JBEI Research Highlights - January 2018

1. Gene stacking of multiple traits for high yield of
fermentable sugars in plant biomass
Background
• Increasing the ratio of C6 to C5 sugars in the cell wall and
decreasing the lignin content are two important targets in
engineering of plants that are more suitable for downstream
processing for second-generation biofuel production.
Approach
• Plants were engineered to have up to fourfold more pectic
galactan in stems by overexpressing GALS1 (galactan
synthase), URGT1 (UDP-Gal transporter), and UGE2
(UDP-Glc epimerase).
• Furthermore, the increased galactan trait was engineered into
plants that were already engineered to have low xylan content
by restricting xylan biosynthesis to vessels where this
polysaccharide is essential.
• Finally, the high galactan and low xylan traits were stacked
with the low lignin trait obtained by expressing the QsuB gene
encoding dehydroshikimate dehydratase in lignifying cells.
Outcomes and Impacts
• The results show that approaches to increasing C6 sugar
content, decreasing xylan, and reducing lignin content can be
combined in an additive manner.
• Thus, the engineered lines obtained by this trait-stacking
approach have substantially improved properties from the
perspective of biofuel production, and they do not show any
obvious negative growth effects.
• The approach used in this study can be readily transferred to
bioenergy crop plants.
Aznar et al. (2018) Biotechnol Biofuels , doi:
The genes for galactan increase, lignin decrease and low
xylan were combined in different ways. The JBEI ‘jStack’
technology was used to generate the constructs.
Fig 2. The transgenic plants
did not display any negative
effects on growth or
mechanical properties. Some
of the lines where all the traits
are shown here.
Substantial improvement in C6/C5 sugar ratio was obtained in all
the lines, with more than 2-fold increase in the best lines. These
lines also had a 50% decrease in lignin.

2. Constraining genome-scale models to represent
the bow tie structure of metabolism
Background
• Mapping metabolic flux is critical for efficient engineering of microbes for
the production of biofuels and bioproducts.
• Our approach is based on simplifying approximation that metabolic flux
from peripheral metabolism into central “core” carbon metabolism is
minimal, and can be omitted when modeling isotopic labeling in core
metabolism.
• Validity of this “two-scale” or “bow tie” approximation is supported both
by the ability to accurately model experimental isotopic labeling data,
and by experimentally verified metabolic engineering predictions using
these methods.
Approach
• Systematically calculate flux bounds for any specified “core” of a
genome-scale model so as to satisfy the bow tie approximation
• Automatically identify an updated set of core reactions that can satisfy
this approximation more efficiently.
• Ran both algorithms on the iJR904 E. coli genome scale model, using
exchange and biomass fluxes from previously published sample data.
Outcomes and Impacts
• These methods accelerate and automate the identification of a
biologically reasonable set of core reactions for use with 13
C MFA or
2S-13
C MFA.
• These methods accelerate and automate the identification of a
biologically reasonable set of core reactions.
• We have dan open source Python implementation of these algorithms at
https://github.com/JBEI/limitfluxtocore that is available to the broader
community.
Backman et al. (2018) Metabolites, doi: 10.3390/metabo8010003
The bow tie structure of metabolism. The bow tie structure
of metabolism describes how numerous different types of
carbon sources (left side of figure) are funneled into a small
set of precursor metabolites (middle of figure) in central
carbon metabolism that are subsequently processed into a
large set of peripheral metabolites (right side of figure).

