Summaries of the research papers published by the Joint BioEnergy Institute (JBEI) in August 2022

1. Office of Biological and Environmental Research
Synthetic gene circuits take root
Background/Objective
A long-standing aim of synthetic biology has been to engineer
genetic circuits that are able to confer prescribed spatiotemporal
patterns of gene expression. This paper is a Perspective piece on
Brophy et al. 2022, which reports a strategy to engineer spatial
transcriptional patterns in the root of the model plant Arabidopsis
thaliana to alter its morphology.
Significance/Impacts
Synthetic biology approaches tailored to embrace the complexity of
developing organisms are uniquely positioned to provide
sophisticated and quantitative tools to dissect the inner workings of
genetic networks specifically in an organismal context.
Engineering complex circuits in plants may enable our ability to
engineer feedstock crops with more complex traits.
Alamos S., Shih P. (2022) “Synthetic gene circuits take root.” Science. doi: 10.1126/science.add6805

2. Office of Biological and Environmental Research
Multiscale Molecular Simulations Strategies for Understanding
the Delignification Mechanism of Biomass in Cyrene
Background/Objective
• Lignin, one of the major constituents of the lignocellulosic biomass along with
cellulose and hemicellulose, is the most-abundant bio-renewable source for
aromatics.
• Due to the recalcitrance, heterogeneity, strong interactions, and hydrophobicity
of lignin, fractionation of lignin remains a major challenge for biorefineries.
• Recent findings showed that the Cyrene solvent has the potential to be used in
biomass pretreatment and higher biomass delignification
Approach
• To understand the delignification mechanism of biomass, we applied multiscale
molecular simulation approaches such as classical molecular dynamics (MD)
simulations and the COSMO-RS model to examine the structure of lignin in Cyrene
and Cyrene-water cosolvent environments.
Results
• From the experiments, Cyrene and Cyrene/water mixtures dissolve ~95% of
organosolv lignin and 70% of lignin from poplar biomass, respectively.
• With increasing Cyrene concentration, the Rg and SASA values increase, indicating
that the lignin adopts a more extended polymer-like structure.
• We have also developed a machine learning model for the prediction of Kamlet-
Taft Parameters
Mohan et al. ACS Sustainable Chem. Eng. 2022, 10, 33, 11016–11029
0.1 0.3 0.5 0.7 0.9 1.1
0.1
0.3
0.5
0.7
0.9
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0.1
0.3
0.5
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0.9
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Beta_12
Linear Fit of Sheet1 D
95% Confidence Band of D
95% Confidence Band of D
beta
Predicted,
Experimental,
Inter cept 0.00 958 ± 0.012
Slope 0.98 41 ± 0 .0229
Resid ual Sum of Squa 0.0 4358
Pear son's r 0.9 8665
R-Sq uare (CO D) 0.9 7347
Adj. R-Squ are 0.9 7294
R2
= 0.97
(a)
D MI
Predicted,
Experimental,
R2
= 0.98
(b)
DMI

3. Office of Biological and Environmental Research
Background/Objective
• Rubisco is responsible for nearly all organic carbon on Earth
• Understanding the biophysical constraints of the enzyme may reveal new
paths to improving and engineering rubisco
Approach
Rubisco can assemble as several different oligomeric states; however, we know
little about how these structural changes effect the kinetics of the enzyme
Results
We retrace the evolution of ancestral and extant form II rubiscos,
revealing a complex and diverse history of oligomerization. We further
use these principles to engineer and demonstrate how changes in
oligomerization can be mediated by relatively few mutations.
Significance/Impacts
We demonstrate that rubisc kinetics can be tuned based on quaternary
structure outlining a path to understanding the physical constraints to plant
type rubisco and providing a potential path for future engineering.
Liu A.K., Pereira J.H., Kehl A.J., Rosenberg D.J., Orr D.J., Chu S.K.S., Banda D.M., Hammel M., Adams P.D., Siegel J.B., Shih P.M. (2022) “Structural
plasticity enables evolution and innovation of rubisco assemblies.” Science Advances. doi: 10.1126/sciadv.adc9440
Structural plasticity enables evolution and
innovation of rubisco assemblies

