Summaries of research published from the Joint BioEnergy Institute in January 2021.

1. Expression of a bacterial 3-dehydroshikimate dehydratase
(QsuB) reduces lignin and improves biomass saccharification
efficiency in switchgrass (Panicum virgatum L.)
Approach
• QsuB from Corynebacterium glutamicum was
expressed in the bioenergy crop switchgrass (Panicum
virgatum L.) using the stem-specific promoter of an O-
methyltransferase gene (pShOMT) from sugarcane.
Outcomes and Impacts
• We show that independent QsuB switchgrass lines
display 12-21% reduction in lignin, a 2-3-fold increase
in the bioaccumulation of PCA and a 5-30% increase
in saccharification efficiency (p < 0.05).
• We are currently field testing our engineered QsuB
switchgrass to assess its agronomic performance and
resilience to environmental stress.
Hao et al. (2021) BMC plant biology. doi: 10.1186/s12870-021-02842-9
Lignin reduction
PCA accumulation
C
O
N
T
R
O
L
Higher sugar yields
C
O
N
T
R
O
L
*p < 0.05
QsuB
C
O
N
T
R
O
L
C
O
N
T
R
O
L
C
O
N
T
R
O
L
QsuB QsuB QsuB
QsuB
C
O
N
T
R
O
L
QsuB
PCA E4P + PEP
3-dehydroshikimate
LIGNIN
Background
• Lignin negatively affects biomass conversion into advanced
bioproducts. Therefore, there is a strong interest in developing
bioenergy crops with reduced lignin content.
• Accumulation of bioproducts is another desired trait for
bioenergy crops.
• Expression of a 3-dehydroshikimate dehydratase (QsuB) in
plants offers the potential for decreasing lignin content and
overproducing a value-added metabolic coproduct suitable for
biological upgrading (i.e., protocatechuate –PCA).

2. Multi-Omics Driven Metabolic Network Reconstruction and Analysis
of Lignocellulosic Carbon Utilization in Rhodosporidium toruloides
Background
• An oleaginous yeast Rhodosporidium toruloides is a promising host
for converting lignocellulosic biomass to bioproducts and biofuels
• In this study, we performed multi-omics analysis and reconstructed
the genome-scale metabolic network of R. toruloides to study the
utilization of carbon sources derived from lignocellulosic biomass
Approach
• Reconstruction of the genome-scale metabolic network, manual
curation of the reconstructed network, and validation of the
metabolic model were performed using Jupyter notebooks in a fully
reproducible manner
• A multi-omics dataset including transcriptomics, proteomics,
metabolomics, lipidomics, and RB-TDNA sequencing was
generated and integrated with the metabolic model to investigate
lignocellulosic carbon utilization
Outcomes and Impacts
• A large and comprehensive multi-omics dataset was generated for
R. toruloides grown on glucose, xylose, arabinose, or p-coumaric
acid as carbon sources found after deconstruction of lignocellulose
• An accurate genome-scale metabolic network was developed for
R. toruloides and validated against high-throughput growth
phenotype and functional genomics data
• The multi-omics dataset and genome-scale metabolic model will be
utilized to maximize the use of the carbon in lignocellulosic
biomass feedstocks and improve the production of biofuel and
bioproduct precursors
Kim et al. (2021) Front. Bioeng. Biotechnol. 8:612832, doi: 10.3389/fbioe.2020.612832
The metabolic reactions, genes, and their localization
for p-coumaric acid degradation pathway in
R. toruloides were proposed using the multi-omics
dataset and metabolic network reconstruction

