JBEI Research Highlights - February 2019

1. Engineering robust production microbes
for large-scale cultivation
Background
• Strain engineering in the laboratory often does not
consider process requirements in larger-scale
bioreactors
• In this review paper, we evaluate the differences
between laboratory-scale cultures and larger-scale
processes to provide a perspective on characteristics of
microbial production hosts that are especially important
during scaling
Highlights
• Synthetic and systems biology tools are increasingly
enabling characterization of strain physiology under
industrially relevant conditions in a bioreactor
• Miniaturization and automation of strain design, strain
construction, and high-throughput measurements
provide the data and data-driven approaches to
translate production performance from laboratory to pilot
and commercial scales.
• Commercial microbial platforms should be selected and
developed based on their relevance to final process
goals
• Systems and synthetic biology can be applied to design
microbial strains that allow reliable and robust
production on a large scale
Wehrs et al. (2019) Trends in Microbiology, doi: 10.1016/j.tim.2019.01.006

2. A rapid methods development workflow for high-
throughput quantitative proteomic applications
Background
• Quantification of proteins is important for bioenergy research of plants
and microbes
• Methods development for the analysis of new proteins from many
different organisms is time consuming and causes bottlenecks in the
conversion of biomass-to-biofuels process
• Methods to rapidly develop reproducible and high-throughput
quantitative assays are needed
Approach
• Figure summarizes the workflow using in-house generated data and
data from online proteomic repositories to inform target peptide
selection, predict chromatographic retention times, and begin
analyzing hundreds of samples in only a few minutes
• The workflow utilizes highly reproducible liquid chromatography
systems to accurately predict peptide retention times.
Outcomes and Impacts
• This workflow simplifies validation of peptides identified from shotgun
proteomic experiments and significantly reduces development time of
high-throughput quantitative targeted proteomic assays to quantify
novel protein targets
• We demonstrated this workflow by developing targeted proteomic
assays to quantify proteins of metabolic pathways from multiple
microbes under different experimental conditions
• This workflow facilitates the development of robust, reproducible, and
high throughput proteomic assays to aid biofuel research applications
Chen et al. (2019) PLoS ONE, doi: 10.1371/journal.pone.0211582.
Workflow to rapidly develop targeted proteomic
methods from shotgun proteomic data.

3. Supply and value chain analysis of mixed biomass
feedstock supply system for lignocellulosic sugar
production
Background
• Implementing a year-round feedstock switching strategy or utilizing
an optimal mixture of corn stover, miscanthus, and switchgrass
could de-risk seasonal feedstock availability, affordability, and
sustainability.
• This study considers opportunities for bridging gaps in feedstock
supply logistics systems by (i) implementing a year-round
feedstock switching strategy based on the harvesting seasons for
corn stover, miscanthus, and switchgrass, and (ii) utilizing a
mixture of corn stover and energy crops, such as miscanthus and
switchgrass, as inputs to the biorefinery.
Approach
• We have performed a comprehensive supply and value chain
analysis for each stage of the system that includes both the mixed
feedstock supply logistics and the downstream biorefinery
conversion process through production of lignocellulosic sugars.
Outcomes and Impacts
• A mixing ratio of corn stover, miscanthus, and switchgrass of 36,
50 and 14%, respectively, minimizes the selling price of sugar.
• Utilizing the optimal mixture of corn stover, miscanthus, and
switchgrass reduces cost of sugar production by 13.6%, when
compared to a baseline sugar price of 441.9 $/metric ton (t)
attributed to a fixed corn stover feedstock basis.
• Results suggest that a high-quality feedstock at high carbohydrate
content is an important metric for consideration, beyond strictly the
feedstock cost, to reduce the selling price of sugar and its
uncertainties.
Baral et al. (2019) Biofuels. Bioprod. Bioref., doi: 10.1002/bbb.1975
Annual average optimal mixing ratio for three feedstocks studied.
Minimum selling price of sugar with annual average optimal mixing
ratio. In this figure, curve (–) indicates cumulative probability, and ‘UL-
95%’ refers to the upper limit at 95% certainty.
M = Mode value
f = Frequency of
the mode

4. Transcriptome analysis of rubber biosynthesis in
guayule (Parthenium argentatum Gray)
Background
• Natural rubber is currently produced nearly exclusively from latex of the
Para rubber tree, Hevea brasiliensis.
• The desire to reduce the environmental cost of rubber production, fears
of pathogen susceptibility in clonal Hevea plantations, volatility in the
price of natural rubber, and increasing labor costs have motivated efforts
to diversify the supply of natural rubber by developing alternative rubber
crops such as guayule (Parthenium argentatum Gray).
• Guayule is an American plant that grows well in semi-arid parts of the
south-west USA
Approach
• To better understand the enzymology and regulation of guayule rubber
biosynthesis and to identify genes with potential uses in the improvement
of rubber yields, we conducted de novo transcriptome assembly and
differential gene expression analysis in guayule.
• This analysis supports a role for rubber in the defense against
pathogens, identified new enzymes potentially involved in the
biosynthesis of rubber as well as transcription factors specifically
expressed in rubber-producing tissues.
Outcomes and Impacts
• Data presented here will be useful in the improvement of guayule as an
alternative source of natural rubber and in better understanding the
biosynthesis of this critical polymer.
• In particular, some of the candidate transcription factors are likely to
control the rubber biosynthesis pathway and are good targets for
molecular breeding or engineering of guayule plants with higher and
more consistent production of rubber.
Stonebloom et al. (2019) BMC. Plant Biol., doi: 10.1186/s12870-019-1669-2
Genes differentially expressed in guayule. Cold-
induced stems is the condition where rubber
biosynthesis is induced. The other conditions are
controls where rubber biosynthesis is low.
Differentially expressed
transcription factors. The
genes in the yellow box are all
specifically induced under
conditions of high rubber
biosynthesis. These
transcription factors are
candidates for master
regulators of rubber
biosynthesis and their
overexpression in guayule is a
strategy to increase rubber
biosynthesis in the crop.

