Development of an E. coli strain for One-pot

Biofuel Production from Ionic Liquid

Pretreated Cellulose and Switchgrass

Approach• An IL-tolerant production host was equipped with IL-tolerant cellulases to produce the advanced biofuel

precursor d-limonene from cellulose and hydrolysate in the presence of IL

Outcomes• Spontaneous resistance of E. coli to an a widely used IL was determined to be due to a knock out

phenotype in a single gene, rcdA.

• This strain was able to produce limonene using saccharified IL-pretreated hydrolysate as carbon source

• Equipping this strain with an IL-tolerant cellulase (J29) allowed production of limonene from

unsaccharified cellulose in the presence of 100 mM [C2C1im][OAc]

M. Frederix et al. (2016). "Development of an E. coli strain for one-pot biofuel production from ionic

liquid pretreated cellulose and switchgrass,” Green Chemistry, DOI: 10.1039/C6GC00642F.

Background• Due to the reduced cost of IL synthesis

significant amounts of this deconstruction

reagent will remain in the sugar stream

used for microbial biofuel production in an

industrial process.

• Pathways for microbial production of

advanced biofuels have been established

but typically evaluated using defined media

and pure sugars.

• IL tolerant cellulases are also known but

have never been consolidated into a

microbe for a one-pot production process.

Significance• This work represents the necessary next step in the development of one-pot biofuel production

processes in the presence of ionic liquids

pLimonene

J29 cellulase rcdA ybjJ *

ILs *

IL tolerance

Figure-1 (Mukhopadhyay)

Switchable Ionic Liquids for One-Pot

Conversion of Biomass to Isopentanol

Outcomes• The IL [Ch][Glu] was used to reversibly adjusts the pH and generated

fermentable sugar yields of 91%,

• These sugars were converted directly into the advanced biofuel

isopentenol through fermentation at a titer of 1.2 g/L, corresponding to

34% of maximum theoretical yield, with no further purification or

separation steps.

• TEA suggests ~$1/gal reduction in the minimum biofuel selling price

compared to biorefineries designed to use [C2C1Im][OAc].

Figure 1. Schematic of a One-Pot Switchable DCA-IL Process. Pretreatment, enzymatic

saccharification and fermentation are integrated into a single reactor (one-pot) process in which

the pH is made favorable for pretreatment by using a 2:1 ratio of Ch to Glu, favorable for

saccharification by addition of one equivalent of Glu to reduce the pH to levels at which

Novozymes Ctec2 and Htec2 are optimally active, and addiction of a small amount of Ch to

slightly increase the pH levels optimal for fermentation.

M. Liszka et al. (2016) ”Switchable ionic liquids based on di-carboxylic acids for one-pot conversion

of biomass to an advanced biofuel,” Green Chemistry, DOI: 10.1039/C6GC00657D.

Background• Ionic liquids based on widely available and

inexpensive choline cations and amino acid-

based anions have been shown to effectively

pretreat biomass.

• Aqueous solutions of these ILs are too basic to

use directly in later enzymatic saccharification

and fermentation steps.

Significance• These are seminal advances in ionic liquid pretreatment that

overcome the pH mismatch between pretreatment, saccharification

and fermentation, facilitating one-pot conversion processes.

Figure 2. Glucose (panel A) and xylose (panel B) yields.

Larger scale one-pot pretreatment with [Ch]2[Glu] and

saccharification reactions in [Ch][Glu] after addition of one

equivalent of [Glu]. Reaction conditions: Solid Circles: 10%

biomass, 25% [Ch]2[Glu], 120ºC, 3hrs; Solid Squares: 10%

biomass, 25% [Ch]2[Glu], 150ºC, 3hrs.

Approach• We developed a process using ionic liquids

based on di-carboxylic acids.

• The two ionization states of di-carboxylic acids

allow switching from a basic pH favorable for

pretreatment to a slightly acidic pH favorable for

cellulases and fermentative organisms.

Enrichment of Microbial Communities Tolerant to

the Ionic Liquids Tetrabutylphosphonium Chloride

and Tributylethylphosphonium Diethylphosphate

Background• Phosphonium ILs have been shown to make cellulose more accessible

without degrading it and efficiently extract of lignin. Several studies have

demonstrated compatibility between microorganisms and phosphonium ILs.

This feature could be beneficial during the biological conversion steps in

biofuel production.

Approach• The aims for this study were to identify thermophilic microbial communities

tolerant to phosphonium ILs that can efficiently decompose lignocellulose in a

high-solids environment and to evaluate cryogenic preservation methods for

the enriched community.

Outcomes• High-solids incubations with stepwise increases in IL concentration

enriched for thermophilic IL-tolerant communities that decomposed

green waste. 16S rRNA sequencing of enriched communities revealed

microorganisms capable of tolerating high levels of IL.

