and renewable resources.

Chapter 6: Materials and experimental procedures

57


Materials

Synthetic oligonucleotides

The oligonucleotide primers used to carry out the work described in this thesis are listed in

table 5.

Table 5: Oligonucleotide primers

Primer Sequence Length [bp] Description

cel5A fw TCC ATG GCA CAG CAG ACT GTC

TGG GGC

29 amplification of TrCel5A

adding NcoI site

cel5A rv TGC GGC CGC CTT TCT TGC GAG

ACA CG


adding NotI site

cel5Acd fw AAC CAT GGA AGG GGT CCG ATT

TGC CGG CG


catalytic domain adding

NcoI site

cel5A seq CTC TGG GCG CAT ACT GCA 18 sequencing TrCel5A

sso1354 His6 fw TCA TGA AAC ATC ACC ATC ACC

ATC ACG CGG CCG CTC AGT CTC

TCA GCG TTA AAC CCG T

58 amplification of sso1354

adding His6 and BspHI site

sso1354 rv AAC TCG AGT TAG AGG AGA GTT

TCA GAA AAG


adding XhoI site

sso1354 KDEL rv AAC TCG AGC TAG AGC TCA TCT

TTG AGG AGA GTT TCA GAA AAG


adding KDEL and XhoI site

mCherry fw GCG GCC GCA ATG GTG AGC

AAG GGC GAG GAG G

31 amplification of mCherry

adding NotI site

mCherry his6 rv CTC GAG CTA GTG ATG GTG ATG

GTG ATG AGC GGT CGC CTT GTA

CAG CTC GTC CAT G

55 amplification of mCherry

adding His6, NotI and XhoI

sites

alcAmin35S-fw AAG GAT CCA CCC GGG TGG CTA

GAA ATA TTT GCG ACT CTT CTG

42 amplification of alcAmin35s

adding BamHI and BstXI

sites

alcAmin35S rv AGC GGC CGC GTT TAA ACC AAT

TGG TCC TCT CCA AAT GAA ATG

AAC TTC C

49 amplification alcAmin35s

adding MfeI site

pSS-3 AGA GAG AGA TAG ATT TGT AGA

GA

23 3´ primer for pSS vector

pSS-5 ATC CTT CGC AAG ACC CTT CCT

CT

23 5´ primer for pSS vector


58

Plasmids

Plant expression vectors for transient expression and generation of transgenic plants were

based on pTRAk, a derivate of pPAM (GenBank no. AY027531). The vectors pTRAkc-AP

and pTRAkc-ER featured constitutive gene expression cassettes controlled by a duplicated

Cauliflower mosaic virus (CaMV) 35S promoter (P35SS). A plant-codon-optimized

signal peptide from the murine mAb24 heavy chain gene (Maclean et al., 2007) and the

sequence for a C-terminal His6 tag were also included. The ER retention signal (KDEL) was

also present in some constructs (Munro and Pelham, 1987). All vectors included a nos

expression cassette containing the nptII gene for kanamycin resistance.

Figure 25: Expression vector pTRAkc-rfp-his6-ERH for heterologous expression and ER

localization in plant cells. ColE1 ori, E. coli replicon; RK2 ori, A. tumefaciens replicon; bla, gene for

β-lactamase (AmpR); RB, right border; LB, left border; SAR, scaffold attachment region; pnos/pAnos,

nos expression cassette; p35SS/pA35SS, CaMV 35SS expression cassette; nptII, gene for neomycin

phosphotransferase II (KanR); DsRed, gene for Discosoma sp. red fluorescent protein; CHS, 5'-UTR

from P. hortense chalcone synthase gene; LPH, plant-codon-optimized signal peptide of murine

antibody mAb24; His6, hexahistidine tag; KDEL, signal for ER retention.

Bacterial strains

Escherichia coli strain DH5α [recA1, endA1, hsdR17 (rK-, mK+), ∆(lacZYA-argF)U169,

supE44, Φ80dlacZ∆M15, thi-1, gyrA96, relA1, deoR] was used for general cloning and

expression because of its modified recombination system (recA1) and the missing

endoglucanase (endA1).


59

For transient expression and stable transformation of tobacco, we used

Agrobacterium tumefaciens strain GV3101::pMP90RK (Koncz and Schell, 1986), which

contains the disarmed Ti helper plasmid pMP90RK conferring resistance against gentamicin

and kanamycin, and containing a RK2 origin of replication as well as virulence (vir) and

transfer (tra) genes essential for the horizontal transfer of T-DNA.

