JBEI highlights March 2016

1. 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

2. 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

3. 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?

4. 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.

5. 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 thioredoxin
domain.

6. 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

7. 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

8. 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.