5. Studies of the protein complexes involving cis-
prenyltransferase in guayule (Parthenium argentatum)
Background
• Guayule (Parthenium argentatum) is a perennial shrub in the
Asteraceae family and synthesizes a high quality,
hypoallergenic cis-1,4-polyisoprene (or natural rubber; NR).
• Despite its potential to be an alternative NR supplier, the
enzymes for cis-polyisoprene biosynthesis have not been
comprehensively studied in guayule.
Approach
• Transcriptome analysis was used to identify guayule genes likely
to be involved in NR biosynthesis
• Three cis-prenyltransferases (PaCPT1-3) and one CPT binding
protein (PaCBP) were identified and expressed in yeast.
• Activity and protein-protein interactions were investigated with the
expressed guayule proteins
Outcomes and Impacts
• Co-expression of PaCBP and each of the CPTs complemented a
dolichol-deficient yeast, whereas the individual expressions could
not.
• Microsomes from the PaCPT/PaCBP-expressing yeast efficiently
incorporated 14C-isopentenyl diphosphate into dehydrodolichyl
diphosphates
• The CPTs and CBP organize biosynthetic complexes
• The comprehensive analyses of CPTs and PaCBP provide the
foundational knowledge to generate a high NR-yielding guayule
Lakusta et al. (2019) Front. Plant Sci., doi: 10.3389/fpls.2019.00165
Transcript level of the four genes determined by RNAseq.
They are all expressed at low levels in leaves (L) while CBP
and CPT3 are highly expressed in stems (S). These two
genes are also induced under cold treatment (black bars)
consistent with the production of NR in stems in winter.
Activity assay using
microsmes from yeast
expressing CBP and CPT.
The substrate was 14C-IPP
and a range of polymers of
isoprene were produced in
all cases. No product was
observed if CBP or CPT was
expressed alone.

6. Techno-economic analysis and life-cycle greenhouse
gas mitigation cost of five routes to bio-jet fuel
blendstocks
Background
• Biological routes ability to produce naphthenes (whereas most other
alternative jet fuel processes produce paraffins) with attractive
properties for aviation applications.
• Past studies have focused largely on thermochemical routes to bio-jet
fuels
• This paper presents a detailed TEA and sensitivity analysis, including
estimated minimum selling price (MSP), and life-cycle greenhouse gas
(GHG) mitigation costs for five routes to four potential bio-jet fuel
molecules – limonane via limonene, limonane via 1,8-cineole,
tetrahydromethylcyclopentadiene dimer (RJ-4), bisabolane, and epi-
isozizaane.
Approach
• We developed stochastic TEA and lifecycle assessment (LCA) models
to quantify the MSP and GHG mitigation costs.
• We identified performance targets needed to reach a targeted selling
price of $0.66/L ($2.50/gal) of bio-jet fuel.
Outcomes and Impacts
• The evaluated jet fuel molecules could reach an MSP of about $1/L-
Jet A-equivalent in optimized future cases, without a lignin-derived co-
product. To reach $0.66/L-Jet A ($2.50/gal), lignin-derived products
would need to be sold for at least $1.9/kg.
• The minimum achievable carbon mitigation cost relative to
conventional Jet-A is $29/metric ton CO2e.
• Commercial airlines may be willing to pay a 4-14 cent/L premium for
these bio-jet fuels based on their higher density and heating values,
because this allows aircraft to fly farther on the same tank of fuel.
Baral et al. (2019) Energy Environ. Sci., doi: 10.1039/C8EE03266A
Most influential input parameters to MSP
Likelihood of reaching different target prices for each
blendstock with potential future improvements
Legend ($/L-Jet A)
≤ 2
>2 and ≤ 2.75
>2.75 and ≤ 3.5
>3.5 and ≤ 4.25
>4.25 and ≤ 5
>5

7. Engineering Corynebacterium glutamicum to
produce biofuels from sorghum biomass
Background
• We sought to understand the advantages from using
alternative microbial hosts which could tolerate more taxing
growth conditions, rather than their accessibility for genetic
manipulations. Isopentenol (3-methyl-3-buten-1-ol), a
biogasoline candidate, has an established heterologous
gene pathway but is toxic to several microbial hosts.
• Reagents used in the pretreatment of plant biomass, such as
ionic liquids, may also inhibit growth of many host strains
such as E. coli or S. cerevisiae.
Approach
• We assessed C. glutamicum for its ability to produce
isopentenol using a synthetic gene pathway and its ability to
survive in the presence of ionic liquids.
• Bottleneck reactions were identified for subsequent study
and optimization.
Outcomes and Impacts
• We successfully expressed a heterologous, mevalonate-
based pathway in the industrial microorganism, C.
glutamicum, for the production of the biogasoline candidate,
isopentenol to produce over 1.1 g/L using sugars from
sorghum biomass.
• We identified critical genetic factors to harness the
isopentenol pathway in C. glutamicum. This work represents
a fifty-fold titer improvement over other terpene molecules
produced in C. glutamicum to date.
Sasaki et al. (2019) Biotechnology for Biofuels, 12(1), doi: 10.1186/s13068-019-1381-3
Upper Panel: A graphical depiction of the five genes expressed in
C. glutamicum to produce the biofuel candidate, isopentenol
(chemical structure on right hand side). Lower Panel: Proteomic
analysis of engineered isopentenol producer strains cultivated
under different conditions and the corresponding protein
abundance of each individual pathway protein.