Plant material

Transient expression and stable nuclear transformation of tobacco plants was carried out in

Nicotiana tabacum cv. Petit Havana SR1.

Experimental procedures

Molecular biology procedures were carried out essentially as previously described

(Sambrook et al., 1989). Restriction endonucleases and DNA-modifying enzymes were used

according to the supplier’s instructions (New England Biolabs, Ipswich, MA USA).

Cloning

Construction of plant expression vectors for TrCel5A

The gene encoding TrCel5A (EGR51020.1, EMBL-CDS) was amplified by PCR from a cDNA

preparation of T. reesei QM9414 (kindly provided by Armin Merckelbach, Institute of

Molecular Biotechnology, RWTH Aachen University, Germany) using primers cel5A fw and

cel5A rv to amplify the whole gene except the first 21 codons comprising the fungal signal

peptide, or primers cel5Acd fw and cel5A rv to amplify the catalytic domain only. The purified

products were first transferred to the pCR2.1 vector (Invitrogen, Darmstadt, Germany) by TA-

cloning to generate the constructs pCR2.1 TrCel5A and pCR2.1 TrCel5Acd respectively.

Digestion with NcoI and NotI released a cassette which was then inserted into the NcoI-NotI

linearized vectors pTRAkc-TP for the whole enzyme and pTRAkc-ER for the catalytic domain

(Maclean et al., 2007). Additional constructs pTRAkc-AP-TrCel5A and pTRAkc-ER-TrCel5A

used in this study were prepared in earlier studies (Klose, 2009; Werthmann, 2009).

For the generation of TrCel5A/mCherry fusion constructs, the mCherry gene was amplified

from pmCherry-C1 (kindly provided by Christina Dickmeis, Institute of Molecular

Biotechnology, RWTH Aachen University, Germany) using primers mCherry fw and

mCherry rv. The purified products were digested with NotI and XhoI and inserted into the

similarly linearized vectors pTRAkc-AP-TrCel5A generating the construct pTRAkc-AP-

TrCel5AmCherry. Construct pTRAkc-ER-mCherry (Christina Dickmeis, Institute of Molecular

Biotechnology, RWTH Aachen University, Germany) was digested with NcoI and XhoI and

the resulting fragment was transferred into the NcoI-XhoI-linearized vector pTRAkc-AP-

TrCel5AmCherry to yield pTRAkc-ER-TrCel5AmCherry.


60

To generate the ethanol inducible expression construct (Caddick et al., 1998) the

alcAmin35S promoter was amplified from pTRAkt-alcR-alcA-stppc (Schirmacher, 2006) using

the primers alcAmin35S-fw and alcAmin35S-rv. The MfeI-PmeI fragment was transferred into

EcoRI-PmeI-linearized pTRAkc-AP-Cel5A and then subcloned as a BstXI-PmeI fragment into

FseI-PmeI-linearized pTRAkt-alcR (kindly provided by Dr. HJ Hirsch, Institute of Botany,

RWTH Aachen University, Germany).

Construction of plant expression vectors for SSO1354

The coding sequence of the SSO1354 catalytic domain (AAK41590.1, EMBL-CDS) was

amplified by PCR from S. solfataricus P2 genomic DNA (kindly provided by Raffaele Cannio,

Institute for Microelectronics and Microsystems, Naples, Italy) using the primers

sso1354 His6 fw and sso1354 rv or sso1354 KDEL rv to generate the His6-sso1354

products. The purified fragments were digested with BspHI and XhoI and inserted into the

NcoI/XhoI-digested pTRAkc-AP and pTRAkc-ER vectors. Additional constructs without a His6

tag (pTRAkc-AP-SSO1354oH and pTRAkc-ER-SSO1354oH were derived from earlier

studies (Röder, 2010).

