January 2023 - Summary slides of Joint BioEnergy Institute (JBEI) research on biofuels and bioproducts.

1. Office of Biological and Environmental Research
Perspectives in growth production trade-off in
microbial bioproduction
Background/Objective
• To produce renewable fuels and bulk commodities, the maximum conversion
of starting materials and stable phenotypes in a bioreactor are vital for an
economically viable process.
Approach
• This review highlights advances in efficient and robust strain engineering
with recent examples of growth coupling, growth decoupling, regulatory
control and use of non-metabolic cellular functions in the context of
microbial host physiology.
Results
• For the maximum conversion and ideal hosts, products, substrates, and scale
agnostic bioconversion success of any type, the trade-off introduced through
strain or host engineering can be addressed with the right combination of host
and product, and thus these methods need to be more widely applied.
Significance/Impacts
• This work is in alignment with the UN Sustainable Development Goals
including affordable and clean energy, responsible consumption and
production, and climate action.
Banerjee, D., et al. RSC Sustainability, doi: 10.1039/D2SU00066K
(A) Although robust growth is required for efficient production, both
growth and production also utilize the same pool of starting
materials, creating a trade-off. (B) Engineering approaches to better
utilize the trade-off towards bioconversion to commodity chemicals.
A
B

2. Office of Biological and Environmental Research
Multiscale molecular simulations for the
solvation of lignin in ionic liquids
Background/Objective
Finding suitable solvents for lignin dissolution and
depolymerization, are major obstacles in the
conversion of lignin to value-added products
Approach
COnductor-like Screening MOdel for Real
Solvents (COSMO-RS) model was used to screen
5670 ionic liquids
Results
Anions such as acetate and methyl carbonate in
combination with cations like tetraalkylammonium are
predicted to be suitable solvents for lignin dissolution
Significance/Impacts
This approach enables rapid screening thousands of
solvents to find those capable of solvating lignin
Mohan, M. et al Sci Rep. https://doi.org/10.1038/s41598-022-25372-2
Figure 1: COSMO-RS predicted sigma potential of (a) lignin and anions, and (b) cations of ILs at 363.15 K.
COSMOTherm version 19.0.1 was used to calculate the sigma potentials

3. Office of Biological and Environmental Research
Development of genetic tools for heterologous protein expression
in a pentose-utilizing environmental isolate of Pseudomonas putida
Background/Objective
• Pseudomonas putida is a promising host for the conversion of biomass-derived
sugars and aromatics to biofuels and bioproducts.
• P. putida is not capable of metabolizing pentose sugars, which can constitute up to
25% of biomass hydrolysates.
Approach
• Genetic tools were developed for P. putida M2, a soil isolate that can metabolize
pentose sugars.
Results
• The functionality of five inducible promoter systems and 12 ribosome binding sites was
assessed to regulate gene expression, which were used to produce indigoidine.
• Chromosomal integration and expression of non-native genes was achieved by using
chassis-independent recombinase-assisted genome engineering (CRAGE)
Significance/Impacts
• These genetic tools provide a foundation to develop hosts complementary to P.
putida KT2440 and expand the ability of this versatile microbial group to convert
biomass to bioproducts.
Figure 1. Flaviolin production from glucose and
xylose using CRAGE- integrated PKS gene in P.
putida KT2440 and M2
Gauttam R., et al, Microbial Biotechnology. doi: 10.1111/1751-7915.14205

4. Office of Biological and Environmental Research
Mass spectrometry imaging-based assays for aminotransferase activity reveal
a broad substrate spectrum for a previously uncharacterized enzyme
Background/Objective
• Aminotransferases (ATs) are a highly versatile class of enzymes that
transfer an amino group from an amino donor to a keto acceptor substrate.
• Conventional AT assays use the intrinsic absorbance or fluorescence of
the substrates or reaction products as the readout for AT activity.
• Direct analysis of AT activity can be performed by HPLC, CE, CD, NMR etc.
However, these techniques are time consuming and have a limited throughput.
Approach
Developed a mass spectrometry-based high throughput assay using
Oxime-NIMS to quantify both substrates and products for AT reactions
and using MSI for results readout
Results
The oxime-MSI AT assay has been developed and are shown to be suitable
for high-throughput screening of AT amino donor and keto acceptor
specificity. This new technology enabled screening of substrate specificity,
And revealed the AtTAR1 can use 13 amino acids as donor.
Significance/Impacts
This novel technology unlocks an exciting opportunity to expand our
knowledge of AT characteristics and functions in nitrogen metabolic networks.
De Raad et al. Journal of Biological Chemistry, DOI: https://doi.org/10.1016/j.jbc.2023.102939
Overview of oxime-MSI AT assay: A) Oxime tagging to detect AT activity;
B) Steps in the oxime-MSI AT assay
MS image of NIMS surface with
printed AtTAT1 reactions,
The relative reaction rate of AtTAT1 with
Tyr and ɑ-KG

