JBEI Research Highlights - February 2018

1. Outcomes and Impacts
• Muconic acid produced in plants is easily recovered from biomass upon conventional biomass pretreatments such as dilute
alkaline, and advanced pretreatments such as ionic liquids.
• Bioenergy crops that accumulate muconic acid will serve a dual purpose by providing not only monosaccharides for microbial
fermentation but also supplying an added-value co-product.
• Using such engineered plant biomass as feedstock for the microbial synthesis of muconic acid will enhance overall titers of
biomanufactured muconic acid.
• Bioenergy crops of the Salicaceae family (e.g. poplar, willow, aspen), which accumulate high level (>10% DW) of metabolites
derived from salicylic acid, represent adequate chassis for the production of muconic acid using the proposed metabolic
pathway.
Eudes et al. (2018) Metab. Eng., doi: 10.1016/j.ymben.2018.02.002
Background
• Adding value to bioenergy crops is essential to achieve economic
sustainability of biorefineries.
• Using metabolic engineering, valuable co-products such as platform
chemicals can be produced inexpensively in plant biomass feedstock.
• Muconic acid represents such an added-value co-product since it is a
precursor to several commodity chemicals such as adipic acid,
terephthalic acid, and caprolactam.
Approach
• As a proof-of-concept, we engineered Arabidopsis as a model for the
phytoproduction of muconic acid.
• Bacterial enzymes were targeted to plastids to enhance the synthesis
of salicylic acid and reroute its pool towards muconic acid.
• A 50-fold increase in muconic acid titer (>600 µg/g DW) was achieved
as compared to the initial engineered line.
Production of muconic acid in plants
0
100
200
300
400
500
600
700
Muconicacid(µg/gDW)
nahG-CatA
nahG-CatA-Irp9
nahG-CatA-Irp9-AroG
Plot showing the increase of muconic acid titers in plant biomass
after three successive rounds of metabolic pathway engineering.
PEP
E4P
DAHP CHOR
Salicylic
acid
CAT
Muconic
acid
De-novo metabolic pathway implemented in plants for the synthesis
of muconic acid via salicylic acid.
Engineered plant lines

2. Improving methyl ketone production in E. coli by
heterologous expression of NADH-dependent FabG
Outcomes and Impacts
• The best methyl ketone performance occurred in strains
expressing FabG derived from Acholeplasma laidlawii
(Al_FabG).
• Fed-batch fermentation revealed a 40% titer improvement
(up to 6 g/L) in an optimized Al_FabG strain compared to a
base strain and comparative proteomic data during
fermentation revealed physiological changes related to
NADPH availability .
• Overall, the results suggest that heterologous expression of
NADH-dependent FabG in E. coli may improve sustained
production of fatty acid-derived renewable fuels and
chemicals.
Goh et al. (2018) Biotechnology and Bioengineering, doi: 10.1002/bit.26558/pdf
Background
• We previously engineered E. coli to overproduce medium- to long-chain saturated and monounsaturated methyl ketones,
which could potentially be applied as diesel fuel blending agents or in the flavor and fragrance industry.
• Fatty acid-derived pathways, such as methyl ketone biosynthesis, face the metabolic challenge of high NADPH demand,
partly resulting from the key fatty acid biosynthetic enzyme, FabG (β-ketoacyl-ACP reductase).
Approach
• We attempted to mitigate cofactor imbalance by
heterologously expressing NADH-dependent, rather than
NADPH-dependent, versions of FabG that we identified in
previous studies.
• We evaluated the performance of engineered strains with
fed-batch fermentation in 2-L reactors by measuring
concentrations of methyl ketones and short-chain fatty
acids, cell dry weight, and shotgun proteomic profiles.
.
Summary comparison of methyl ketone-producing strains of E. coli
with and without expression of A. laidlawii FabG (Al_FabG). The
Al_FabG strain showed higher methyl ketone titer and lower pyruvate
accumulation (upper) and proteomic results at 50 hr showed
differential expression of proteins associated with NADPH production.
0
2
4
6
8
10
0
1
2
3
4
5
6
0 50 100 150 200 250
OrganicacidsandCDW
METHYLKETONES(g/L)
Time (hr)
MK with Al_FabG
MK
without
Al_FabG
FED-BATCH FERMENTATION
1.E-07
1.E-06
1.E-05
1.E-04
1.E-03
1.E-02
1.E-01
1.E+00
-10 -8 -6 -4 -2 0 2 4 6 8
p-value Log2 Fold Change
PROTEOMICS @ 50 hr
With Al_FabGWithout Al_FabG
MaeB
PGI

