JBEI Research Highlights - March 2017

1. Production of odd-carbon dicarboxylic acids in
E. coli using an engineered biotin−fatty acid
biosynthetic pathway
Haushalter et al. (2017) “Production of Odd-Carbon Dicarboxylic Acids in Escherichia coli Using an Engineered Biotin−Fatty Acid
Biosynthetic Pathway” Metab. Eng. 39:247-256 doi: 10.1016/j.ymben.2016.12.008.
Background
• Dicarboxylic acids are commodity chemicals used in the production of plastics,
polyesters, nylons, fragrances, and medications.
• Bio-based routes to dicarboxylic acids are gaining attention due to
environmental concerns about petroleum-based production of these
compounds.
• Here we report a novel pathway to odd-carbon dicarboxylic acids directly from
glucose in Escherichia coli by employing an engineered pathway combining
enzymes from biotin and fatty acid synthesis.
Significance
• Optimization of the pathway will lead to industrial strains for the production
of valuable odd carbon diacids as potential biorefinery products.
Approach
Outcomes
• Both fatty acid and biotin biosyntheses begin with malonyl-ACP. This
intermediate is either directly used by FAS, or methylated by BioC and
extended by the same FAS complex to generate pimeloyl-ACP methyl ester.
• Overexpression of BioC increases metabolic flux down this pathway, while
knockout of BioH allows extension beyond the C7 pimeloyl intermediate.
• The extended ACP-bound methyl ester is cleaved off of ACP via thioester
hydrolysis catalyzed by ˈTesA, which appears to play a role in hydrolyzing the
resulting monomethyl esters to dicarboxylic acids.
• When bioC and ˈtesA were coexpressed, odd-carbon DCAs ranging from C9 to C15
were produced with negligible amounts observed in the control expressing ˈtesA and
rfp.
• Most observed products were DCAs, with Brassylic acid (C13) comprising of 80−90%
of DCAs produced.
• BioC genes from B. cereus, P. putida, and Kurthia were each coexpressed with ˈtesA
yielding higher titers in B. cereus and Kurthia.

2. Chemoselective methylation of phenolic hydroxyl
group prevents quinone methide formation and
repolymerization during lignin depolymerization
K. Kim, T. Dutta, E. Walter, N. Isern, J. Cort, B. Simmons, S. Singh (2017) “Chemoselective methylation of
phenolic hydroxyl group prevents quinone methide formation and repolymerization during lignin
depolymerization”. ACS Sustainable Chemistry & Engineering, DOI: 10.1021/acssuschemeng.6b03102
Background
• In lignin depolymerization, the presence or absence of various
functional groups significantly affect the reaction and the
distribution of products
• The phenolic hydroxyl group (Ar-OH) is one of the most
reactive functional groups in lignin, such blocking groups could
confer unique advantages to lignin depolymerization reaction
Approach
• Native lignin (milled wood lignin) obtained from Pine as well as
dimeric model compounds were used to prove the hypothesis
• Both lignin and structurally modified lignin were
thermochemically depolymerized at 300 °C and the resulting
products were analyzed
Outcomes
• Chemoselective masking of the Ar-OH group by methylation
was found to suppress secondary repolymerization and
charring during depolymerization
• Methylation of Ar-OH prevents formation of reactive quinone
methide intermediates, which are partly responsible for
undesirable secondary reactions
Significance
• This work demonstrate that structural modification of lignin is
desirable for production of low Mw phenolic products
• This approach could be directed toward alteration of natural
lignification processes to produce biomass more amenable to
depolymerization in a biorefinery setting
• Solid yield significantly
decreased from
methylated lignin
Selective methylation of pine milled wood lignin (MWL)

