Alteration of of polyketide stereochemistry from

Anti to Syn by a ketoreductase domain exchange

in a type I modular polyketide synthase subunit

Outcomes• LipPks1+TE contains an A2-type KR domain and specifically yielded

the (2S,3S) configuration (or Anti) in the acid products (Figure B).

• We exchanged the original A2-type KR domain with three different

types of B-type KR domains (B, B-1, and B-2) but none of these

attempts were successful. The reason is still unclear.

• We exchanged the original A2-type KR domain with an A1-type KR

domain and were able to successfully convert the stereochemistry at

the C2 position to generate the Syn products (Figure B).

Eng et al. (2016) “Alteration of of polyketide stereochemistry from anti to syn by a ketoreductase domain

exchange in a type I modular polyketide synthase subunit” Biochemistry, 55, pp. 1677-1680.

Background• Polyketides made by type I polyketide synthases (PKSs) are typically

rich in stereocenters.

• Altering the stereochemistry is an important method to increase

chemical diversity.

Significance• These results demonstrate a novel alteration of polyketide product

stereochemistry from Anti to Syn that may prove useful for biofuels

and renewable chemicals.

Approach• Exchange ketoreductase (KR) domains in a model PKS system

(LipPks1+TE).

• KR domains are responsible for altering the stereochemistry in both the

b-hydroxy and a-alkyl groups.

• A naming conversion has been established to describe b-hydroxy (A or

B) and a-substituent (1 = nonepimerized, 2 = epimerized) as shown in

Figure A.

A A1

B1

A2

B2

A

B

KR domain exchange

B

A2

A1

Anti

Syn

TEACPKRATKSAT ACP

AT ACP KS AT KR ACP TE

Outcomes• A set of 41 mutated lines has been sequenced.

• Over 2400 mutations were identified and 1284 genes

were mutated in the 41 mutants.

• Established a pipeline for analysis of sequences of

mutated lines.

Li, et al. (2016) “Genome-wide sequencing of 41 rice (Oryza sativa L.)

mutated lines reveals diverse mutations induced by fast-neutron

irradiation” Mol. Plant. doi: 10.1016/j.molp.2016.03.009.

Background• There is a lack of genomics tools available to

determine the function of genes involved in grass cell

wall biosynthesis and other biological processes.

Significance• These results reveal that a diversity of mutations are

induced by fast-neutron irradiation.

• We have established a whole-genome sequenced mutant

collection that will significantly facilitate biofuel research in

grasses, including switchgrass and sorghum.

Genome-wide characterization of mutations in 41 FN-induced rice mutant

lines. (A) Strategy for generating and sequencing the rice FN mutagenized population.

(B) Representation of the diversity of mutations induced by FN irradiation. The inner pie

chart indicates the number of each type of mutations in the 41 sequenced M3 lines. The

outer circle shows the number of genes affected by each type of mutation. SBS: single

base substitutions; DEL: deletions; INS: insertions; INV: inversions; TRA: translocations;

and DUP: tandem duplications. (C) Comparisons between FN-induced SBSs and the

SNPs present between X.Kitaake and the reference genome. (D) Size distribution of FN-

induced deletions the 41 rice mutant lines. (E) Representation of the twelve rice

chromosomes on an Mb scale. (F) Representation of repetitive sequences in the

reference genome in non-overlapping windows (window size = 500 kb). (G) The

sequencing depth of the 42 rice lines. (H) Genome-wide distribution of FN-induced

mutations in non-overlapping 500-kb windows. (I) Translocations.

Approach• Whole-genome sequencing of a model grass mutant

population

• Apply complementary bioinformatics algorithms to

identify genetic mutations

Genome-wide sequencing of 41 rice

(Oryza sativa L.) mutated lines reveals diverse

mutations induced by fast-neutron irradiation

Biomass Modification, Characterization, and

Process Monitoring Analytics to Support Biofuel

and Biomaterial Production

Outcomes• Rapid instrumental techniques have been developed for real-time monitoring of

diverse processes, such as the efficacy of specific pretreatment strategies, or

downstream products, such as biofuels and biomaterials.

• Real-time process monitoring techniques are needed for all stages of the

feedstocks-to-biofuels conversion process to maximize efficiency and lower

costs by monitoring and optimizing performance.

Lupoi, J., Simmons, B., & Henry, R. (2016). ”Biomass Modification, Characterization, and Process Monitoring Analytics to

Support Biofuel and Biomaterial Production". Front Bioeng Biotechnol, 4, 25. doi, 10.3389/fbioe.2016.00025

Background• This Frontiers Research Topic journeys through various challenges

facing researchers seeking to develop fuels and products derived from

lignocellulosic biomass.

