Research summaries for papers published by the Joint BioEnergy Institute in April 2022

1. Office of Biological and Environmental Research
Comparative Study on the Pretreatment of Aspen and Maple With
1-Ethyl-3-methylimidazolium Acetate and Cholinium Lysinate
Background/Objective
• Ionic liquids can be used to dissolve, fractionate, and even convert the
lignocellulosic components of biomass so it can be used a renewable carbon
resource
• The development of pretreatment processes to identify potential woody biomass
feedstock for sustainable future biorefinery and energy applications are needed.
Approach
We compared the feasibility of ionic liquid pretreatment processes for two woody
biomasses, maple and aspen, using two mechanistically distinct ionic liquids at
different pretreatment temperatures.
Results
A significant loss of crystallinity was observed for all [C2mim][OAc]-pretreated
biomasses, whereas the pretreatment with [Ch][Lys] resulted in an increase in the
crystallinity index.
Significance/Impacts
No reports exist in the literature studying the effect of temperature on the
delignification and crystallinity of aspen and maple during their pretreatment with
[C2mim][OAc] and [Ch][Lys].
Choudhary, H.., et al. (2022) Frontiers in Energy Research, doi: 10.3389/fenrg.2022.868181
Sugar yields from (A) aspen and (B) maple after IL pretreatments at
different temperatures. Glucose (dark gray bars) and xylose (light gray bars).
The percentage of lignin removal is shown in black closed circles.

2. Office of Biological and Environmental Research
Complete Genome Sequences of Five Isolated Pseudomonas Strains that Catabolize
Pentose Sugars and Aromatic Compounds Obtained from Lignocellulosic Biomass
Background/Objective
• Pseudomonas putida KT2440 is a promising host for the production of
biofuels and bio-based chemicals
• However, P. putida KT2440 lacks the native ability to metabolize
pentose sugars
Approach
• A method was developed to isolate Pseudomonas putida isolates from
soils that metabolize pentose sugars (Figure 1)
Results
• Five isolates were obtained that grew on aromatics and xylose,
two isolates also grew on arabinose
• Two isolates were had >96% amino acid identity to P. putida KT2440;
complete genomes were obtained for all five isolates
Significance/Impacts
• These isolates may provide new pentose conversion pathways and
be alternative hosts to P. putida KT2440
Park, Mee-Rye, et al Microbiology Resource Announcements (2022): e00987-21. doi.org/10.1128/mra.00987-21
Temp. = 30°C
pH = 7.0 ± 0.1
Genomic analysis
(PacBio seq. & illumina seq.)
Xylose M9 agar
Growth kinetics in M9 media
with carbon source
Environmental soil samples
collection
Pseudomonas isolation agar (PIA)
1st
Screening
p-Coumarate M9 agar
Growth test in liquid media
2nd
Screening
Xylose M9 agar
Figure 1. Isolation protocol for P. putida isolates that grow on pentose sugars

3. Office of Biological and Environmental Research
A new approach to zip-lignin: 3,4-dihydroxybenzoate
is compatible with lignification
Background/Objective
• Lignin renders plant biomass recalcitrant to enzymatic conversion into
simple sugars
• Expression of bacterial 3-dehydroshikimate dehydratase (QsuB) is known
to reduce lignin in Arabidopsis and switchgrass
Approach
QsuB was transferred to the bioenergy crop poplar to reduce lignin
Results
-> QsuB poplar trees contain up to 30% less lignin
-> DHB incorporates lignin and creates cleavable bonds (‘zips’)
-> QsuB biomass displays higher saccharification efficiency
Significance/Impacts
The improvement of poplar biomass quality via the QsuB
engineering strategy has the potential to improve the economics
of advanced bioproducts
Unda et al., 2022 New Phytol. https://doi.org/10.1111/nph.18136
QsuB
DHB E4P + PEP
3-dehydroshikimate
LIGNIN
DHB
(%
biomass)
WT QsuB-1 QsuB-2
Sugars
released
(%
biomass)
0
5
10
15
20
25
Line
1
Line
2
Line
35
Line
10
Line
15
Line
15
WT
Lignin
(%
biomass)
*p<0.05
*
* * * *
*p<0.05
Low lignin
High sugar yields
High DHB

4. Office of Biological and Environmental Research
Downregulation of Squalene Synthase Broadly
Impacts Isoprenoid Biosynthesis in Guayule
Background/Objective
• Guayule is a natural rubber-producing plant species.
• Production of natural rubber by guayule requires increased yield for economic
sustainability.
Approach
An RNAi gene silencing strategy was used to engineer isoprenoid biosynthesis by
downregulation of squalene synthase to initiate natural rubber synthesis.
Results
Downregulation of squalene synthase in guayule successfully increased natural
rubber production. Analysis of mevalonic acid and methylerythritol phosphate
pathway intermediates has provided the first evidence of cellular crosstalk between
these essential metabolic pathways in guayule.
Significance/Impacts
This study identified insights and strategies for engineering isoprenoid production in
guayule.
Placido, D., et al. (2022) Metabolites, doi: 10.3390/metabo12040303
Relative expression of metabolic and putative NR biosynthesis and structural
genes in SQSi lines’ background. Plant phenotypes for controls and SQS
lines, RT-PCR for the isoprenoid pathway, and natural rubber biosynthesis-
associated genes.