3. Production of jet fuel precursor mono-
terpenoids from engineered Escherichia coli
Mendez-Perez et al. (2017) “Production of jet fuel precursor monoterpenoids from
engineered Escherichia coli,” Biotechnology and Bioengineering, doi: 10.1002/bit.26296
Background
• Monoterpenes are isoprenoids commonly used in fragrances, flavors
and pharmaceuticals. Monoterpene-derive molecules are promising
alternatives to petroleum-derived jet fuels since they have similar
carbon chain length, heat of combustion and freezing point.
• Monoterpenes are synthesized from geranyl diphosphate (GPP),
which is also the precursor for farnesyl diphosphate (FPP), essential
for the biosynthesis of cell-wall polysaccharides (Figure 1).
Fig 1. Monoterpene biosynthesis and competing pathway
Fig 2. Production of 1,8‐cineole and metabolomics
analysis in the engineered strains
Approach
• Monoterpene production was achieved by expressing a nine-enzyme
mevalonate pathway organized in three operons expressed from a
single plasmid (1P) or two plasmids (2P).
• A mutation in E. coli’s native FPP synthase (IspA*) was tested to
improve availability of GPP for the production of monoterpenes (1,8-
cineole and linalool).
Outcomes
• Monoterpene production in the strain with limited FPP biosynthesis
(DH1*) results in selective pressure against production when the
monoterpene synthases are expressed from a high copy plasmid.
• Monoterpene production at high levels required not only the
optimization of GPP levels but also a basal level of FPP to maintain
growth (Figure 2).
Significance
• The optimized strains produced 653 mg/L of 1,8-cineole and 505 mg/L
of linalool, the highest reported titers for monoterpene production in
bacteria.

4. Ternary ionic liquid–water pretreatment systems
of an agave bagasse and municipal solid waste
blend
Perez-Pimienta et al. (2017). "Ternary ionic liquid-water pretreatment systems of an agave bagasse
and municipal solid waste blend," Biotechnol Biofuels, 10(1), 72. doi, 10.1186/s13068-017-0758-4
Background
• There are several challenges that need to be addressed for IL
pretreatment to become viable for commercialization, including IL cost
and recyclability
• One approach to potentially reduce IL cost is to use a blend of ILs at
different concentrations in aqueous mixtures
• It is unclear whether ILs can maintain process performance when
utilizing low-cost, low-quality biomass feedstocks such as the paper
fraction of municipal solid waste (MSW), which are readily available in
high quantities
Glucan and xylan conversion of pretreated agave bagasse
using recycled IL on blended MSW‐AGB feedstocks
Approach
• We evaluated 14 IL-water systems with mixtures of 1-ethyl-3-
ethylimidazolium acetate ([C2C1Im][OAc]), 1-butyl-3-ethylimidazolium
acetate ([C4C1Im][OAc]), and water that were used to pretreat MSW
blended with agave bagasse (AGB)
Outcomes
• The AGB/MSW (1:1) blend demonstrated a glucan conversion of
94.1% and 83.0% using IL systems with ~10 and ~40% water content,
respectively
• The glucan and xylan hydrolysis yields obtained from recycled IL
exhibited a slight decrease in pretreatment efficiency (less than 10%
in terms of hydrolysis yields compared to that of fresh IL)
Significance
• Our results demonstrated that mixing ILs and blending the paper
fraction of MSW with agricultural residues, such as AGB, may
contribute to lower the production costs while maintaining high sugar
yields
Schematic of overall workflow used in the study