Protein production in tobacco plants

Agroinfiltration of Nicotiana tabacum

Electrocompetent Agrobacterium tumefaciens cells, strain GV3101::pMP90RK GmR, KmR,

RifR (Koncz and Schell, 1986), were transformed by electroporation (Shen and Forde, 1989)

and selected on YEB medium plates (1% (w/v) Bacto tryptone, 1% (w/v) yeast extract, 0.5 %

(w/v) NaCl, 1.5% (w/v) agar, pH 7.0) supplemented with 100 mg ml-1 carbenicillin. Colonies

were transferred to liquid YEB medium containing kanamycin (50 mg ml-1), rifampicin (50 mg

ml-1) and carbenicillin (100 mg ml-1) for 36–40 h (26°C, 180 rpm) for selection. The

suspension was then supplemented with 10 µM acetosyringone, 10 mM MES (pH 5.6) and

10 mM glucose and the bacteria were incubated overnight. The OD600 of the culture was

adjusted to 1.0 with 2x infiltration medium (0.86% (w/v) MS salts, 10% (w/v) sucrose, 0.36%

(w/v) glucose, pH 5.6) and the adjusted suspension was supplemented with 200 µM

acetosyringone prior to incubation for approximately 45 min at room temperature. Before

infiltration, tobacco leaves were sprayed with water to open the stomata. The adaxial surface

of the leaves was scratched before the suspension was injected into the intercostal fields

using a syringe without a cannula.


61

Stable nuclear transformation of Nicotiana tabacum

Transgenic tobacco lines (Nicotiana tabacum L. cv. Petit Havana SR1) were established

using the leaf disc transformation method (Horsch, 1985). T0 plants were initially grown on

Murashige-Skoog medium containing 3% (w/v) sucrose, 100 mg l-1 kanamycin and 200 mg l-1

claforan, then transferred to soil in the greenhouse and selfed to produce T1 and T2

generation plants. The cell culture was kindly carried out by Flora Schuster, Institute of

Molecular Biotechnology, RWTH Aachen University.

Cultivation of tobacco plants

For the detailed analysis of recombinant protein expression and cell wall properties, plants

were grown in soil without the addition of fertilizer (Einheitserde classic, Patzer GmbH,

Sinntal-Altengronau) in the greenhouse at a constant 22°C (dark period) or 25°C (light

period) and 70% of relative humidity. Illumination was provided by Philips IP65 400 W lamps

(180 µmol s-1 m-2; λ= 400–700 nm) in a 16/8-h light/dark cycle with daily watering. The plants

were cultivated by Ibrahim Alamedi, Institute of Molecular Biotechnology, RWTH Aachen

University.

Ethanol-induced protein production in transgenic tobacco plants

Transgenic and wild-type tobacco plants were irrigated once with a defined volume of spilling

water containing concentrations of ethanol ranging from 0.1 to 10% (v/v). Induced plants

were kept separately from non-induced plants to avoid induction via ethanol vapor.

Isolation and analysis of plant genomic DNA

Genomic DNA was isolated from transgenic plants with the Wizard® Genomic DNA

Purification Kit (Promega, Mannheim, Germany). The transgene was detected by PCR using

the primers listed above.

Extraction and purification of recombinant proteins

Tobacco leaves from the transient expression experiments or transgenic plants were ground

in liquid nitrogen and homogenized in phosphate-buffered saline (PBS) at pH 7.0,

supplemented with 1 mM phenylmethylsulfonyl fluoride (PMSF). The extract was centrifuged

at 15,000 x g for 20 min at 4°C followed by filtration to remove suspended particles from the

extract.

The His6-tagged proteins were purified from the total soluble protein by Ni–NTA agarose

affinity chromatography (Qiagen, Hilden, Germany) following the manufacturer’s protocol.

Imidazole was removed using a Roti®Spin cut off column (Roth, Karlsruhe, Germany) with a

MWCO of 10 kDa. Total protein levels were determined using the Bradford method

(Bradford, 1976) with bovine serum albumin (Roth, Karlsruhe, Germany) as the standard.


62

Hyperthermophilic proteins without a tag were purified by removing plant bulk proteins in a

heat precipitation step at 85°C for 10 min and then separating small molecules such as

sugars from the recombinant enzyme by gel filtration with PD 10 Columns (GE Healthcare

Bioscience, Pittsburgh, PA USA).

Protein chemical methods

Protein separation by discontinuous SDS polyacrylamide gel electrophoresis

Protein samples were mixed 1:5 with reducing loading buffer (62.5 mM Tris-HCl (pH 6.8), 4%

(w/v) SDS, 0.05% (w/v) bromphenol blue, 10% (v/v) 2-mercaptoethanol, 30% (v/v) glycerol).

