1. Endoribonuclease-Based Two-Component
Repressor Systems for Tight Gene Expression Control in Plants
Outcomes
• A two-component expression−repression system based
on the activity of Csy4 endoRNase was developed and
validated in dicot and monocot plants.
• The repressor system was shown to be highly efficient
and versatile. It can be used for single-gene repression,
synchronized multigene repression, and conditional
repression. Using bioinformatics analysis, additional
endoRNase-based two-components system were
isolated and validated in planta.
Liang et al. (2017). “Endoribonuclease-Based Two-Component Repressor Systems for Tight Gene
Expression Control in Plants.” ACS Synth Biol, DOI: 10.1021/acssynbio.6b00295
Background
• Tools for precise regulation of gene expression are largely lacking, which
imped progress in trait improvement of crops and plant metabolic
engineering.
Significance
• The endoRNase based repression system represented a robust, versatile repression system for precise regulation of
transgenes in plants.
• The system supports metabolic engineering and the development of complex traits in crops
Approach
• A two-component repressor system was developed using the Csy4
endoRNase and its target sequence
• Tobacco and rice based transient expression systems and stable transgenic
lines were used to validate this new expression repression tool
• Fluorescence and expression analysis were used to
quantify the efficiency of the repression system
2. Structural Analysis of Cell Wall
Polysaccharides Using PACE
Mortimer, J. C. (2017). "Structural Analysis of Cell Wall Polysaccharides Using
PACE". Methods Mol Biol, 1544, 223-231. doi, 10.1007/978-1-4939-6722-3_16
Background
• The plant cell wall is composed of many complex
polysaccharides.
• The composition and structure of the polysaccharides affect
various cell properties including cell shape, cell function and cell
adhesion. It is also critical to understanding biomass composition
for deconstruction.
• Many techniques to characterize polysaccharide structure are
complicated, requiring expensive equipment and specialized
operators e.g. NMR, MALDI-MS.
Significance
• This book chapter provides a detailed, easy-to-implement
protocol for establishing PACE as a method, with extensive
trouble-shooting notes.
• It provides an updated method from the original methods paper
(Goubet et al. 2002) using reagents with significantly lower
toxicity.
Approach and Outcomes
• PACE (Polysaccharide Analysis using Carbohydrate gel
Electrophoresis) uses a simple, rapid technique to analyze
polysaccharide quantity and structure (Goubet et al. 2002).
• Whilst the method here describes xylan analysis, it can be applied
(by use of the appropriate glycosyl hydrolase) to any cell wall
polysaccharide.
Above: Example PACE gel showing xylanase
digestion of Arabidopsis stem cell wall,
revealing xylan structure. These gels can be
used to quantitate xylan quantity and degree of
branching.
3. Bioenergy Potential from
Food Waste in California
Outcomes
• Between 10% and 99% of gross HMS and can be
digested using state AD infrastructure and in the same
month of production, and between 10% and 66% can be
digested in-county using AD infrastructure and in the
same month of production.
• Only 55% of gross LMS can be converted to energy
using excess capacity at CA solid biomass power plants
1) Breakdown of high moisture waste availability by Month in
California.
The is the first study to examine the month-by-month availability of high-moisture
biomass feedstock for energy production, which is critical because long-term
storage of such waste can be costly and impractical in many cases,
Breunig et al. (2017). "Bioenergy Potential from Food Waste in California". Environ Sci
Technol. doi, 10.1021/acs.est.6b04591 https://www.ncbi.nlm.nih.gov/pubmed/28072520
Background
• The U.S. generated approximately 38 million tonnes of
municipal food waste in 2014, approximately 95% of
which was landfilled.
• Food waste represents an opportunity to recover energy
and have an outsized impact on greenhouse gas
emissions because of its disproportionately large
contribution to landfill methane emissions.
Significance
• Determined scale of food waste available and
identified urgent need for new food waste-to-
energy facilities, particularly in light of recent
and upcoming biomass power plant closures
2) Locations where new waste-to-energy capacity exists, and where
new capacity is needed in California.
Capacity at existing dry and wet waste-to-energy facilities in many parts of the state
means that little or no new capacity is required. However, in agricultural regions
where the population is small but waste sources are substantial (culled produce, food
processor waste), new facilities are required.
Approach
• We determined the quantity, locations, and temporal
variation in food waste generation, use these results to
model regional and sub-annual electricity and heat
generation potential, and gain insight into challenges
associated with food waste utilization.