5. Plant cell wall: never too much acetate
Outcomes
• BS1 appears to reside in the Golgi rather than being secreted to the
wall similarly to most polysaccharide hydrolases. Polysaccharide
hydrolases in the Golgi are likely playing an editing role, for instance,
by correcting ‘mistakes’ made by biosynthetic enzymes
• BS1 appears to function in removing excess acetylation to ensure the
right amount of xylan acetylation.
• Xylans in bs1 mutant plants have a substantial amount of acetylated
arabinose, which is not usually found in the wild-type plants, and BS1
is also playing a role in removing this unusual acetylation.
Figure 1 | Xylans are cell wall polysaccharides that are made from xylose and usually
acetylated, with acetyl groups on some of the backbone xylose residues. However,
esterases such as BS1 may be required to trim excess acetylation from the nascent
xylan. In the absence of BS1 activity, rice xylans are more heavily acetylated and
surprisingly contain acetylated arabinose residues, which are not usually present.
Xylans are very important components of secondary cell walls and essential for proper
function and development of plant vasculature. The right amount of xylan acetylation
is required; too much or too little acetylation has adverse effects on plant growth and
development.
Background
• Plant cell walls incorporate a variety of acetylated polysaccharides. In addition to enzymes catalyzing acetylation
(acetyltransferases), plants produce enzymes to remove acetyl groups (acetylesterases).
• A new xylan acetylesterase has now been identified, which belongs to a new family of carbohydrate esterases.
• The paper from JBEI is an opinion piece describing the significance of a primary paper in the same journal issue
(Zhang et al. 2016, Nat Plants 3)
Significance
• Xylan acetylation is the major source of acetate in biomass, and is important because acetate is a strong
inhibitor of biofuel production in yeast.
• The work of Zhang et al. is important because it identifies a new family of carbohydrate esterases and it
suggests that polysaccharide deacetylation may take place in the Golgi prior to deposition of the nascent
polymer in the plant cell wall.
Approach
• The new esterase is important in plants; bs1 mutants have elevated
xylan acetylation and defects in plant growth and development.
• The broad specificity of esterases in vitro is a major challenge in
assigning their functions, but in the case of the Zhang et al. study,
the xylan acetylesterase activity in vitro is consistent with the
phenotype of the mutant plants, which provides confidence in the
functional assignment.
Scheller, H. V. (2017). "Plant cell wall: Never too much acetate”. Nat Plants, 3, 17024. doi, 10.1038/nplants.2017.24

6. Advanced biodiesel and biojet fuels
from lignocellulosic biomass
Tian, T., & Lee, T. S. (2017). Advanced Biodiesel and Biojet Fuels from Lignocellulosic Biomass. In Consequences of Microbial
Interactions with Hydrocarbons, Oils, and Lipids: Production of Fuels and Chemicals (pp. 1‐25), S. Y. Lee (Ed.), Springer International
Publishing.
Background
• Global energy demands have stimulated interest
in renewable, carbon-neutral diesel and jet fuel
from biomass. Genetically engineered microbial
hosts can utilize sugars hydrolyzed from
lignocellulosic biomass as a carbon source to
biosynthesize a broad panel of bioproducts
including fatty acid-, isoprenoid-, and alcohol-
derived compounds, which can be used as
precursors or directly as fungible alternatives to
diesel and jet fuel.
Significance and perspectives
In this chapter, we focus on advanced biodiesel
and biojet fuels produced by genetically
engineered hosts capable of utilizing lignocellulosic
precursors.
• First, we briefly discuss the selection criteria of the
molecules targeted for microbial production.
• Then, the biosynthesis of each fuel type are discussed
based on pathway construction, host selection, and
strain optimization. We also highlight some recently
developed synthetic biology tools and their significance
in pathway engineering and genomic regulation.
• Finally, we briefly describe and compare various
lignocellulosic biomass deconstruction technologies
and report several commercialization examples of
biodiesel and biojet fuel production from biomass. Microbial conversions of glucose to diverse biofuel products via 2‐ketoacid pathway,
isoprenoid pathway and fatty acid pathway