3. Employing a biochemical protecting group for a
sustainable indigo dyeing strategy
Background
• 50,000 tons of indigo are synthesized annually, of which 95% is
used to dye the over 4 billion denim garments manufactured
annually.
• Indigo is chemically synthesized on an industrial scale from aniline, a
toxic compound derived from the petroleum product benzene.
• There is a compelling need for an environmentally sustainable
approach that achieves production of indigo as a valuable
bioproduct.
Approach
• Developed a sustainable dyeing strategy that not only circumvents
the use of toxic reagents for indigo chemical synthesis but also
removes the need for a reducing agent for dye solubilization.
• This alternative indigo dyeing process that mimics the natural
biochemical protecting group strategy employed by the Japanese
indigo plant P. tinctorium.
• Goal was to gain temporal control over the regeneration of the
indoxyl intermediate to achieve indigo crystallization in the cotton
fibers.
Outcomes and Impacts
• We identified the gene sequence of a novel glucosyltransferase from
P. tinctorium (PtUGT1) with activity on indoxyl, characterized the
enzyme structurally, and successfully co-expressed it with the
oxygenase FMO in E. coli to produce up to 2.9 g/L indican in vivo
from fed tryptophan.
• This bioindican was shown to be an effective fabric dye when
hydrolyzed by a β-glucosidase.
Hsu et al. (2018) Nature Chemical Biology , doi: 10.1038/nchembio.2552
Overall GT-B fold of PtUGT1 (PDB ID 5NLM). The N-terminal
Rossmann domain (blue) consists of a seven-stranded parallel β-sheet
surrounded by nine α-helical segments, and the C-terminal Rossmann
domain (red) consists of a six-stranded parallel β-sheet and five
α-helices.
(a) Top row, Pure indican with no β-glucosidase (BGL); pure indican with
β-glucosidase; bio-indican with β-glucosidase. Bottom row, Indigo,
non-reduced; indigo, reduced with sodium dithionite. (b) Scarf (100% white
cotton gauze fabric) dyed with indican.

4. Forward genetics screen coupled with whole-genome
resequencing identifies novel gene targets for improving
heterologous enzyme production in Aspergillus niger
Background
• Efficient expression of recombinant enzymes capable of
hydrolyzing polysaccharides present after pretreatment is a critical
need at JBEI.
• Aspergillus niger is a promising recombinant host under
development at JBEI to meet this need.
• We have previously identified several enzymes capable of
maintaining high activity in the presence of ionic liquids used in
biomass pretreatment that we would like to express in A. niger.
Approach
• We developed a forward genetics screen coupled with
whole-genome resequencing to identify specific lesions responsible
for a protein hyper-production phenotype in A. niger.
• Use bioinformatics to identify loci associated with heterologous
enzyme hyper-production, including those that appear to be
specific to the heterologous protein expression construct used.
Outcomes and Impacts
• Our strategy successfully identified novel targets, including a
low-affinity glucose transporter, MstC, whose deletion significantly
improved secretion of recombinant proteins driven by a
glucoamylase promoter. Deletion of mstC loci improved
heterologous enzyme production driven by a glucoamylase
promoter (PglaA) fourfold.
• Mutations in the Ypt/Rab GTPase-activating protein found in BOTH
strain J03 2.8 and NAT strain J03 4.3 might further increase
production of heterologous BG if introduced into one HET strain.
Reilly et al. (2018) Applied Microbiology and Biotechnology , 102(4), 1797-1807, doi: 10.1007/s00253-017-8717-3
Mutations in J03-derivative strains associated with
hyper-production of heterologous and/or native
beta-glucosidase.
Loss of the mstC locus enhances heterologous enzyme
production.

5. Isolation and characterization of novel mutations in
the pSC101 origin that increase copy number
Background
• Synthetic biology is predicated on predictable levels of gene
expression. This is often achieved through the use of plasmids with
specific copy numbers.
• Isolating novel copy number variants, and precisely characterizing
their expression profiles may enable finer tuning of synthetic genetic
circuits.
Approach
• To Identify high copy variants of the pSC101 origin, we leveraged
random mutagenesis and plate based selections.
• We modelled the location of the most common mutations to predict
other potential residues that control copy number.
• To evaluate the relationship between copy number and fluorescent
protein production strength we created site-directed mutants within
pSC101 vectors expressing RFP controlled by an arabinose-inducible
promoter.
Outcomes and Impacts
• We identified 10 previously unidentified mutations within the pSC101
origin that increase plasmid copy. Theses residues are likely to
contribute to RepA dimerization.
• We identified two specific mutations within the RepA protein that result
in copy numbers comparable to other commonly used medium and
high copy vectors in Escherichia coli.
• By combining mutations we were able to create super-high copy
vectors, with copy numbers reaching ~500 copies/cell.
Thompson et al. (2018) Sci Rep, doi: 10.1038/s41598-018-20016-w