3. Identification of an algal xylan synthase indicates
that there is functional orthology between algal and
plant cell wall biosynthesis
Background
• Insights into the evolution of plant cell walls have important
implications for comprehending these diverse and abundant
biological structures.
• In order to understand the evolving structure–function
relationships of the plant cell wall, it is imperative to trace the
origin of its different components.
• 1,4-β-xylan is one of the most abundant components in plant
biomass.
Approach
• The present study is focused on plant 1,4-β-xylan, tracing its
evolutionary origin by genome and transcriptome mining followed
by phylogenetic analysis, utilizing a large selection of plants and
algae. It substantiates the findings by heterologous expression
and biochemical characterization of an alga xylan synthase.
Outcomes and Impacts
• Of the 12 known gene classes involved in 1,4-β-xylan formation in
plants, XYS1/IRX10, IRX7, IRX8, IRX9, IRX14 and GUX occurred
for the first time in charophyte algae. An XYS1/IRX10 ortholog
from Klebsormidium flaccidum, KfXYS1, possesses 1,4-β-xylan
synthase activity, and 1,4-β-xylan occurs in the K. flaccidum cell
wall.
• These data suggest that plant 1,4-β-xylan originated in
charophytes and shed light on the origin of one of the key cell wall
innovations to occur in charophyte algae, facilitating
terrestrialization and emergence of polysaccharide-based plant
cell walls.
Jensen et al. (2018) New Phytologist, doi: 10.1111/nph.15050
Phylogeny of the
green lineage
(Viridiplantae). The
key genes required
for synthesis of the
hemicellulose 1,4-β-
xylan evolved in a
common ancestor of
Klebsormidiophyceae
and land plants.
• Top: Enzymatic assay showing
that KfXYS1 from
Klebsormidium flaccidum has
xylan synthase activity in vitro
and can add xylose residues
to a 1,4-β-xylotetraose
acceptor substrate. Bottom:
Cell walls from K. flaccidum
contain 1,4-β-xylotetraose as
showed by digestion with
endo-xylanase and
Polysaccharide Analysis using
Carbohydrate gel
Electrophoresis (PACE). Lane
2 is a no enzyme control, and
the other lanes are
combinations of xylanase and
other enzymes. The pattern
shows the presence of
arabinofuranose substitutions
and the absence of
glycoronosyl substitutions.

4. Bi-functional glycosyltransferases catalyze both
extension and termination of pectic galactan
oligosaccharides
Background
• Pectins are the most complex polysaccharides of the plant cell
wall.
• Based on the number of methylations, acetylations, and glycosidic
linkages present in their structures, it is estimated that up to 67
transferase activities are involved in pectin biosynthesis.
• Pectic galactans constitute a major part of pectin in the form of
side chains of rhamnogalacturonan-I.
• In Arabidopsis, Galactan Synthase 1 (GALS1) catalyzes the
addition of galactose units from UDP-Gal to growing β-1,4-
galactan chains.
• However, the mechanisms for obtaining varying degrees of
polymerization remain poorly understood.
Approach
• In this study, we show that AtGALS1 is bifunctional, catalyzing
both the transfer of galactose from UDP-α-D-Gal and the transfer
of an arabinopyranose from UDP-β-L-Arap to galactan chains.
• The two substrates share a similar structure, but UDP-α-D-Gal is
the preferred substrate, with a tenfold higher affinity.
• Transfer of Arap to galactan prevents further addition of galactose
residues, resulting in a lower degree of polymerization.
Outcomes and Impacts
• We show that this dual activity occurs both in vitro and in vivo.
• The herein described bi-functionality of AtGALS1 suggest that
plants can produce the incredible structural diversity of
polysaccharides without a dedicated glycosyltransferase for each
glycosidic linkage.
Laursen et al. (2018) Plant J., doi: 10.1111/tpj.13860.
Schematic structure of pectic galactans. The GALS1
enzyme is responsible both for elongation of chains with
galactose residues and for termination by arabinose
residues.
Enzyme assay showing
the addition of galactose
and arabinopyranose to
galacto-pentaose by
GALS1. Arabinofuranose
is not transferred by
GALS1. GALS1 was
expressed in tobacco and
a fluorescent labeled
galactopentaose was
used as acceptor
substrate.
Kinetic analysis of the
transfer of galactose to
galactopentaose by
GALS1 shows a
sigmoid curve
indicating more than
one substrate binding
site.