7. Transgenic expression of fungal accessory
hemicellulases in Arabidopsis thaliana triggers
transcriptional patterns related to biotic stress and
defense response
Outcomes
• Transcripts for genes involved in plant defense and biotic stress were
significantly increased in the transgenic lines regardless of the tissue or
transgene expressed, with great overlap with the plant-pathogen interaction
pathway (KEGG: ATH04626). The defense genes were further categorized as
siacylic acid signalling.
• Transcripts for genes involved in photosynthesis (KEGG: ATH00196) were
reduced in all transgenic line examined.
Tsai et al. (2017) ”Transgenic expression of fungal accessory hemicellulases in Arabidopsis thaliana triggers
transcriptional patterns related to biotic stress and defense response.” PLoS One, DOI: 10.1371/journal.pone.0173094
Background
• Plants transgenically expressing microbial cell wall degrading enzymes can
lead to alter cell wall properties that are beneficial for lignocellulosic
applications
• Such expression strategies may lead to undesirable plant phenotype for
bioenergy feedstocks (ex. dwarf, necrotic leaves). This study investigate the
plant host responses as the result of microbial transgene expression.
Significance
• An understanding of underlying molecular mechanism and regulatory pathway
resulted from transgenic expression of microbial hemicellulases in planta
facilitates better engineering of bioenergy feedstocks
Approach
• Performed RNAseq on basal and mid stems collected from Arabidopsis thaliana
constitutively expressing Aspergillus nidulans α-arabinofuranosidase (AnAF54)
and Phanerochaete carnosa glucuronoyl esterase (PcGCE).
Heat maps illustrate relative transcript
abundance in transgenic lines in plant‐
pathogen interaction pathway (top) and
photosynthesis (bottom)

8. Determination of glycoside hydrolase
specificities during hydrolysis of plant cell
walls using glycome profiling
Walker et al., (2017) "Determination of glycoside hydrolase specificities during hydrolysis of plant
cell walls using glycome profiling,” Biotechnol Biofuels, 10, 31. doi, 10.1186/s13068-017-0703-6
Background
• Collaboration between BESC, GLBRC, and JBEI.
• The general biochemical reactions of GH enzymes are
understood, but the substrate specificity and mechanistic
understanding as a function of GH designation remains unclear
Significance
• The identification of GH specificities for a wide diversity of
polysaccharide structures provided by glycome profiling AND
oxime-NIMS offers a unique combination to understand the
hydrolytic capabilities and constraints of individual enzymes as
they interact with plant biomass that may benefit commercial
enzyme mixtures
Approach
• To better understand the specificity of enzyme hydrolysis within the
complex matrix of polysaccharides found in the plant cell wall, we
studied the reactions of individual enzymes using glycome
profiling, and quantitative nanostructure initiator mass spectrometry
(oxime-NIMS) to determine soluble sugar products of their
reactions
Outcomes
• Single, purified enzymes from the GH5_4, GH10, and GH11
families of glycoside hydrolases hydrolyzed hemicelluloses as
evidenced by the loss of specific epitopes from the glycome
profiles in enzyme-treated plant biomass
• The GH10 enzyme proved to be reactive with the broadest
diversity of xylose-backbone polysaccharide epitopes, but was
incapable of reacting with glucose-backbone polysaccharides
• GH5 and GH11 enzymes studied have the ability to react with both
glucose- and xylose-backbone polysaccharides Oxime-NIMS analysis of enzyme activity (μmol of reducing
sugar released per μmol of pure enzyme per hour at 55 °C)
for different classes of reducing sugar products obtained from
enzymatic hydrolysis of (a) AFEX-corn stover or (b) AFEX-
switchgrass. Hexose products are derived from cellulose,
while pentose and mixed pentose/hexose products are from
various hemicellulose polysaccharides.