4. Office of Biological and Environmental Research
On the structures of lignin subunits and lignin nanoparticles
Background/Objective
• Lignin nanoparticles (LNPs) have become an important platform to valorize
lignin
• The structures of lignin molecules (subunits) and nanoparticles in solution
are not well understood
Approach
We used small angle neutron scattering (SANS) to investigate solution
structures of lignin subunits and the LNPs. The chemical structure of
the lignins was characterized by 31P NMR and GPC.
Results
Lignin aggregates were found in studied solutions, and they consisted of high
molecular weight lignin subunits. The LNPs were shown as solid spheres
surrounded by a large hydration shell.
Significance/Impacts
Mechanistic investigations of the LNP formation may consider the aggregates
that already exist in the initial lignin solutions. The size, shape, and
compactness are important factors that affect the uses of the LNPs, which were
obtained from the SANS data for the first time
Xin Zhang, Jinxu Zhang, Hua Yang, Chunyong He , Yubin Ke, Seema Singh, and Gang Cheng, ChemSusChem, DOI: 10.1002/cssc.202201230
Figure 1. SANS data of kraft lignin in DMSO-d6, EG-
d6 and 0.1N NaOD
Figure 2. SANS data of kraft lignin in DMSO-d6 and
the kraft LNPs in D2O

5. Office of Biological and Environmental Research
Transcriptional and metabolic changes associated with internode development
and reduced cinnamyl alcohol dehydrogenase activity in sorghum
Background/Objective
• Sorghum is a promising biomass crop.
• The secondary cell wall forms the majority of the biomass.
• Sorghum secondary cell wall biosynthesis is still poorly understood.
Approach
• In a study led by the Cesarino lab at the University of Sao Paulo,
Brazil, secondary cell wall deposition was analyzed along the
developing internode, by pairing transcriptomics with
metabolomics in both wild type and a low lignin (bmr6) line.
Results
• Gene modules were identified that correlate with secondary cell
wall deposition
• bmr6, which is perturbed in cinnamyl alcohol dehydrogenase
(CAD) resulted in distinct detoxification routes in sorghum
internodes
Significance/Impacts
• Our systems biology approach provides a landscape of the metabolic and transcriptional
changes associated with internode development and with reduced CAD activity in sorghum
Ferreira et al. 2022, J. Ex. Bot, doi.org/10.1093/jxb/erac300
Dynamics of metabolite accumulation along the
elongating internode of WT and bmr6 plants.

6. Office of Biological and Environmental Research
Translating advances in microbial bioproduction
to sustainable biotechnology
Background/Objective
• The success of a drop-in bioproduct is contingent on market competition with petrochemical analogues and weighted
upon relative economic and environmental metrics.
• While the quantification of comparative trade-offs is critical for accurate process-level decision making, the translation
of industrial ecology to synthetic biology is often ambiguous and assessment accuracy has proven challenging.
Approach
In this review, we explored strategies for evaluating industrial biotechnology through life cycle
and techno-economic assessment, then contextualized how recent developments in synthetic
biology have improved process viability by expanding feedstock availability and the
productivity of microbes.
Results
By juxtaposing biological and industrial constraints, we highlighted major obstacles
between the disparate disciplines that hinder accurate process evaluation.
Significance/Impacts
Effective integration of synthetic biology with environmental and economic assessments is
critical for actualizing industrial biotechnology and decarbonizing bulk chemical production.
Carruthers D.N., Lee T.S. (2022) “Translating advances in microbial bioproduction to sustainable biotechnology."
Front. Bioeng. Biotechnol. doi: 10.3389/fbioe.2022.968437
FIGURE 1. The integration of LCA and
synthetic biology within a circular
bioeconomy.

7. Office of Biological and Environmental Research
JBEI Enabled Publications

8. Office of Biological and Environmental Research
Biofilm disruption enhances growth rate and
carbohydrate-active enzyme production in anaerobic fungi
Background/Objective
• Anaerobic gut fungi (AGF) are lignocellulose degraders that form
biofilms in the rumen and in all standard culture techniques.
• Biofilm formation complicates laboratory analysis and scale-up of AGF
cultures, and it may alter gene expression relative to non-biofilm culture.
Approach
Using the non-rhizoidal AGF, Caecomyces churrovis, we quantify the
impacts of biofilm formation on AGF gene expression, metabolic flux,
growth rate, and xylan degradation rate to inform future industrial scale-up.
Results
C. Churrovis grew faster and upregulated catabolic CAZymes in stirred
culture relative to biofilm culture, with no change in metabolic flux.
Significance/Impacts
Stirred culture of non-rhizoidal AGF offers a way to accelerate
CAZyme production and biomass valorization without altering the
fermentation product profile.
Leggieri P.A., Valentine M.T., O'Malley M.A. (2022) “Biofilm disruption enhances growth rate and carbohydrate-active enzyme production in anaerobic fungi.” Bioresour Technol. doi: 10.1016/j.biortech.2022.127361
Stirred
Biofilm
Stirred
Biofilm
RNA harvest