4. Deciphering flux adjustments of engineered E.
coli cells with changing growth conditions
He, et al., “Deciphering flux adjustments of engineered E. coli cells during
fermentation with changing growth conditions” Metab. Eng. 39:247-256 doi:
10.1016/j.ymben.2016.12.008.
Background
• Cell metabolism involves coordination of cellular processes at multiple levels,
making the complexity of biological systems challenging to understand.
• This can be addressed by studying metabolic adaptations towards
environmental and genetic perturbations, which requires information from
multiple omics techniques and/or computational simulations.
• 13C-metabolic flux analysis is the only method to quantify in vivo enzymatic
reaction rates in a network, with the resulting flux distributions reflecting the
functional outputs of gene-protein-metabolite interactions.
Significance
• This work shows that maintaining metabolic robustness is a primary choice for
E. coli cells under environmental stresses or enzyme overexpression, while
adaptability is an alternative, but necessary and beneficial choice in some
cases.
• Knowledge and techniques deployed advance the state-of-the-art in metabolic
engineering for the production of biofuels and bioproducts.
Approach
• 13C-metabolic flux analysis was used to characterize two violacein-producing
E. coli strains with vastly different productivities, and to profile their metabolic
adjustments resulting from external perturbations during fermentation.
Outcomes
• Results indicate stable flux ratios in central carbon metabolism throughout the
early growth stages.
• In the late stages of growth the high producer rewired its flux distribution
significantly leading to upregulated pentose phosphate pathway, TCA cycle,
and reflux from acetate utilization.
• The low producer, with stronger promoters, shifted its relative fluxes in the late
stages of growth by enhancing the flux through the TCA cycle and acetate
overflow, while exhibiting a reduced biomass growth and a minimal flux
towards violacein synthesis.
5. Engineering glucose metabolism of
E. coli under nitrogen starvation
Background
• Decoupling growth from production is a lesser explored strategy in
metabolic engineering, but may allow a greater flux of carbon to
final products.
• Nitrogen limitation is a desirable way to limit growth during the
production stage.
• E. coli naturally slows its metabolism in N‐limitation. This is caused
by α‐KG regulation of the PtsI protein
Chubukov et al. (2017) “Engineering glucose metabolism of Escherichia coli under nitrogen starvation”.
Systems Biology and Applications 3, Article number: 16035 (2017)doi:10.1038/npjsba.2016.35
Outcomes
• We found increased product yield in nitrogen starvation conditions compared with
exponential growth, and found that the higher glucose uptake rate of PtsI‐
overexpressing cells was maintained even in stationary phase.
• By overexpressing PtsI, we achieved a fourfold increase in metabolic rates although
additional expression of PtsI did not alter the overall production parameters,
presumably due to other rate‐limiting steps in the pathway.
Approach
• We over expressed the ptsI gene cultivated the resulting strains in
nitrogen limitation to limit growth during the production stage.
• We investigated fatty alcohol production during nitrogen starvation
in engineered E. coli strains.
Significance
• Overexpression of PtsI is likely to be a useful tool in bioenergy‐related metabolic
engineering as productivity of engineered pathways becomes limited by central
metabolic rates during stationary phase production processes.
6. Microbial Production of Isoprenoids
Wong, J. et al. (2017). ”Microbial Production of Isoprenoids". In Consequences of Microbial Interactions with
Hydrocarbons, Oils, and Lipids: Production of Fuels and Chemicals, Springer, DOI: 10.1007/978-3-319-31421-1_219-1
Background
• Isoprenoids are among the most diverse groups of
compounds synthesized by biological systems.
• It has been estimated that there are approximately
40,000–70,000 known isoprenoids.
• The chemical structure of isoprenoids provides many
beneficial aspects to act as a fuel alternative.
Significance
• This book chapter provides a detailed overview of the
different applications, routes and organisms for isoprenoid
production.
• Isoprenoids are a versatile class of compounds that have
multiple roles in bioenergy production, both as a biofuel and
as a bioproduct.
Approach and Outcomes
• This book chapter covers many of the opportunities and
challenges related to the microbial production of
isoprenoids.
• Compares the two primary pathways of terpene production:
mevalonate and DXP.
• Compares and contrasts production in E. coli, S. cerevisiae,
and other hosts.
• Highlights the need for novel process configurations
integrating fermentation and product recovery, cell reuse,
and low-cost technologies for product separation.
Terpene biosynthetic pathways