5. Office of Biological and Environmental Research
Perspectives for self-driving labs in synthetic biology
Background
Self-driving labs (SDLs) combine fully
automated experiments with artificial
intelligence (AI) that decides the next set of
experiments.
Approach
We review discusses challenges and
opportunities in creating SDLs for synthetic
biology.
Results
While SDLs are bound to be costly endeavors, the
expected returns make them worthwhile
undertakings.
Significance/Impacts
A fully functioning network of SDLs would not
only provide significant biological knowledge,
but also fully exploit synthetic biology for
biomanufacturing purposes.
Garcia Martin, H., et al. Biotechnology, https://doi.org/10.1016/j.copbio.2022.102881
Figure 1: SDLs combine automated robotic platforms and data collection
with AI that processes these data to decide the next set of experiments to
perform and, potentially, which hypotheses and theories to test.
bisabolene), transforms them into a host (e.g. a bacteria
Figure 1
Current Opinion in Biotechnology
SDLs combine automated robotic platforms and data collection with AI
that processes these data to decide the next set of experiments to
perform and, potentially, which hypotheses and theories to test.
Figure
2 Systems Biology

6. Office of Biological and Environmental Research
JBEI Enabled Papers

7. Office of Biological and Environmental Research
Greenhouse Gas and Air Pollutant
Emissions from Composting
Background/Objective
• Nearly all bioenergy production processes result in some
quantity of residual organic solid material. Those can be
combusted for heat, land applied, composted, or landfilled
• Uncertainty around the emissions from managing these
residual solids remains a barrier for scaling up bioenergy
Approach
This is a review exploring the greenhouse gas and air pollutant
emissions from composting various organic wastes (untreated and
residual solids remaining after bioenergy production.
Results
Using a biological conversion process (e.g. AD) before
composting residuals can reduce emissions. High aeration rates
minimize methane emissions but can increase ammonia emissions.
Significance/Impacts
The study provides guidelines for estimating emissions from
composting organic wastes and residual solids from bioenergy
facilities and insights into key emissions drivers.
Nordahl, S. L. et al. Environmental Science & Technology. DOI: 10.1021/acs.est.2c05846

8. Office of Biological and Environmental Research
Whole-genome sequencing-based characteristics of
Escherichia coli Rize-53 isolate from Turkey
Background/Objective
Urinary tract infections (UTIs) are one of the most common infectious diseases
identifying genetic determinants of antibiotic resistance in a clinical isolate of UTI-causing Escherichia coli.
Approach
The clinical isolate was obtained from a urine sample of a UTI patient in Turkey and identified via 16S rDNA sequencing
Results
Ten antibiotic resistance genes were identified in the genome of the isolate:
serotype of the isolate were identified as ST2141 and O107/H39, respectively
Significance/Impacts
Genetic determinants of resistance to tetracycline, β-lactam and aminoglycoside antibiotics were identified using
WGS in a uropathogenic E. coli from ST2141 lineage and O107:H39 serotype, isolated in Turkey.
Turumtay (2023) doi: 10.17219/acem/152704

9. Office of Biological and Environmental Research
Crop engineering for human microbiomes
Background/Objective
• Different plant-based foods that people consume can have a significant impact on their gut microbiota
• We advocate for more research into this emerging field.
Approach
We highlight recent studies that investigate the role of microbes on raw plant
foods, as well as molecular composition of different grains, on the human gut.
Results
• Some varieties of sorghum were genetically engineered to positively impact the
human gut microbiome after mock-digestion.
• Microbial communities found on specific raw plants were shown to affect
human metabolism.
Significance/Impacts
In the future, the relationship between human gut microorganisms and plant
biochemistry could play a role in crop breeding and the development of plant-based
foods.
Serrano, K., Bezrutczyk, M. Nat Rev Microbiol https://doi.org/10.1038/s41579-022-00850-6
Figure 1: You are what (plant-associated microbes and secondary
metabolites) you eat

10. Office of Biological and Environmental Research
Photobiological production with
Ca-alginate hydrogels
Background/Objective
• Autotrophs use CO2 and light directly and produce metabolites that can
support the growth of other microbes. Co-culture of such microbes with
engineered production strains can result in useful bioproduction systems.
• However, developing stable multispecies systems is challenging.
Approach
• Encapsulation/ immobilization of strains in calcium alginate hydrogels were
hypothesized to protect individual strains from co-culture stress.
• Mixed culture with the autotrophic sucrose producing Synechococcus
elongatus and encapsulated engineered Yarrowia lipolytica and Pseudomonas
putida that produce b-carotene and indigoidine respectively were used
Results and Significance
• Hydrogel compartmentation led to protective effects on the growth of
engineered P. putida and Y. lipolytica.
• The heterotrophic strains inside the hydrogel produced much higher
concentrations of the colored chemicals than those in free cell co-culture.
• This approach is scalable and provides a route to stable mixed cultures and
improved bioconversion.
Zhao, R., et al. Sci Rep 12, 22163 (2022). https://doi.org/10.1038/s41598-022-26437-y
Improved production of valuable pigments using immobilized production
systems co-cultures with an autotroph.
JBEI P. putida strains engineered to produce Indigoidine provided an ideal
system for this proof-of-concept study conducted at WUSTL