• Cryogenic preservation of enriched communities reduced the IL

tolerance of the community and decreased the relative abundance of IL-

tolerant organisms.

Significance• Gradual increases in phosphonium-based IL concentrations during the enrichment yielded communities capable of thriving

on green waste in a high-solids, thermophilic environment relevant to bioenergy production.

• Current storage techniques appear to be too harsh for total community recovery; there was a substantial decrease in

respiration, which can be attributed to a decrease in activity of IL-tolerant microorganisms.

S. Pace et al. (2016) “Enrichment of microbial communities tolerant to the ionic liquids tetrabutylphosphonium chloride

and tributylethylphosphonium diethylphosphate,” Appl Micro Biotech, DOI: 10.1007/s00253-016-7525-5.

Microbial Activity (CO2 respiration) of community with phosphonium IL

Community structure (16 rRNA gene) ofCommunity with phosphonium IL

A DUF-246 Family Glycosyltransferase-like

Gene Affects Male Fertility and the

Biosynthesis of Pectic Arabinogalactans

Outcomes• NbPAGR-silenced plants exhibited reduced internode and

petiole expansion. Cell wall materials from NbPAGR-silenced

plants had reduced galactose content compared to the control.

• Arabidopsis lines overexpressing PAGR exhibit pleiotropic

developmental phenotypes and the loss of apical dominance as

well as an increase in RG-I type-II arabinogalactan content.

VIGS of PAGR in

Nicotiana bethamiana.

The PAGR gene is

essential in Arabidopsis

and knock-out mutants could

not be obtained. Instead the

orthologs were silenced in

tobacco. The plants exhibit

reduced expansion of

internodes. A: Control, B:

silenced plants.

S. Stonebloom et al. (2016) "A DUF-246 family glycosyltransferase-like gene affects male fertility and the

biosynthesis of pectic arabinogalactans,” BMC Plant Biol, 16(1), 90, DOI: 10.1186/s12870-016-0780-x.

Background• Pectins are a group of structurally complex plant cell wall

polysaccharides whose biosynthesis and function remain

poorly understood.

• To date few enzymes involved in the biosynthesis of

pectin have been described.

Significance• These results support a function for PAGR in the biosynthesis of

RG-I arabinogalactans and illustrate the essential roles of these

polysaccharides in vegetative and reproductive plant growth that

ultimately may play a role in increasing bioenergy crop yields.

Approach• T-DNA insertions in PAGR were identified in Arabidopsis

thaliana and were found to segregate at a 1:1 ratio of

heterozygotes to wild type.

• To characterize a loss-of-function phenotype for PAGR,

the Nicotiana benthamiana orthologs, NbPAGR-A and

NbPAGR-B, were transiently silenced using Virus

Induced Gene Silencing.

A B A B C

Overexpression of PAGR in

Arabidopsis. The plants exhibit reduced

growth, loss of apical dominance and

swollen pedicels. A,D: Wild-type, B,C,E,F:

Overexpressing plants.

D E F

Wild Type

35S:line 6

35S:line 9

Control

NbPAGR-

silenced

Glycoprofiling of isolated RG-I (rhamnogalacturonan I) from

silenced and overexpressing plants. Silenced plants show

decreased presence of several RG-I arabinogalactan epitopes. In

contrast overexpressing plants show increased presence of RG-I

epitopes. RG-I was isolated by size-exclusion chromatography and

profiled using monoclonal antibodies.

Synthetic and Systems Biology for Microbial

Production of Commodity Chemicals

Outcomes• Challenges start at molecule selection, where a difficult balance between

economic potential and biological feasibility must be struck.

• Choosing optimal relative protein expression levels for maximum production is still

the subject of heuristic, non-systematic approaches.

• Toxic metabolic intermediates and proteins can significantly affect production, and

dynamic pathway regulation emerges as a powerful but yet immature tool to

prevent it.

• Host engineering arises as a much needed complement to pathway engineering

for high bioproduct yields.

• A final, and often underestimated, challenge is the successful scale up of

processes to commercial volumes.

Integrated Fluid Dynamics (IFD) merges traditional

fluid dynamics analysis describing fluid flow with

models of bacterial metabolism.

V. Chubukov et al. (2016) “Synthetic and systems biology for microbial production of commodity

chemicals,” Nature Systems Biology & Applications, 2, 16009, DOI: 10.1038/npjsba.2016.9

Background• The combination of synthetic and

systems biology is powerful

framework to study fundamental

questions in biology.

• However, we cannot yet engineer

biological systems as easily and

precisely as we engineer physical

systems.

• In this review, we describe the path

from the choice of target molecule to

scaling production up to commercial

volumes, along with the associated

challenges.The process of bioengineering strains for commodity chemicals from

initial concept (target molecule selection) to scale up (process engineering

and implementation), along with a selection of tools applicable to each step

and the grand challenges that need to be met.