Separation was carried out under reducing conditions in 12% (w/v) polyacrylamide gels. Gels

were run in a Tris-glycine-SDS running buffer (25 mM Tris-HCl (pH 8.3), 0.2 M glycine,

0.25% (w/v) SDS) at a constant voltage of 200 V for 45 min. The gels were either stained

with 0.25% (v/v) Coomassie Brilliant Blue G-250, 50% (v/v) methanol, 10% (v/v) acetic acid

and then destained in 5% (v/v) methanol, 7.5% (v/v) acetic acid) or used for western blotting

(see below).

Protein analysis by Western blot

Proteins separated by SDS-PAGE were electrotransferred in blotting buffer (25 mM Tris,

192 mM glycine, 20% (v/v) methanol) for 60 min at a constant 100 V to a nitrocellulose

membrane, and blocked for 30 min at room temperature with 5% (w/v) skimmed milk

dissolved in PBS. His6-tagged proteins were detected with a polyclonal anti-His5 preparation

(Rockland Immunochemicals, Gilbertsville, USA) and a secondary, alkaline-phosphatase-

conjugated goat anti-mouse antibody (Dianova, Hamburg, Germany). T. reesei cellulases

were detected with polyclonal antibodies recognizing Trichoderma viride cellulase

(antibodies-online, Aachen, Germany) and a secondary alkaline-phosphatase-conjugated

goat anti-rabbit antibody (Dianova, Hamburg, Germany). The signal was visualized with

nitroblue tetrazolium chloride/5-bromo-4-chloro-3-indolyl phosphate p-toluidine salt

(NBT/BCIP; Roth, Karlsruhe, Germany).

Enzymatic assays

Protein analysis by activity staining

Protein samples were separated by SDS-PAGE in 12% polyacrylamide gels containing

0.15% (w/v) carboxymethylcellulose (CMC). The proteins were then renatured by washing

twice at room temperature for 15 min with 50 mM potassium acetate buffer (pH 4.8)

containing 20% (v/v) propan-2-ol followed by washing twice for 30 min with the same buffer

without propan-2-ol. The samples were incubated for 30 min in the potassium acetate buffer


63

at 50°C for mesophilic enzymes or 90°C for hyperthermophilic enzymes. The reactions were

stopped by incubating in 50 mM Tris-HCl (pH 7.5) for 30 min at room temperature. The gels

were stained for 30 min in 0.1% (w/v) Congo Red (Sigma-Aldrich, Seelze, Germany) and

destained in 1 M NaCl. To achieve a higher contrast between degradation and non-

degradation areas, the gel was incubated in 0.5% (v/v) acetic acid after destaining.

Activity against 4 methylumbelliferyl-β-D-cellobioside

The activity of cellulases against 4 methylumbelliferyl-β-D-cellobioside (4MUC) was carried

out as previously described (Ziegelhoffer et al., 2001). Each 5-µl sample aliquot was assayed

in 100 µl 50 mM sodium acetate (pH 4.8) containing 0.5 mM 4MUC in a 96-well plate. Plates

were covered with adhesive lids to prevent evaporation and incubated for 60 min either at

55°C or 90°C. The reaction was stopped by adding 100 µl 0.15 M glycine (pH 10.0). The

fluorescence was determined on a Tecan Infinite M200 (Tecan Deutschland GmbH,

Crailsheim) at 465 nm using an excitation wavelength of 360 nm. Fluorescence data

representing 4MUC activity was corrected by subtracting the average data from wild-type

crude plant extracts (n = 3). Conversion rates were calculated from corrected data based on

a series of 4MU standards (1–10 nM).

Activity against azo-CM-cellulose

The soluble chromogenic substrate azo-CM-cellulose (Megazyme, Bray, Ireland) was used

to determine the temperature and pH tolerance of the recombinant enzymes as previously

described (Jørgensen and Olsson, 2004), with five replicates taken at each measurement

point. Adsorption data were corrected by subtracting the average data from wild-type extracts

(n = 5). Temperature dependence was determined using 50 mM acetate buffer (pH 4.5) at

temperatures of 20–70°C. The pH tolerance was determined at 90°C using 50 mM citrate

buffer (pH 2.0–3.5), 50 mM acetate buffer (pH 3.5–5.5) and 50 mM phosphate buffer (pH

5.5–7.0). Salt tolerance was determined using 50 mM acetate buffer (pH 4.5) at 90°C

supplemented with up to 4.6 M NaCl.