Significance• This collection of papers demonstrates how advances at multiple levels are likely

to contribute to the successful industrial scale production of biofuels and

biomaterials

Approach• These challenges include: the rapid quantification of plant cell wall chemistry,

enabling yields of potential monomeric sugars to be assessed, identification of

plants possessing ideal trait that can be brought to the forefront of research

efforts; once the native plant chemistry is known, how can yields be improved

by chemically or genetically altering plant cell walls to reduce recalcitrance;

does genetic modification of plants to increase accessibility to saccharification

enzymes hinder the plant’s growth and/or function; are the innovative methods

identified by researchers cost-effective and scalable to a commercial level?

Engineering cellular metabolism

Outcomes• Most strains used for industrial production require

a large number of genetic modifications, not only

in the pathways of interest, but also in other

pathways in order to efficiently redirect metabolic

flux.

• It is often necessary to combine overexpression

of specific enzymes with deregulation of the

pathway in order to ensure high flux through the

pathway of interest.

Nielsen, J., & Keasling, J. D. (2016). "Engineering Cellular Metabolism". Cell, 164(6), 1185-1197.

Background• Despite the advanced systems and synthetic

biology technologies now available for detailed

phenotypic characterization of cells and genome

editing, developing new cell factories that meet the

economic requirements for industrial scale

production is still challenging.

• Engineering a cell factory involves several rounds

of the so-called ‘design-build-test’’ cycle.

Significance• The development of a solid knowledge base for such new platform cell factories will obviously

be time-consuming, but using the scaffold for knowledge integration established through

BioCAD, it will be possible to advance rapidly in the development of bioenergy solutions.

(A) According to the bow-tie structure of metabolism, all carbon sources are

converted to 12 precursor metabolites that are used for biosynthesis of all secreted

metabolites. The precursor metabolites are also used for the biosynthesis of all

building blocks that are needed for synthesizing macromolecules making up the

biomass of the cell. (B) Illustration of how an acetyl-CoA over-producing strain can

be used as a platform strain for production of a range of different molecules.

Approach• Review paper discusses the principles and current

challenges of metabolic engineering, focusing on

how metabolism can be engineered for industrial

level production of specific chemicals.

Structural characterization of enzymes in

the lignin β-aryl ether

cleavage pathway

Outcomes• Nine X-ray crystal structures and kinetic characterization of β-etherase LigD, LigO, LigL and LigG

from Sphingobium.

• Detailed information on the cofactor and substrate binding sites, and on the catalytic mechanisms

of these enzymes.

Significance• The structure-function relationships and biochemistry of these enzymes

can inform future efforts in producing advanced biofuels and chemicals from lignin-derived materials.

Pereira et al. (2016). “Structural and Biochemical Characterization of the Early and Late Enzymes in the Lignin β-aryl Ether Cleavage Pathway from Sphingobium sp SYK-6” Journal of Biological Chemistry doi, 10.1074/jbc.M115.700427

Background• The production of fuels and chemicals

from lignocellulosic biomass has

created new opportunities for

developing high value products from

lignin, the most abundant aromatic

polymer in nature.

• Sphingobium sp. SYK-6 degrades

aromatic oligomers derived from lignin.

Approach• Using X-ray crystallography, enzyme

kinetic assays, we have characterized

bacterial enzymes that cleave β-aryl

ether bonds, the most abundant inter-

unit linkage in lignin.

FIGURE 1. a) Cartoon and molecular surface representations of apo-LigL and the LigL-NADH-(αS,βR)-GGE complex. b) The active site of LigL in complex with NADH showing the interactions involving the co-substrate NADH. c) The substrate binding site for LigL-NADH-(αS,βR)-GGE showing residues Asp95, Ser144, Tyr158, Pro188 and Arg222 that interact directly with the GGE substrate. d)Active site of LigL-NADH-(αS,βR)-GGE showing the catalytic tetrad N115-S144-Y158-K162, and a water molecule (W75) involved in the extended proton relay system.

FIGURE 2. a) Overall cartoon representation of the LigG-GS-AV complex dimer. b)Superposition of the GSH binding site of apo-LigG (magenta) and LigG-GSH (PDB ID 4G10) (orange) structures. c) Molecular surface representation of the LigG monomer in complex with the GS-AV substrate analog. d) Active site of the LigG-GS-AV complex. The glutathionyl moiety of the GS-AV substrate sits on the top of the four β-strands of the N-terminal thioredoxindomain.