5. Office of Biological and Environmental Research
Machine-learning from 321 Gene Expression Profiles of Pseudomonas putida
KT2440 Unveils its Genome-scale Transcriptional Regulatory Network (TRN)
Approach
• We generated putidaPRECISE321, a compendium of 321 high-
quality RNA-Seq samples, and performed independent
component analysis (ICA) to analyze it.
Results
• We obtained 84 groups of independently modulated genes
(iModulons) that explain 75.7% of the total variance in the set.
• We identified the regulatory functions of 39 important TFs
(e.g., HexR), associated with iModulons.
• Complex transcriptional changes driven by utilization of different
carbon sources, the presence of stressors, and evolution-derived
mutations were explained with iModulon activity changes.
Significance/Impacts
• The structure of the P. putida TRN that governs its metabolism,
stress responses, and other cellular functions has now been revealed.
Lim et al. (2022) Metabolic Engineering, doi: 10.1016/j.ymben.2022.04.004
Background/Objective
• Elucidating the TRN is fundamental to understanding bacterial
physiology and engineering it for practical use. TRNs are poorly
understood in DOE hosts of interest such as P. putida.

6. Office of Biological and Environmental Research
Artificial Intelligence for Synthetic Biology
Background/Objective
• Biology has dramatically changed in the last two decades.
• The combination of high-throughput phenotypic data with precision DNA
editing provides a unique opportunity to link changes in DNA to phenotype.
• Artificial intelligence (AI) is ideally posed for this task.
Approach
In this article, we provide AI practitioners interested in working on
synbio an overview of:
• The potential of synthetic biology.
• Initial successes of AI in synbio.
• Main challenges faced.
Results
• Sociological, as well as technological, challenges abound.
• AI solutions that incorporate causal reasoning, interpretability, robustness, and
uncertainty estimation are needed.
• Synbio can also inspire new AI approaches.
Significance/Impacts
AI can radically enhance synbio and enable its full impact.
Eslami, Mohammed, et al. "Artificial intelligence for synthetic biology." Communications of the ACM 65.5 (2022): 88-97

7. Office of Biological and Environmental Research
JBEI Enabled Papers

8. Office of Biological and Environmental Research
Recent advances in biological activities of lignin and
emerging biomedical applications: A short review
Background/Objective
• As an abundant biopolymer, lignin gains interest owing to its renewable nature
and polyphenolic structure.
• Studies are being carried out to relate the biological activities to the
polyphenolic structures.
Approach
This review summarizes latest research results on the biological activities of lignin
and highlights potential applications exampled by selective studies.
Results
We analyzed and summarized lignin’s various applications, including antibacterial,
antiviral, and antifungal activity, antioxidant activity, drug delivery, wound healing,
antidiabetic activity, and other emerging applications.
Significance/Impacts
Lignin has various potential applications, ranging from food and cosmetics to
pharmaceuticals and biomedical applications. Further research should be performed
to highlight the potential of this biomolecule
Ullah, I., et al. (2022) International Journal of Biological Macromolecules, doi: 10.1016/j.ijbiomac.2022.03.182
Overall scheme on lignin fractionation and its antioxidant and antimicrobial
activities.

9. Office of Biological and Environmental Research
Corynebacterium glutamicum as an Efficient Omnivorous
Microbial Host for the Bioconversion of Lignocellulosic Biomass
Background/Objective
• Corynebacterium glutamicum has been successfully employed for the industrial
production of amino acids and other bioproducts
• However, several challenges still need to be overcome for efficient
bioconversion of biomass to bioproducts.
Approach
The omnivorous strain C. glutamicum ATCC 13032 was investigated for production
of mixed acids from sugars and aromatics present in lignocellulosic biomass.
Results
By using C. glutamicum as a microbial host, we demonstrate bioproduction of L-
lactate, succinate, and acetate from corn stover biomass hydrolysates with
simultaneous utilization of sugars and aromatics. This demonstrated C. glutamicum
as an efficient microbial host for utilizing diverse carbon substrates present in
biomass hydrolysates, such as glucose, arabinose, and xylose, in addition to its
natural ability to assimilate lignin-derived aromatics.
Significance/Impacts
Our findings open the door to valorize all the major carbon components of biomass
hydrolysate by using C. glutamicum as a microbial host for biomanufacturing.
Mhatre, A.., et al. (2022) Frontiers in Bioengineering and Biotechnology, doi: 10.3389/fbioe.2022.827386
Schematic representation of the overall process employed in this study. Corn
stover was subjected to deacetylation and mechanical refining (DMR) to
obtain liquid biomass hydrolysate. The hydrolysate constituting glucose,
xylose, arabinose, and aromatics was used as fermentation feedstock for C.
glutamicum to produce mixed acids having various applications.

10. Office of Biological and Environmental Research
Optimization of Heterologous Glucoraphanin Production In Planta
Background/Objective
Glucoraphanin is a plant specialized metabolite found in cruciferous vegetables that has long been
a target for production in a heterologous host because it can subsequently be hydrolyzed to form
the chemopreventive compound sulforaphane before and during consumption.
Approach
Previous studies have only been able to produce small amounts of glucoraphanin in heterologous
plant and microbial systems compared to the levels found in glucoraphanin-producing plants,
suggesting that there may be missing auxiliary genes that play a role in improving production in
planta.
Results
We leveraged transient expression in Nicotiana benthamiana to screen a
combination of previously uncharacterized coexpressed genes and
rationally selected genes alongside the glucoraphanin biosynthetic
pathway. This strategy alleviated metabolic bottlenecks, which improved
glucoraphanin production by 4.74-fold.
Significance/Impacts
Our study describes a method to systematically identify novel genes
involved in improving the yields of bioproducts in plants. This approach
can be used to further optimize production of natural products, but also
commodity chemicals and potential biofuels.
Barnum CR*, Endelman BJ*, Ornelas IJ, Pignolet RM, Shih PM. (2022) Optimization of heterologous glucoraphanin production in planta. ACS Synthetic Biology, accepted