6. Engineering E. coli for simultaneous glucose-xylose
utilization during methyl ketone production
Outcomes and Impacts
• Two strategies were effective at enhancing methyl ketone
titer: (1) chromosomal deletion of pgi to increase intracellular
NADPH supply and (2) downregulation of CRP (cAMP
receptor protein) by replacement of the native RBS.
• Combining these strategies resulted in the most favorable
overall phenotypes (see strains XW1074 and especially
XW1075 in figure at right).
• This work demonstrated a strategy for engineering
simultaneous utilization of C6 and C5 sugars in E. coli
without sacrificing production of methyl ketones (diesel fuel
blending agents) from cellulosic biomass.
Wang et al. (2018) Microbial Cell Factories , doi: 10.1186/s12934-018-0862-6
Background
• We previously developed an E. coli strain that efficiently converts glucose to methyl ketones (MK) for use as diesel fuel
blending agents.
• Since lignocellulosic biomass contains a substantial portion of xylose (typically the second most abundant sugar after
glucose), we re-engineered this strain to more efficiently metabolize sugars by facilitating simultaneous, rather than the
native sequential (diauxic), utilization of glucose and xylose.
Approach
• Genetic manipulations were performed to alleviate carbon
catabolite repression in our most efficient MK-producing
strain (EGS1895).
• A strain engineered for constitutive expression of genes
involved in xylose transport and metabolism showed
synchronized C6-C5 sugar consumption, but a 5-fold
decrease in MK production. Further efforts were made to
improve MK production in this strain (XW1014).
.
Summary comparison of methyl ketone production and sugar
consumption for engineered strains. Methyl ketone yield, methyl
ketone productivity, and sugar consumption period are each
normalized to the maximum value among the six strains. Strain
XW1075 performed best overall.

7. Biomass pretreatment using deep eutectic solvents
(DESs) from lignin derived phenols
Outcomes and Impacts
• Demonstrated that novel DESs formed from a
renewable-derived resource, lignin, were prepared by mixing
ChCl and HBD at an appropriate molar ratio.
• A fresh batch of the DES, ChCl–PCA, resulted in
approximately 87% glucose yield, and the yield decreased to
78% and 70% for 2nd and 3rd runs, respectively.
• Ease of synthesis, availability and economic viability of the
components make DESs attractive and a potential
compelling alternative to ILs.
Kim et al. (2018) Green Chemistry , doi: 10.1039/C7GC03029K
Background
• DESs have been gaining attention as inexpensive
alternatives to conventional ILs used in biomass
pretreatment.
• Lignin-derived compounds can serve as raw materials for
DES synthesis and use in a biorefinery context.
Approach
• Ten phenolic compounds were tested as a hydrogen bond
donor to synthesize novel DESs at varying molar ratios
with ChCl.
• After initial screening, four DESs were selected chosen
and they were used for biomass pretreatment of
switchgrass.
• After pretreatment, the residual DESs were washed out to
remove potential inhibitory effects on enzymatic hydrolysis
and subsequent fermentation steps by phenolic
compounds.
Saccharification yield from the DES pretreated switchgrass
(expressed as the percentage of the theoretical maximum based
on the initial glucan and xylan contents in the starting biomass).
HBA: hydroxybenzoic acid; CAT: catechol; VAN: vanillin; PCA:
p-coumaric acid.
Biorefinery concept using renewable DESs derived from lignin