3. Efficient production of oxidized terpenoids via
engineering fusion proteins of terpene synthase and
cytochrome P450
Background
• The functionalization of terpenes using cytochrome P450 enzymes
is a versatile route to the production of useful derivatives that can
be further converted to value-added products.
• Many terpenes are hydrophobic and volatile making their
availability as a substrate for P450 enzymes significantly limited
during microbial production.
Approach
• This work developed a strategy to improve the accessibility of
terpene molecules for the P450 reaction by linking terpene
synthase and P450.
• As a model system, fusion proteins of 1,8-cineole synthase (CS)
and P450cin were investigated via experimental and structural
analysis.
Outcomes and Impacts
• Fusion proteins of CS and P450cin showed an improved
hydroxylation of the monoterpenoid 1,8-cineole up to 5.4-fold.
• Structural analysis by SEC-SAXS indicated the linker length
affects the flexibility, which eventually affects the catalytic activity,
of the fusion enzymes.
• The application of fusion strategy to the biosynthetic pathway for
oxidized epi-isozizaene products resulted in a 90-fold increase.
• This strategy could be widely applicable to improve the
biosynthetic titer of the functionalized products from hydrophobic
terpene intermediates.
Wang et al. (2021) Metabolic Engineering, 64: 41–51. (doi.org/10.1016/j.ymben.2021.01.004)
Enzyme fusions were engineered to improve substrate
availability as terpenes are hydrophobic and easily lost by
phase separation. Fusion proteins showed improved
production of oxidized mono- and sesquiterpenoids.

4. Genomic mechanisms of climate adaptation in
polyploid bioenergy switchgrass
Background
• Switchgrass (P. virgatum) is both a promising biofuel crop and an
important component of the North American tallgrass prairie.
• Biomass production is the principal breeding target for switchgrass
as a forage and bioenergy crop
Approach
• Deep PacBio long-read sequencing coupled with deep short-read
polishing and bacterial artificial chromosome (BAC) clone validation
produced a highly contiguous ‘v5’ AP13 genome assembly.
• Analysis of biomass and survival among 732 resequenced
genotypes, which were grown across 10 common gardens that span
1,800 km of latitude to find evidence of climate adaptation.
Outcomes and Impacts
• Highly accurate and complete reference genome for tetraploid
switchgrass was generated using long-read DNA sequencing
technology
• With the help of whole genome sequence it was estimated that the
two parental species of switchgrass diverged from a common
ancestor about 6.7 million years ago, and that the two genomes
came back together in a whole-genome duplication at least 4.6
million years ago
• Investigating patterns of climate adaptation, the genome resources
and gene–trait associations developed here provide breeders with
the necessary tools to increase switchgrass yield for the sustainable
production of bioenergy.
Geographical distribution of common gardens (n = 10)
and plant collection locations (n = 700 georeferenced
genotypes), and spatial distribution models of each
ecotype. The ecotype color legend accompanies the
representative images of each ecotype to the right of
the map (images were taken at the end of the 2019
growing season and the background was removed
with ImageJ (https://imagej.nih.gov/ij)). White-outlined
points (coloured by ecotype, or in white if no ecotype
assignment was made) indicate the georeferenced
collection sites of the diversity panel. The labeled white
circles with black crosses indicate the locations of the
10 experimental gardens. Scale bars, 1 m.
Lovell, J.T., MacQueen, A.H., Mamidi, S. et al. Nature (2021). https://doi.org/10.1038/s41586-020-
03127-1

5. Technoeconomic analysis for biofuels and
bioproducts
Background
• This article provides a review of current literature on Technoeconomic
analysis (TEA), which is an approach for conducting process design and
simulation, informed by empirical data, to estimate capital costs,
operating costs, mass balances, and energy balances for a commercial
scale biorefinery
• TEA serves as a useful method to screen potential research priorities,
identify cost bottlenecks at the earliest stages of research, and provide
the mass and energy data needed to conduct life-cycle environmental
assessments.
Approach
• We reviewed recently published work on TEA applied to biofuel and
bioproduct production
• We reviewed the challenges of integrating conventional process
simulation software with uncertainty analysis and life-cycle assessment
and noted recent examples of good implementations of this approach
• We also reviewed the types of financial metrics that are most commonly
used in industry to evaluate potential projects, in contrast with metrics
used in academic and research settings
Outcomes and Impacts
• Recent studies have produced new tools and methods to enable faster
iteration on potential designs, more robust uncertainty analysis, and
greater accessibility through the use of open-source platforms.
• There is also a trend toward more expansive system boundaries to
incorporate the impact of policy incentives, use-phase performance
differences, and potential impacts on global market supply.
• Recent advances in high-throughput experimental pipelines have great
potential if integrated with TEA to generate insights about commercial-
scale implications.
Scown et al. (2021) COBIOT, doi: 10.1016/j.copbio.2021.01.002
Figure 1. Scope of well-conducted
technoeconomic analyses for biofuels and
bioproducts