This document summarizes the development and testing of a quorum sensing (QS)-mediated gene expression system to control bisabolene production in engineered E. coli. Researchers developed a QS system using the LuxI/R genes from Vibrio fischeri to induce expression of the bisabolene production pathway without the need for external inducers. The best QS strain, with the sensor genes integrated into the genome and an optimized response plasmid, produced 1.1 g/L of bisabolene, a 44% improvement over previous inducible systems. This QS-based system provided defined and homogeneous gene expression and production compared to inducible controls.

1. Outcomes and Impacts
• Knocking out the gene corresponding to the RPE reaction is predicted to increase
acetate production by 6.2% according to the MoMA methodology and by 60.0% when
using the ROOM methodology. As can be seen with an RPI knockout, both MoMA and
ROOM predict a decline in acetate production.
Ando & Garcia (2018) Synthetic Metabolic Pathways: Methods and Protocols, https://doi.org/10.1007/978-1-4939-7295-1_21
Background
• Accelerating the Design–Build–Test–Learn (DBTL) cycle in synthetic biology is critical
to achieving rapid and facile bioengineering of organisms for the production of, e.g.,
biofuels and other chemicals.
• The learn (L) phase of the DBTL cycle and is, arguably, the hardest and most weakly
supported step in current metabolic engineering practice.
• The measurement of intracellular metabolic fluxes is specifically noteworthy as providing
a rapid and easy-to-understand picture of how carbon and energy flow throughout the
cell.
Approach
• Here, we present a detailed guide to performing metabolic flux analysis in the
Learn phase of the DBTL cycle, where we show how one can take the isotope labeling
data from a 13C labeling experiment and immediately turn it into a determination of
cellular fluxes that points in the direction of genetic engineering strategies that will
advance the metabolic engineering process.
• We will show how to use metabolomic data obtained from 13C labeling experiments to
generate actionable items to increase acetate production in E. coli.
• For our modeling purposes we use the Joint BioEnergy Institute (JBEI) Quantitative
Metabolic Modeling (jQMM) library, which provides an open-source, python-based
framework for modeling internal metabolic fluxes and making actionable predictions on
how to modify cellular metabolism for specific bioengineering goals.
Overview of the workflow for
13C Two-Scale Metabolic
Flux Analysis. The Test phase
of the workflow is in blue
while the Learn phase steps
are in red.
Two-scale 13C metabolic flux analysis for
metabolic engineering

2. A bacterial pioneer leaves a complex legacy
Outcomes and Impacts
• A defined community succession was observed during 15
L and 300 L cultivations at ABPDU
• Appearance of cellulase activity in the medium correlated
with relative decline of pioneer population
• This study used scaled community cultivations and
genome-resolved metagenomics to identify new cellulase
complexes distinct from cellulosomes and underscores
the importance of extracellular glycoside hydrolases as
“public goods” for biomass-deconstructing communities
Background
• In previous work at JBEI, cellulolytic consortia were used
to produce Jtherm, an thermo/IL tolerant cellulase cocktail
• Scaling of cellulolytic consortium to 300 L provided
sufficient enzyme to identify active component(s) of
Jtherm
Approach
• Time-resolved metagenomics was applied to 15 L and
300 L cultivations (14 days) to determine changes in
community structure. Genome-resolved metagenomics
was used to recover genomes in consortium. Biochemical
purification and heterologous expression were used to
characterize the active cellulases
The bacterial pioneer population is an uncultivated Firmicutes
that contains unique gene clusters with multi-domain cellulases
and LPMOs. A subset of these cellulases (CelABC) were purified
as multi-protein complexes; glycosylation appeared to be key to
complex formation and stability; the complexes provided the
majority of the activity in the Jtherm cocktail
Kolinko et al. (2017) Nature Microbiology, doi: 10.1038/s41564-017-0052-z.

3. Automated lab evolution (ALE) generates a
platform strain for IL tolerance
Outcomes and Impacts
• MG1655 and DH1 clones were obtained with tolerance
to [C2C1mim][OAc] at 6% concentration.
• Sequencing revealed common mutations in intergenic
region adjacent to mdtJI (SMR promoter-pump pair)
and coding region of yhdP (predicted transporter)
• MG1655 adapted clones significantly outperformed
JBEI IL tolerant E. coli strains established to date
• This study establishes ALE as an effective platform to
generate strains that are tolerant to ionic liquids.
Mohamed et al. (2017) Microbial Cell Factories, 16, 204, doi: 10.1186/s12934-017-0819-1
Background
• Previous work at JBEI has developed IL tolerant
E. coli strains using functional genomics and
isolating spontaneous mutants
• Automated lab evolution (ALE) is a promising
technique to adapt microbes to grow under stress
conditions (i.e. IL challenge)
Approach
• ALE was performed for 40 days on E. coli DH1
and MG1655 in the presence of increasing
amounts of [C2C1mim][OAc] and [C4C1mim]Cl
• Adapted clones were isolated, tested in
secondary screens and sequenced
Comparison of TALE evolved IL tolerant clones
MG1655#4.7 and MG1655#3.10 to previously engineered
tolerant strains JBEI-13314 and JBEI-10101 in LB (a, b)
and M9 (c, d) media containing either 300 mM of
[C2C1Im][OAc] [5.1% (w/v)] or [C4C1Im]Cl [4.4% (w/v)].
TALE evolved clones exhibited improved growth compared
to rationally-designed strains, particularly in M9, where
JBEI-13314 and JBEI-10101 were severely inhibited