Determination of reducing sugars

The reducing sugar content was measured using the p-hydroxybenzoic acid hydrazide

(PAHBAH) assay, by mixing one volume of sample with two volumes of PAHBAH reagent

(Lever, 1972). The solution was incubated at 100°C for 10 min and absorbance was

measured at 410 nm. Defined concentrations of glucose were used for calibration. Five

replicates were measured at each time point.

Determination of activity in ionic liquids

The conversion of pure cellulose or tobacco cell wall polysaccharides by the

hyperthermophilic endoglucanase was measured in high concentrations of ionic liquids. The


64

tobacco cell wall polysaccharides were prepared as previously described (Kawazu et al.,

1999). Therefore tobacco leaves were harvested and ground to powder under liquid nitrogen

followed by two washes with 25 ml distilled water and centrifugation (20,000 x g, 4°C, 15

min). Insoluble cell wall material was washed several times with methanol-chloroform (1:1)

until the green color disappeared completely. The organic solvent was then removed by

evaporation.

For activity determination, 0.5% (w/v) of the dried cell wall polysaccharides or Avicel was

dissolved in either 80% (v/v) 1-ethyl-3-methylimidazolium acetate (EMIM-Ac) or 80% (v/v)

1,3-dimethylimidazolium dimethylphosphate (MMIM-DMP). The aqueous-buffered enzyme

was added to a final concentration of 0.2 g l-1 and 20% (v/v) buffer in the reaction mixture.

The reaction mixture was incubated on a shaking platform at 90°C and samples were taken

at intervals to determine reducing sugar levels.

The reaction was stopped by cooling each sample and the cellulose was precipitated by

adding one volume of water followed by centrifugation (15,000 x g, RT, 2 min). Three phenol-

chloroform (1:1) extractions steps and one chloroform extraction were carried out to separate

the hydrolysis products and proteins from the ionic liquid. The reducing sugar content was

measured using the PAHBAH assay.

Chemical analysis of plant cell wall polymers

Extraction of alcohol-insoluble residues

Ten leaf tissue samples were taken from 6–7-week-old plants representing each transgenic

line and wild-type tobacco. To determine differences in the cellulose content, alcohol-

insoluble residues (AIRs) were prepared as previously described (Foster et al., 2010).

Approximately 50 mg per sample was ground to a fine powder under liquid nitrogen and plant

cell walls were isolated by washing with different organic solvents. Starch was removed by

hydrolysis with amylase and pullulanase (Sigma-Aldrich, Seelze, Germany). The remaining

AIRs were extracted with acetone, dried and weighed.

Analysis of matrix polysaccharides

The matrix polysaccharides were characterized as previously described (Albersheim et al.,

1967). We hydrolyzed 1–4 mg of AIR in 250 µl 2 M trifluoroacetic acid (TFA) for 90 min at

121°C. The cooled samples were centrifuged (10,000 x g, 4°C, 10 min) and 100 µl of the

supernatant was used for the alditol acetate derivatization procedure (Foster et al., 2010).

Insoluble material was washed three times with isopropanol and used to analyze the

crystalline cellulose content (see below). The cell wall matrix sugars were identified by

recording their mass profile or retention time. GC/MS was kindly carried out by Markus Günl

(Institute of Bio- and Geosciences, Forschungszentrum Jülich).


65

Measurement of crystalline cellulose

The crystalline cellulose content was determined as previously described (Updegraff, 1969b)

after hydrolysis of the non-crystalline cellulosic polysaccharides with acetic and nitric acid.

The remaining crystalline cellulose residues were hydrolyzed with 72% (w/v) sulfuric acid and

glucose was measured using the anthrone assay (Scott and Melvin, 1953).

Histological methods

Immunolabeling of TrCel5A

10-µm sections were prepared from transgenic and wild type leaf material using a Leica

Cryostat Microtome (Leica, Wetzlar, Germany). The sections were incubated in acetone for

30 min at room temperature, blocked in PBS containing 1% (v/v) goat serum (Sigma-Aldrich,

Seelze, Germany) and incubated with the primary antibody diluted (1:50) in PBS containing

1% (v/v) goat serum for 45 min. T. reesei cellulases were detected with polyclonal antibodies

recognizing Trichoderma viride cellulase (1 mg/ml, antibodies-online, Aachen, Germany),

additionally KDEL fused cellulases were detected with monoclonal antibodies recognizing the

KDEL sequence (1 mg/ml, Enzo Life Science, Lörrach, Germany). The sections were then

washed three times in PBS containing 0.05% (v/v) Tween-20 (Roth, Karlsruhe, Germany)

and incubated with the secondary antibody diluted (1:200) in PBS containing 1% (v/v) goat

serum for 45 min. For secondary antibody fluorescein isothiocyanate-conjugated goat anti-

rabbit or goat anti-mouse antibodies (1.5 mg/ml, Dianova, Hamburg, Germany) were used.