9. Office of Biological and Environmental Research
A microbial supply chain for production of the
anti-cancer drug vinblastine
Background/Objective
• Monoterpene indole alkaloids (MIAs) are a diverse family of complex
plant secondary metabolites.
• The world’s supply chain for vinblastine relies on low-yielding
extraction and purification of the precursors vindoline and catharanthine
Approach
• We engineered yeast with a very long biosynthetic pathway including 30
enzymatic steps, taken from several plants, beyond the yeast native
metabolites geranyl pyrophosphate and tryptophan.
Results
• The engineered yeast produces catharanthine and vindoline, which were
then chemically coupled to produce vinblastine
Significance/Impacts
• As the vinblastine pathway is one of the longest MIA biosynthetic
pathways, this study positions yeast as a scalable platform to produce
more than 3,000 natural MIAs and a virtually infinite number of new-to-
nature analogues
Zhang et al. 2022 Nature doi: 10.1038/s41586-022-05157-3.
Article
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Ergosterol
biosynthesis
GPP (1)
Tryptophan (2)
Stemmadenine
acetate (18)
Catharanthine (22)
Tryptamine (13)
Loganic acid (10)
7-Deoxyloganic
acid (9)
7-Deoxylognetic
acid (8)
7-Deoxylognetic
alcohol (7)
Nepetalactol (6)
8-Oxogeranial (5)
8-Hydroxygeraniol (4)
Geraniol (3)
FPP
FPSN144W
GPPS2
ERG20F96W, N127W
DMAPP
IDI1
O
P
O
O
P
-
O
O
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IPP
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NH2
TDC
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Shikimate
pathway
MVA
pathway
PRX1
T3O
T3R
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Stemmadenine (17) Geissoschizine (16) Strictosidine aglycone (15) Strictosidine (14) Secologanin (12)
Tryptamine (13)
Loganin (11)
Precondylcarpine
acetate (open) (19)
Dihydroprecondyl-
carpine acetate (20)
Tabersonine (21)
16-Hydroxy-
tabersonine (23)
16-Methoxy-
tabersonine (24)
Vinblastine (29)
Vindoline (28) Desacetylvindoline (27) Deacetoxyvindoline (26)
3-Hydroxy-16-methoxy-2,3-
dihydrotabersonine (25)
Semisynthesis by
chemical coupling
Fig.1|Completebiosyntheticpathwayfortheproductionofvinblastinein
yeast.Yeastgenes(orange)overexpressed,dynamicallyknockeddownor
deletedareindicatedbygreenarrows,redarrowsandredcrosses,respectively.
Abbreviationsnotalreadydefinedinthetextareasfollows:IPP,isopentenyl
pyrophosphate;DMAPP,dimethylallylpyrophosphate;GPPS,GPPsynthase;
stemmadenine-O-acetyltransferase;CS,catharanthinesynthase;TS,
tabersoninesynthase;T16H,tabersonine16-hydroxylase;16OMT,tabersonine
16-O-methyltransferase;T3O,tabersonine3-oxygenase;T3R,16-methoxy-
2,3-dihydro-3-hydroxytabersoninesynthase;NMT,3-hydroxy-16-methoxy-
2,3-dihydrotabersonine-N-methyltransferase;D4H,deacetoxyvindoline

10. Office of Biological and Environmental Research
CRISPR-RNAa :Targeted activation of translation using
dCas13 fusions to translation initiation factors
Background/Objective
• A cornerstone of bioengineering are techniques to maximize
expression of desired genes, to boost productivity
• No tools exist to enhance the final step: rates of RNA translation
Approach
We tethered translation initiation factors (IFs 1 and 3) onto RNA-
targeting dCasRx, to promote translation of RNA into protein.
Results
dCasRx-IF3 enhanced gene expression of a demonstrative gene (RFP)
up to 21.3-fold, without substantial detrimental fitness impacts.
Significance/Impacts
Bioengineers can utilize this tool to maximize expression of key genes,
potentially leading to higher productivity of biomaterials (indigoidine),
biofuels (terpenoids), or biopharmaceuticals (insulin).
Otoupal, P. B., Cress, B. F., Doudna, J. A., & Schoeniger, J. S. (2022). CRISPR-RNAa: targeted activation of translation using dCas13
fusions to translation initiation factors. Nucleic acids research, 50(15), 8986–8998, https://doi.org/10.1093/nar/gkac680
New Tool
More Pink =
More Gene Expression