JBEI Research Highlights - December 2017

1. Towards industrial production of isoprenoids in
Escherichia coli using CRISPR-Cas9
Background
• Escherichia coli has been the organism of choice for the production of
different chemicals by engineering native and heterologous pathways.
• However, there are several issues to using E. coli as an industrial
biofuel producing host, including susceptibility to phage contamination,
inefficiency in growing on certain desirable carbon sources (especially
sucrose), and low recombination capacity of DNA into its
chromosome.
Approach
• Address some of the main issues associated with E. coli as an
industrial platform for isoprenoids.
• As a proof of concept, we generated an E. coli DH1 strain able to
produce the isoprenoid bisabolene from sucrose by integrating the
cscAKB operon into the chromosome and by expressing a
heterologous MVA pathway under stress-responsive control.
• In order to optimize the chromosomally integrated MVA pathway, we
established a CRISPR-Cas9 system to rapidly and systematically
replace promoter sequences.
Outcomes and Impacts
• When the entire pathway was integrated into the chromosome,
production levels dropped dramatically relative to plasmid-mediated
expression.
• CRISPR-Cas9 based optimization strategy led to higher pathway
expression and a 5-fold improvement in bisabolene production.
• Based on these results, we also proposed directions for chromosomal
metabolic engineering.
Alonso-Gutierrez et al. (2017) Biotechnol Bioeng, doi: 10.1002/bit.26530)

2. Expressing thermophilic cellulases using
Aspergillus niger
Outcomes and Impacts
• Expression constructs for 25+ IL-tolerant enzymes were
built an transformed into A. niger. Almost all of the enzymes
expressed, indicating that this organism is amenable to
expressing heterologous enzymes
• One enzyme, a beta-glucosidase, was characterized in
detail and shown to be functionally equivalent to the same
enzyme expressed in E. coli.
• Biomass deconstruction technologies that depend on novel
enzymes cannot be realized unless they can be produced in
an industrial host. This study is a first step toward that goal.
Release of sugars during saccharification of IL pretreated switchgrass
using identical enzymes expressed in E. coli or A. niger.
Background
• Many IL-tolerant cellulases have been identified at JBEI
from previous studies of microbial consortia.
• However, they are expressed at low titers using standard
hosts. To be commercially viable, they need to be
produced in an industrial host capable of producing high
titers of enzymes.
• Aspergillus niger is a filamentous fungus that is known to
produce high titers of native enzymes and has been shown
to be effective at recombinant protein expression.
Approach
• Expressed 20 IL-tolerant, thermophilic enzymes in A. niger
to assess its potential as a enzyme production host.
IL, pH, and temperature profiles of a beta-
glucosidase expressed in E. coli or A. niger
Campen et al. (2017) PLOS ONE, doi: 10.1371/journal.pone.0189604
RecoveredSugars(g/L)
Incubation Time (Hours)
0
0.5
1
1.5
2
6 12 24 48 72
E. coli- A. niger-
E. coli- A. niger-
xylose
glucose

3. Outcomes and Impacts
• The length of the GIPC glycan chain was found to be critical. In tomatoes and potatoes, and Arabidopsis, the chain is short
(Series A). In wheat or barley, this chain is longer (Series B and up).
• The cytolysin binds to the GIPC glycan chain. If the chain is too long, the cytolysin cannot interact with the membrane, and
cannot trigger membrane collapse.
• In the future, it may be possible to develop resistant strains of now-sensitive plants by modifying their GIPC chain length.
• Cytolysin-type molecules could be engineered to produce specific herbicides. These would have fewer environmental impacts
than current options such as glyphosphate.
Lenarcic et al. (2017) Science, doi: 10.1126/science.aan6874
Background
• Cytolysin is a toxin produced by some plant pathogens,
such as the causal agent of potato blight, Phytophthora
infestans.
• Cytolysin causes a collapse of the plant cell membrane,
killing the tissue.
• It was not known how cytolysin acted, or why it affected
only some plant species.
Approach
• This work was led by Prof. Thortsen Nürnberger and his
group at Tübingen University in Germany. They proposed
that a class of glycosylated sphingolipids, the GIPCs might
be involved.
• Previously, JBEI had identified and characterized genes
responsible for GIPC glycosylation, and produced plants
with altered GIPC glycan motifs These different types of
GIPCs were isolated and used as tools to test the activity
of the cytolysin.
Eudicot plant-specific sphingolipids determine
host selectivity of microbial NLP cytolysins
Cytolysin mediated
plant leaf necrosis in
different species.
Images were taken
48 hours after
infiltration.
GIPC quantification
of Series A: Series B
GIPCs in these
species (i.e., short
vs longer chain
GIPCs).

4. Production of lathyrane diterpenoids using
engineered Saccharomyces cerevisiae
Outcomes and Impacts
• Our data conclusively show that ElC9OX1p and JcC5OX2p
co-expressed alongside an ADH allow for the biosynthesis
of the important lathyrane precursor jolkinol C.
• The final engineered jolkinol C strain (JWY521) produced
800 mg/L jolkinol C.
• This work represents the highest levels of oxidized
diterpenoids produced to date in any microorganism and
establishes a new yeast chassis for subsequent efforts in
recombinant expression of a wide range of lathyrane
diterpenoids.
(a) Structures of bioactive Euphorbiaceae diterpenes. (b) Proposed
pathway for the production of Euphorbiaceae diterpenoids begins with
geranylgeranyl diphosphate (GGPP) from the DXP pathway in plants,
which is cyclized by casbene synthase (CBS).
Background
• Lathyrane diterpenoids are a group of tricyclic diterpenes
found in plants that are derived from the hydrocarbon
casbene and are a compelling class of compounds due to
their unique properties and wide applications.
• Reconstituting complex oxidized terpenoid biosynthetic
pathways, such as those of lathyranes, in heterologous
microbial hosts has posed numerous problems to date.
• S. cerevisiae is considered a better host for these
compounds than common prokaryotes, such as Escherichia
coli, due to the presence of the endoplasmic reticulum.
Approach
• Engineered S. cerevisiae to produce the lathyrane backbone
casbene at high titers.
• Optimized the expression of P450s and an ADH to produce a
strain with all pathway genes stably integrated as a chassis
for additional gene discovery.
Wong et al. (2017) Metabolic Engineering, doi: 10.1016/j.ymben.2017.12.007
Production of 6,9-hydroxy-5-ketocasbene and jolkinol C with various
combinations of C9OX CYP, C5OX CYP, and ADH variants.