Evaluation of agave bagasse recalcitrance

using AFEXTM, autohydrolysis, and ionic

liquid pretreatments

Outcomes• AFEX completely preserves plant carbohydrates.

• AH solubilized 62% of xylan from untreated AGB.

• A 25% delignification occurred after IL pretreatment.

• Intensity of β-aryl ether units in aliphatic, anomeric and aromatic regions

of NMR spectra decreased in all pretreated samples

• IL pretreatment generated highest sugar yields

A) Aromatic region of HSQC-NMR

spectra of untreated and pretreated

agave bagasse samples. 1) First time that

S/G lignin ratio of untreated Agave tequilana is

calculated. 2) Untreated AGB had a S/G ratio

of 4.3, which is higher than others current

biofuel feedstocks. 3) An increased S/G ratio is

observed in AFEX (5.0) and IL (4.7) but AH

decrease up to 4.2, due to lignin distribution

changes in the plant cell wall.

Pérez-Pimienta, et al. (2016) “Evaluation of agave bagasse recalcitrance using AFEXTM,

autohydrolysis and ionic liquid pretreatments” Bioresource Technology, 211, pp. 216-223.

Background• Agave is a drought-resistant cellulosic

feedstock with high productivities in semiarid

regions and minimal water/nutrients inputs.

• Independent bioconversion studies have been

made on agave bagasse (AGB) to reduce its

recalcitrance for downstream process, but

different experimental conditions were applied.

Significance• These comparative analyses will contribute to a better understanding of

AGB recalcitrance and enable more rational selection of biorefinery

configurations.

B) Glucan conversion of untreated and pretreated

agave bagasse at final saccharification time (72 h) using

a lower and higher biomass loading.1) IL-pretreated AGB achieved the highest glucan conversion of all

three pretreatments. 2) Yields of glucose in the major hydrolysate

stream were 28.2, 24.0 and 29.1 kg per 100 kg of untreated AGB

for AFEX, IL and AH, respectively.

Approach• First direct side-by-side comparative pretreatment

assessment on agave bagasse using ammonia

fiber expansion (AFEX), autohydrolysis (AH) and

ionic liquid (IL).

A

B

Rapid and robust optimization of energy

traits in crops

Outcomes• Progress in plant synthetic biology shows great promises

• Synthetic biology can leverage engineering at the genome; transcription,

translation; protein stability, activity and assembly; and metabolic flux levels

• Plant synthetic biology will support the development of multiple traits that are

needed to reach high product yields in different bioenergy crops

Shih P, Liang Y, Loqué D. (2016) “Biotechnology and synthetic biology approaches for metabolic engineering of

bioenergy crops.” The Plant Journal, doi: 10.1111/tpj.13176

Background• Novel strategies and technologies are needed

to speed up the development of robust and

diverse energy crops to fulfill sustainably and

cheaply U.S. demand in renewable energy.

Significance• This article highlights the importance and promises of synthetic biology to support the

development and optimization of energy and agronomical traits in crops

Approach• Application and development of synthetic

biology tools for plants

• Use of synthetic biology to support rapid and

robust plant engineering

Revealing the thermal sensitivity of lignin

during glycerol thermal processing (GTP)

through structural analysis

Outcomes• NMR results indicate that GTP is very effective at

breaking bonds within lignin.

• GTP generated smaller molecular weights of lignin as

a result of depolymerization.

• GTP generated moieties that were stable up to

temperatures >290 oC

Zhang et al. (2016) "Revealing the thermal sensitivity of lignin during glycerol thermal

processing through structural analysis” RSC Advances, 6(36), 30234-30246

Background• There is a strong specialties market for

selected lignin polymers (e.g., lignosulfonates),

due to their self-assembling properties.

• Kraft lignin has not penetrated commercial

markets due to poor thermal properties.

• Glycerol thermal processing (GTP) offers a

compelling alternative approach.

Significance• GTP generates a lignin product that is suitable for use as a

polymeric co-product comparable to other thermoplastics.

HSQC of EMAL and isolated GTP lignin – aliphatic region (SGL6:

recovered sweet gum lignin at GTP severity log(R0) ¼ 4.61; SGL8:

recovered sweet gum lignin at GTP severity log(R0) ¼ 5.03).

Approach• Comparison of the chemical and thermal

properties of lignin generated by a variety of

processes compared to that generated by GTP.