4. Xylose induces cellulase production
in Thermoascus aurantiacus
Outcomes and Impacts
• Xylose was shown to specifically induce both cellulases
and xylanases in T. aurantiacus; acid hydrolysate from
biomass induced proteins to levels comparable to pure
xylose
• Xylose induction was scaled to 20 L cultures and
saccharification experiments demonstrated that dilute acid-
pretreated corn stover could be saccharified at 60 ℃
• This study demonstrates that dilute acid hydrolysate can be
used for enzyme production, which a promising biorefinery
strategy
Shüerg et al. (2017) Biotechnology for Biofuels, 10, 271, doi: 10.1186/s13068-017-0965-z
Background
• Previous work demonstrated that Thermoasucs
aurantiacus produced a highly active cellulase mixture
that saccharified biomass at 70℃
• This cellulase mixture was generated by growth on
switchgrass substrates
Approach
• To identify sugars that may induce cellulase
production, fed-batch cultures were set-up in shake
flasks and bioreactors to determine whether biomass-
derived sugars induced the expression of cellulases
Batch FB
XyloseXylan
2% 2% 0.5% 0.5%
Batch FB
GlucoseXylan
2% 2% 0.5% 0.5%
T. aurantiacus protein production with glucose and xylose
SDS-PAGE (a), protein concentration (b). Batch cultures
were performed by adding glucose and xylose at the
beginning of the cultivation and fed-batch cultures were
performed by adding the sugars continuously using a
peristaltic pump. Shift cultures with 2% beechwood xylan
as the substrate were used as positive controls for protein
production. Batch cultures are underlined in red and fed-
batch cultures in blue

5. Cascade production of lactic acid from
universal types of sugars catalyzed by La(OTf)3
Outcomes and Impacts
• We report a universal method of converting sugars to lactic with
high yield ~70%
• This is the first work to convert pyrolytic sugar into lactic acid by
chemocatalysis and also lignocellulosic sugars are converted to
lactic acid without hydrolysis
• This approach could potentially be extended to other lignocellulosic
sugars after simple removal of lignin from biomass pretreatment,
rendering moderate to high yield of lactic acid
Liu et al. (2017) ChemSusChem, doi: 10.1002/cssc.201701902
Background
• Lactic acid is on the list of the DOE’s top chemical opportunities
from carbohydrates and the market for lactic acid is increasing
rapidly due the the expanding use of lactic acid to produce plastics
• The current process of lactic acid production is dominated by
fermentation, which requires monosaccharides
• The chemocatalytic process to produce lactic acid offers high
reaction rates and simple separation process
Approach
• Universal types of sugars obtained from pyrolysis, ionic liquid
pretreatment were prepared and subjected to catalytic conversion
using La(OTf)3
Proposed mechanism of aldo sugars
conversion to lactic acid in the
presence of Lewis acid.
Lignocellulosic biomass upgrading
through thermochemical conversion
process

6. High loading solubilization and upgrading of
polyethylene terephthalate in low cost
bifunctional ionic liquid
Outcomes and Impacts
• Low cost (~$1.2/kg) biocompatible IL, cholinium phosphate
([Ch]3[PO4]) plays bifunctional roles in PET solubilization and
glycolysis degradation
• High loading of PET (10 wt%) is readily dissolved in [Ch]3[PO4] at low
temperatures (120 °C, 3h) and even in water-rich conditions
• Acid precipitation yields terephthalic acid as the dominant
depolymerized monomer with a theoretical yield of ~95%
• In the presence of ethylene glycol, [Ch]3[PO4] catalyzed glycolysis of
PET resulted in ~100% PET conversion and ~60.6% bis(2-
hydroxyethyl)terephthalate (BHET) yield
Jian et al. (2017) ChemSusChem, doi:10.1002/cssc.201701798.
Background
• Concerns on the depletion of fossil reserves, environmental pollution
and more efficient & circular carbon economies have highlighted the
importance of recycling polyethylene terephthalate (PET)
• High solubilization of PET is critical as a first step in recycling, but is
challenging in toxic solvents
• Achieving PET solubilization at high loading under mild conditions by
using relatively nontoxic and lower cost solvents is highly desirable
Approach
• Biocompatible IL was developed by changing imidazolium ring based
cation to cholinium cation
• Hydrogen bond functionalization was used to improve the activity of
IL in PET solubilization under relatively low temperature
In situ confocal fluorescence microscopy
images of PET solubilization in
[Ch]3[PO4] as a function of temperature
Proposed mechanism of PET
glycolysis catalyzed by [Ch]3[PO4]

7. Autonomous control of metabolic state by a quorum
sensing (QS)-mediated regulator for bisabolene
production in engineered E. coli
Background
• Inducible gene expression systems have disadvantages such as leaky expression, lack of dynamic control, and the prohibitively
high costs of inducers associated with large-scale production
• Quorum sensing (QS) systems in bacteria control gene expression in response to population density, and the LuxI/R system from
Vibrio fischeri is a well-studied example
• QS system could be ideal for biofuel production strains as it is self-regulated and does not require the addition of inducer
compounds, which reduce operational costs
Approach
• QS system was developed for inducer free production of the biofuel compound bisabolene from engineered E. coli.
• Seven variants of the pSensor plasmid, which carry the luxI-luxR genes, and four variants of the pResponse plasmid, which carry
bisabolene producing pathway genes under the control of the PluxI promoter
• A chromosome-integrated QS strain was engineered with the best combination of Sensor and Response plasmid
Outcomes and Impacts
• The best combination of pSensor integrated in the genome and re-engineered pResponse plasmid produced bisabolene at a titer of
1.1 g/L without addition of external inducers.
• This is a 44% improvement from our previous system
• QS strain also displayed higher homogeneity in gene expression and isoprenoid production compared to an inducible-system strain
Genome Plasmids SApSensor pResponseSummary: using QS-mediated regulator
to control bisabolene synthesis
Kim et al. (2017) Metabolic Engineering, 44:325-336, doi: 10.1016/j.ymben.2017.11.004
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