5. Synthetic biology of polyketide synthases
Background
• Complex reduced polyketides represent the largest class of
natural products that have applications in medicine,
agriculture, and animal health, and are produced naturally by
polyketide synthases (PKS).
• We have used the nomenclature of synthetic biology to
classify polyketide synthases as parts and devices,
respectively, and have generated detailed lists of both.
• Provides a common reference tool for the broader synthetic
biology community.
Approach
• Developed a list of common synthetic biology syntax for
PKSs – parts, devices and chassis.
• Review of current approaches to engineer PKSs
• Review of ClusterCAD, an online database to facilitate
identification and selection for targeted PKSs for a particular
pathway or pdoruct.
Outcomes and Impacts
• it should be possible to design a polyketide of desired
structure, write out the list of parts and devices required,
employ ClusterCAD or similar software to design the devices,
synthesize the DNA of the required sequence, introduce the
DNA into a desired chassis, and express it under a selected
means of regulation.
• The ability to link modules in sequence to generate
completely new polyketide structures without losing
biochemical activity is still extremely challenging.
Yuzawa et al. (2018) Ind Microbiol Biotechnol., doi: 10.1007/s10295-018-2021-9
(A) Example chemical target used to demonstrate guiding chimeric
PKS engineering with the ClusterCAD software tool. (B) A
maximum common substructure (MCS) plot highlighting the
common regions between the example chemical target (on right),
and narbonolide, a chemically similar natural PKS found in
ClusterCAD (on left). The non-highlighted regions represent
engineering modifications necessary to produce the example
product.
Structures of malonyl-CoA analogs that are incorporated
during biosynthesis of naturally occurring polyketides.

6. Outcomes and Impacts
• A mixture of 42 wt.% [C2C1Im][OAc] and 58 wt.% DMSO is proposed as the optimal pretreatment solution and the recycling
and reuse of the mixture of solvents are also studied.
• The fermentability of the hydrolysates generated after pretreatment is evaluated using an E. coli strain engineered to
produce isoprenol, and the highest isoprenol yields generated were 24.5 g/kg switchgrass.
• This study suggests an avenue for developing more efficient and cost-effective IL-based processes for the production of
biofuels and bioproducts.
Wang et al. (2018) ACS Sustainable Chem. Eng., doi: 10.1021/acssuschemeng.7b04908
Background
• The production cost and viscosity of certain ionic liquids
(ILs) are among the major factors preventing the
establishment of economically viable IL-based biomass
pretreatment technologies.
Approach
• One of the approaches to reduce the cost of IL
pretreatment has been the use of mixtures of ILs with
inexpensive solvents such as dimethyl sulfoxide
(DMSO)
• We conducted a mechanistic study of DMSO-assisted
IL pretreatment of switchgrass
• An engineered E. coli strain, KG1R10, containing a
heterologous mevalonate-based isoprenol pathway was
used for isoprene production.
Dimethyl sulfoxide assisted ionic liquid
pretreatment of switchgrass for isoprenol production