9. Cyanobacterial carbon metabolism: fluxome
plasticity and oxygen dependence
Wan et al., (2017) “Cyanobacterial Carbon Metabolism: Fluxome Plasticity and Oxygen
Dependence” Biotechnol. Bioeng. DOI 10.1002/bit.26287.
Background
• Synechocystis sp. strain PCC 6803 has been widely used as a photo-
biorefinery chassis.
• Based on its genome annotation, this species contains a complete TCA cycle,
an Embden-Meyerhof-Parnas pathway (EMPP), an oxidative pentose
phosphate pathway (OPPP), and an Entner–Doudoroff pathway (EDP).
• In this study, we evaluated Synechocystis 6803 carbon metabolism based on
fluxome plasticity and oxygen dependence.
Significance
• This work demonstrates how genetic profiles do not always reflect actual
metabolic flux through native or heterologous pathways.
Approach
• To evaluate how Synechocystis 6803 catabolizes glucose under heterotrophic
conditions, we performed 13C metabolic flux analysis, metabolite pool size
analysis, gene knockouts, and heterologous expressions.
Outcomes
• Cyanobacteria cannot grow with glucose under anaerobic conditions in the
absence of sufficient light.
• Oxidative phosphorylation appears to be essential for cell metabolism in the
absence of sufficient light.
• Cyanobacteria employ the OPP pathway and oxidative phosphorylation as the
key pathways for production of energy and reducing equivalents during dark
and heterotrophic conditions.
• TCA cycle provides the building blocks for growth, but it maintains a low flux
rate.
• The discrepancy between genome scale model predictions and our
experimental results demonstrates that many hidden enzymatic constraints or
reaction thermodynamics may force microbes to select unpredictable
metabolic topologies.
Synechocystis 6803 fluxomes under different growth conditions. Key
reactions in glycolysis (A), the Calvin cycle (B), the TCA cycle (C), and the
OPPP were compared: under dark condition (D), photoheterotrophic
condition (H; DCMU-induced or atrazine-induced), photomixotrophic condition
(M), and photoautotrophic condition (A).
Synechocystis 6803 growth under aerobic and anaerobic
conditionafter an 8-day cultivation.

10. Characterization of white poplar and eucalyptus after
ionic liquid pretreatment as a function of biomass
loading using x-ray diffraction and small angle neutron
scattering
Outcomes
• SANS data show that smaller pores contributed less to
the increased porosity than larger pores after IL
pretreatment
• XRD data indicate that native cellulose crystalline
structure of eucalyptus sample was distorted, possibly
due to intercalation of IL molecules between (110) planes
and (11 ̅0) planes
Yuan et al. (2017) ”Characterization of white poplar and eucalyptus after ionic liquid pretreatment as a function of biomass loading
using x-ray diffraction and small angle neutron scattering,” Bioresource Technology, DOI: 10.1016/j.biortech.2017.02.014
Background
• Pretreatment conditions and biomass types significantly affect the
biomass structures and sugar conversion as a function of biomass
loading
Significance
• Further insights into IL pretreatment processes and refined
current mechanistic understanding on this bioenergy
technology
Approach
• A side-by-side comparison of physical structural changes of two
hardwood biomasses that exhibited different response to IL
pretreatment as a function of biomass
• SANS detected pores of radii ranging from ~25 to 625 Å, enabling
assessment of contributions of pores with different sizes to increased
porosity after pretreatment
Plots of (a) SANS data of IL pretreated white poplar samples ; (b) relative
increase in intensity at various q values for white poplar samples

11. Biomass pretreatment using dilute aqueous ionic
liquid (IL) solutions with dynamically varying IL
concentration and its impact on IL recycling
Yuan et al. (2017) “Biomass pretreatment using dilute aqueous ionic liquid (IL) solutions with dynamically varying IL
concentration and its impact on IL recycling.” ACS Sustainable Chem. Eng., DOI: 10.1021/acssuschemeng.7b00480
Background
• The cost of IL pretreatment process relative to other
pretreatment technologies has been a challenge
Approach
• A distillation apparatus was used in the pretreatment,
allowing utilization of aqueous IL solutions with initially
low IL concentrations that were not considered as
effective pretreatment media previously
Outcomes
• Aqueous [C2C1Im][OAc] solutions with initial
concentrations of 7 and 15 wt.% were tested to
pretreat white poplar samples at 130C for 3h. After
pretreatment, sugar conversion was found to account
for 72-77 % of the sample pretreated in neat
[C2C1Im][OAc].
• The encouraging results of this operation prompted a
new approach to recycle and reuse of [C2C1Im][OAc]:
use the liquor collected after biomass pretreatment in
neat [C2C1Im][OAc] without firstly separating water
from it.
Significance
• The potential of this approach of biomass
pretreatment, IL recycling and reuse may stimulate the
design of new IL pretreatment technologies suitable for
a biorefinery.
Effect of pretreatment liquor reuse and [C2C1Im][OAc]
concentration in aqueous solutions on sugar conversion of
pretreated white poplar samples.