After three further washes in PBS containing 0.05% (v/v) Tween-20, the sections were

analyzed using a Leica DRM light microscope (Leica, Wetzlar, Germany).

Conversion of transgenic tobacco leaf material

To estimate the glucan content, tobacco leaf material was hydrolyzed in 72% sulfuric acid for

45 min at room temperature and hydrolyzed carbohydrates were measured using the

anthrone assay (Scott and Melvin, 1953). For the conversion of leaf material, 50-100 mg of

fresh tobacco leaf tissue was ground to powder under liquid nitrogen and resuspended in

50 mM sodium acetate (pH 4.8) containing 15 Filter paper units (FPU) CTec2 (Novozymes,

Bagsvaerd, Denmark) per gram of glucans. The reaction was stopped by boiling for 10 min

followed by centrifugation (10,000 x g, RT, 10 min) To determine the rate of hydrolysis, the

remaining crystalline cellulose was measured as previously described (Foster et al., 2010;

Scott and Melvin, 1953). Final values were based three independent experiments each

comprising three replicates.


66

Statistical methods

Arithmetic mean of a statistical sample

μ = 1��

Equation 1: Calculation of the arithmetic mean µ of a statistical sample. Where a is defined as

the value and n is the number of values.

Standard deviation of a statistical sample

= � 1� − 1�(� − μ)�

Equation 2: Calculation of the standard deviation σ of a statistical sample. Where a is defined as

the value, µ is the arithmetic mean and n is the number of values.

Statistical difference between different groups of samples

One-way analysis of variance (ANOVA) followed by Student’s pairwise t test was used to

determine the significance of differences between groups, with a threshold of P ≤ 0.05 or

even P ≤ 0.01, using Microsoft Excel 2010.