8. Linking secondary metabolites to gene
clusters in six diverse Aspergillus species
Background
• The genus of Aspergillus contains fungi that are relevant to plant
and human pathology, food biotechnology, enzyme production,
model organisms, and a selection of extremophiles.
• Filamentous fungi produce a diverse range of secondary
metabolites (SMs), including compounds that can serve as
chemicals, pharmaceuticals and related bioproducts.
• No comparative analysis across genomes for the identification of
gene clusters that produce SMs has been conducted previously.
Approach
• Four diverse Aspergillus species (A. campestris, A.
novofumigatus, A. ochraceoroseus, and A. steynii) have been
whole-genome PacBio sequenced to provide genetic references
in three Aspergillus sections and coupled with two existing
genomes.
• Thirteen Aspergillus genomes were analyzed with comparative
genomics to determine phylogeny and genetic diversity, and link
secondary metabolites of interest to specific fungi.
Outcomes and Impacts
• We have identified putative SM clusters for aflatoxin,
chlorflavonin, and ochrindol in A. ochraceoroseus, A. campestris,
and A. steynii, respectively, and novofumigatonin,
ent-cycloechinulin, and epi-aszonalenins in A. novofumigatus.
• Provides new insight into how biological, biochemical, and
genomic information can be combined to identify genes involved
in the biosynthesis of specific SMs that may be of interest and
use as bioproducts.
Kjærbølling et al. (2018) Proceedings of the National Academy of Sciences ,
doi: 10.1073/pnas.1715954115
(A) Overview of the SM gene clusters predicted in A.
novofumigatus and their homologs in the reference species. (B)
Overview of the SM gene clusters predicted in A. fumigatus and
their homologs in the reference species. (C) A Venn diagram of the
A. fumigatus and A. novofumigatus SM gene clusters. (D) The
number and different types of SM gene clusters predicted in A.
fumigatus and A. novofumigatus. DMATS, dimethylallyl tryptophan
synthase; NRPS, nonribosomal peptide synthetase; PKS,
polyketide synthase; TC, terpene cyclase.

9. Characterization of lignin streams during bionic
liquid-based pretreatment
Background
• Cholinium lysinate ([Ch][Lys]), an example of a bionic liquid, is
reported to have excellent biomass pretreatment efficiency and
successfully employed in a one-pot IL-based configuration, with
improved economics.
• It is imperative to have a qualitative and quantitative understanding
of the partitioning of lignin in the different process streams by
performing a detailed mass balance analysis and structural
characterization.
Approach
• A complete mass balance analysis of different components after
[Ch][Lys] pretreatment followed by enzymatic saccharification of
switchgrass, eucalyptus, and pine.
• The lignin streams generated were characterized using a
two-dimensional (2D) 1H–13C heteronuclear single quantum
coherence (HSQC) nuclear magnetic resonance (NMR)
spectroscopy, gel permeation chromatography (GPC), and gas
chromatography–mass spectrometry (GC-MS).
Outcomes and Impacts
• Pretreatment of switchgrass resulted in a maximum reduction of
lignin, whereas the lignin in pine remained primarily intact.
• The 2D HSQC-NMR study, coupled with the SEC and compositional
analysis data, revealed more carbohydrate residues after enzymatic
hydrolysis for both eucalyptus and pine, indicating more
recalcitrance, which will demand further process optimization.
• Provides a deeper understanding of the origins of biomass
recalcitrance as a function of genotype and pretreatment process.
Dutta et al. (2018) ACS Sustainable Chemistry & Engineering , doi: 10.1021/acssuschemeng.7b02991
Mass balances (%DM) for the major biomass components of (A)
switchgrass, (B) eucalyptus, and (C) pine during ionic liquid
pre-treatment (IL-PT) with [Ch][Lys] and lignin streams (L1, L2L,
L2S, L3L, L4S, L4L) generated during the process.