References

67

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Appendix

80

Appendix

Abbreviations

4MUC 4-methylumbelliferyl cellobioside

Ac acetate

AFEX ammonia fiber expulsion

AIR alcohol insoluble residues

BGL beta-glucosidase

CaMV cauliflower mosaic virus

CBH cellobiohydrolase

CBM carbohydrate binding module

cd catalytic domain

CDH cellobiosdehydrogenase

CMC carboxymethylcellulose

Da Dalton

DMP dimethylphosphate

DMSO dimethylsulfoxide

EB energy balance

EG endoglucanase

EMIM 1-ethyl-3-methylimidazolium

ER endoplasmic reticulum

FAEA ferulic acid esterase A

FITC fluorescein isothiocyanate

GAX glucoronoarabinoxylan

GC gas chromatography

GH glycosyl hydrolase

GT glycosyl transferase

GX glucoronoxylan

IL ionic liquid

M molar

MAMP microbe associated molecular pattern

MMIM 1,3-dimethylimidazolium

MS mass spectrometry

MU methylumbelliferone

ORF open reading frame

PAGE polyacrylamide gel electrophorese

PAHBAH 4-hydroxybenzoic acid hydrazide

PAL phenylalanine ammonium lyase

PCWS plant cell wall polysaccharides

PGA polygalacturonic acid

Appendix

81

PMO polysaccharide monooxygenases

PMSF phenylmethylsulfonyl fluoride

RG I rhamnogalacturonan I

rpm revolutions per minute

SDS sodium dodecylsufate

TSP total soluble protein

UDP uridine diphosphate

UTR untranslated region

v volume

w weight

XyG xyloglucan

Appendix

82

Register of equipment

Name Type Manufacturer

"-20°C premium "-20°C freezer Liebherr, Germany

Herafreeze "-80°C freezer Heraeus, Germany

Infinite 200 96-well plat reader Tecan, Germany

Waring blender blender Warring, USA

Allegra-X15R centrifuge Beckmann Coulter, Germany

Avanti J 301 centrifuge Beckmann Coulter, Germany

5414 D centrifuge Eppendorf, Germany

Mikrofuge R centrifuge Beckmann Coulter, Germany

Multiporator electroporation device Eppendorf, Germany

Herolab E.A.S.Y Win32 gel documentation Herolab GmbH, Germany

Grant QBT heating device Cambridge, GB

Heraeus B12 incubator Heraeus, Germany

Innova 4340 incubator/shaker New Brunswick Scinetific, Germany

Kuhner ShakerX incubator/shaker Kuhner AG, CH

Leica DRM+DCF microscope Leica, Germany

Olympus Bx4I microscope Olympus, Germany

Leica VT1000S vibratome Leica, Germany

Leica CM3050S cryo microtome Leica, Germany

Primus 96 Plus PCR cycler Eurofins MWG Operon, Germany

Masterflex L/S peristaltic pump Cole Parmer, Germany

Nikon CoolPix 5400 photographic camera Niko, Germany

Biophotometer photometer Eppendorf, Germany

Heraeus HS9 sterile bench Heraeus, Germany

Ikamag REC-G stirrer IKA Labortechnik, Germany

Thermomixer comfort temperature controlled mixer Eppendorf, Germany

Herolab UVT-20M 302nm UV transilluminator Herolab GmbH, Germany

G-560E vortex Scientific inductries, USA

Appendix

83

Table of figures

Figure 1: Global demographic development. .......................................................................................... 2

Figure 2: The primary cell wall of a growing cell as found in most flowering plants. ............................... 4

Figure 3: Schematic representation of hemicellulose polysaccharides................................................... 6

Figure 4: Current overview on fungal enzymatic degradation of cellulose. ............................................. 9

Figure 5: Schematic presentation of plant expression cassettes for the differential targeting of TrCel5A.

........................................................................................................................................................ 18

Figure 6: Transient expression of TrCel5A in N. tabacum. ................................................................... 19

Figure 7: Transient expression of TrCel5A-mCherry fusion in tobacco. ............................................... 20

Figure 8: Localization of transiently-expressed TrCel5A-mCherry fusion in 90-µm sections of tobacco

leaf tissue. ..................................................................................................................................... 20

Figure 9: Analysis of transgenic tobacco expressing TrCel5A. ............................................................. 22

Figure 10: Enzymatic activity of TrCel5A at different pH values and temperatures. ............................. 23

Figure 11: Subcellular localization of recombinant TrCel5AER in transgenic tobacco cells. .................. 24

Figure 12: Subcellular localization of recombinant TrCel5A in transgenic tobacco cells. ..................... 25

Figure 13: Transgenic tobacco plants expressing TrCel5A. ................................................................. 26

Figure 14: Phenotypic analysis of transgenic tobacco plants. .............................................................. 27

Figure 15: Enzymatic hydrolysis of transgenic tobacco leaves. ............................................................ 29

Figure 16: Generation and analysis of transgenic tobacco expressing TrCel5A under the control of an

alcohol-inducible promoter. ............................................................................................................ 36

Figure 17: Inducible TrCel5A expression in tobacco plants. ................................................................. 37

Figure 18: Phenotype of transgenic tobacco plants. ............................................................................. 39

Figure 19: Crystalline cellulose content in transgenic tobacco plants expressing TrCel5A. ................. 40

Figure 20: Schematic representation of the SSO1354 expression cassettes including an N-terminal

His6 tag. .......................................................................................................................................... 44

Figure 21: Substrate SDS-PAGE in the presence of 0.15% (w/v) CMC and extracts from different

SSO1354 transgenic plants. .......................................................................................................... 45

Figure 22: Tobacco plants expressing active SSO1354. ...................................................................... 46

Figure 23: Biochemical characterization of plant-derived SSO1354. .................................................... 47

Figure 24: Activity of SSO1354 in high concentrations of ionic liquids. ................................................ 49

Figure 25: Expression vector pTRAkc-rfp-his6-ERH for heterologous expression and ER localization in

plant cells. ...................................................................................................................................... 58

Appendix

84

Scientific publications

Publications

Klose H, Röder J, Girfoglio M, Fischer R, Commandeur U (2012) Hpyerthermophilic

endoglucanase for in planta lignocellulose conversion. Biotechnology for Biofuels 5:

63.




Garvey M, Klose H, Fischer R, Lambertz C, Commandeur U (2013) Cellulases for

biomass degradation: comparing recombinant cellulase expression platforms. Trends

in Biotechnology, 10: 581-593.

Garvey M, Klinger J, Klose H, Fischer R, Commandeur U (2013) Expression of

recombinant cellulase Cel5A from Trichoderma reesei in tobacco plants. Journal of

Visualized Experiments, in press.

Poster presentations

Klose H, Wennekamp S, Commandeur U (2011) Heterologous expression of fungal

cellulases in plants. First International Working on Walls (WoW) Symposium, 01.-

03.2011, Vancouver, Canada.

Klose H, Röder J, Girfoglio M, Fischer R, Commandeur U (2011) In planta

expression of hyperthermophilic β-glucanase. 10th International Symposium on

Biocatalysis, 02.-06.10.2011, Giardini Naxos (ME), Italy

Klose H, Röder J, Girfoglio M, Fischer R, Commandeur U (2012) Expression of

hyperthermophilic β-glucanase in plants. NSERC Bioconversion Network

Pretreatment Workshop, 04.-06.06.2012, Vancouver, Canada

Appendix

85

Klose H, Klinger J, Röder J, Girfoglio M, Fischer R, Commandeur U (2012)

Hyperthermophilic β-glucanase for advanced in planta biomass conversion. 6th

International Congress on Biocatalysis, 02.-06.10.2012, Hamburg

Schmidt T, Przyklenk M, Girfoglio M, Becker J, Klose H, Fischer R, Commandeur U

(2012) Synergistic effects of combined cellulolytic enzymes active in pure ionic

liquids. 6th International Congress on Biocatalysis, 02.-06.2012, Hamburg.

Klose H, Lambertz C, Günl M, Commandeur U (2013) Inducible expression of

mesophilic cellulases in tobacco. 9th International Conference on Renewable

Resources & Biorefineries, 05.-07.06.2013, Antwerp, Belgium

Appendix

86

Acknowledgement

The work of the last years was a magnificent and challenging experience for me.

During this time many people supported me, both directly and indirectly, with my

academic career. Here, I would like to thank them for their precious help.

I am grateful to Prof. Dr. Rainer Fischer for the opportunity to work at the Institute for

Molecular Biotechnology and his support during my PhD-work.

Dr. Ulrich Commandeur I would like to thank for the allocation of this interesting topic

and for all the lively debates.

To Prof. Dr Björn Usadel I am thankful for the willingness to take the co-review of my

thesis and all the valuable aid in understanding cell wall chemistry.

A big thank goes to Dr. Flora Schuster for her excellent work in the plant cell culture,

Dr. Marcus Günl for his help with the measurement of cell wall sugars, Ibrahim

Alameidi for his care in cultivating the plants, Maria Krämer and Dr. Dmitrij

Hristodorov for their kind help with my histological experiments, Dr. Richard Twyman

for the critical review of my thesis, Dr. Michele Girfoglio for his help with the

extremophilic enzymes and all the delightful discussions (not only on science), Dr.

Megan Garvey for her precious aid with my manuscripts and her invaluable

willingness for scientific discussion especially in the last months.

I am grateful to Karo Richter and Tini Dickmeis not only for sharing office and lab with

me in the last five years but also being such wonderful colleagues!

A big thanks goes to all the coworkers of the Institute (present and former) with whom

I have spent such great times in the last years!

All this work would not have been possible without the constant aid and

understanding from my family, especially my parents, to whom I would like to express

my deepest gratitude.

Appendix

87

Declaration

I hereby declare that all information in this document has been obtained and

presented in accordance with academic rules and ethical conduct. I also declare that,

as required by these rules and conduct, I have fully cited and referenced all material

and results that are not original to this work.

_____________________________

Holger Klose, Aachen 18.12.2013

Appendix

88

Curriculum vitae

Personal data

Name: Holger Klose

Gender: Male

Date of birth: 17th Aug, 1981

Place of birth: Lüdenscheid

Nationality: German

Marital status: Single

Employment

01/2014 Institute of Biology I (Botany/Molecular Genetics), RWTH

Aachen University, Aachen

Doctorate

11/2009 – 12/2013 RWTH Aachen University, Aachen,

Doctoral studies: Biotechnology

Education

10/2007 – 10/2009 RWTH Aachen University, Aachen, Germany

Master of Science in Biotechnology/Molecular

Biotechnology

10/2002 – 09/2007 RWTH Aachen University, Aachen, Germany

Bachelor of Science in Biotechnology/Molecular

Biotechnology

08/1992 – 06/2001 Bergstadt Gymnasium, Lüdenscheid, Germany

“recombinant cellulase production in...

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