Anaerobic fungi produce a wealth of enzymes that can act on cellulose, hemicellulose, and lignin. These enzymes show promise for accelerating the breakdown of lignocellulose for industrial applications like chemical and fuel production. Molecular dynamics simulations provide insights into how these enzymes may interact with lignocellulose and enable its breakdown. This could allow enzymes from anaerobic fungi to be applied industrially.

1. Office of Biological and Environmental Research
Enzyme Discovery in Anaerobic Fungi (Neocallimastigomycota)
Enables Lignocellulosic Biorefinery Innovation
Background/Objective
• Lignocellulose is a highly complex
biopolymer that is difficult to hydrolyze.
• Coordinated action of several diverse
enzymes are required to act on lignocellulose.
Approach
Recently, anaerobic fungi have emerged as
attractive hosts for diverse carbohydrate active
enzymes (CAZYmes) as well as enzyme complexes.
Results
Anaerobic fungi produce a wealth of CAZymes that
act on cellulose, hemicellulose, and even lignin. Many
enzymes remain to be characterized from these fungi.
Significance/Impacts
Enzymes sourced from anaerobic fungi can be
translated into industrial use to accelerate
lignocellulose breakdown and value-added chemical
and fuel production.
Lankiewicz T.S., Lillington S.P., O’Malley M.A. (2022) “Enzyme Discovery in Anaerobic Fungi (Neocallimastigomycetes) Enables Lignocellulosic Biorefinery Innovation.” Applied and Industrial Microbiology.

2. Office of Biological and Environmental Research
Background/Objective
• The carbohydrate D-glucose is the monomer of cellulose, the most abundant biopolymer
found in nature and a major constituent of wood
• Numerous solvents have been explored to study the solubility of glucose, however, the
solubility of glucose is lower in organic solvents
Approach
• The present study aims to enhance the solubility of glucose in imidazolium-based ILs by
adding DMSO as a cosolvent.
• Molecular dynamics (MD) simulations was performed to measure the structural and
dynamic properties of glucose.
Results
• There is a strong correlation between glucose solubility and the MD simulated interaction
energies, and the ranking of glucose solubility is following order: [Emim][MeSO3] >
[Emim][EtSO4] > [Emim][SCN].
• From the MD simulations, it was concluded that anion and DMSO play an important role in
the solubility of glucose than cation.
Significance/Impacts
• [Emim][MeSO3] shows stronger interaction energies and larger contact probability with
glucose than [Emim][EtSO4] and [Emim][SCN], thereby increasing the solubility of glucose
• Adding DMSO to IL (2:1), DMSO forms additional new hydrogen bonds with glucose
without much affecting the anion-glucose interactions
Effect of Cosolvent on the Solubility of Glucose in Ionic Liquids:
Experimental and Molecular Dynamics Simulations
Figure 2. Correlation between experimental glucose solubility
and MD predicted interaction energies between glucose/IL
and glucose-IL/DMSO (1:2 mole ratio) systems.
[Emim][MeSO3] [Emim][EtSO4] [Emim][SCN]
0.08
0.10
0.12
0.14
0.16
0.18
0.20
0.22
0.24
0.26
0.28
Glucose in IL/DMSO
Glucose in Pure IL
MD Sim I.E. in IL/DMSO
MD Sim I.E. in Pure IL
Ionic Liquid
Experimental
solubility
(x
glucose
)
-375
-350
-325
-300
-275
-250
MD
simulated
I.E.
(kJ
mole
-1
)
Figure 1. Correlation between experimental glucose solubility
and MD predicted interaction energies between glucose/IL
and glucose-IL/DMSO (1:2 mole ratio) systems.
Mohan et al. Fluid Phase Equilibria 562 (2022) 113559; doi: 10.1016/j.fluid.2022.113559

3. Office of Biological and Environmental Research
Revisiting theoretical tools and approaches for the valorization of
recalcitrant lignocellulosic biomass to value-added chemicals
Background/Objective
Theoretical frameworks and associated computational
methods play a vital role in developing efficient
conversion of biomass to biofuels and chemicals.
Approach
Surveyed the latest developments in the use of
computational chemistry and biology in the various stages
of producing biofuels and chemicals from lignocellulosic
biomass.
Results
Physics-based modeling approaches have been most
impactful in understanding solvent-biomass interactions
and the structure and function of enzymes, while machine
learning approaches have made important contributions in
synthetic biology and metabolic pathway optimization.
Significance/Impacts
Provides a compendium of computational tools applied to
all stages of biomass valorization.
Pham, L.T.M., Choudhary, H., Gauttam, R., Singer, S.W., Gladden, J.M., Simmons, B.A., Singh, S., Sale, K.L., (2022). Revisiting Theoretical Tools and Approaches
for the Valorization of Recalcitrant Lignocellulosic Biomass to Value-Added Chemicals. Frontiers in Energy Research. 10. doi:10.3389/fenrg.2022.863153.
Computational strategies used in process optimization for lignocellulose
biomass deconstruction and upgrading to valuable fuels and chemicals.

4. Office of Biological and Environmental Research
Comparing in planta accumulation with microbial routes to set targets
for a cost-competitive bioeconomy
Background/Objective
• Plants and microbes can make some of the same bioproducts, yet there is no
systematic way to determine which route is preferable.
• This study aimed to establish break-even yield and accumulation rates.
Approach
The study uses technoeconomic analysis to compare in planta and microbial
production routes for catechol, muconic acid, 4-HBA, and PDC in biorefineries.
Results
Mass accumulation ranging from 0.1 to 0.3 dry weight % can achieve costs
comparable to microbial routes operating at 40 to 55% of maximum theoretical
yields from glucose.
Significance/Impacts
The study provides generalizable insights into breakeven points between plants
and microbes, assuming residual plant biomass can be converted to biofuel.
Both plants and microbes have an important role in producing bioproducts.
Yang, M., Liu, D., Baral, N.R., Lin, C.Y., Simmons, B.A., Gladden, J.M., Eudes, A. and Scown, C.D., 2022. Comparing in planta accumulation
with microbial routes to set targets for a cost-competitive bioeconomy. Proceedings of the National Academy of Sciences, 119(30), p.e2122309119.

5. Office of Biological and Environmental Research
Circularity in Mixed Plastics Chemical Recycling Enabled
by Variable Rates of Polydiketoenamine Hydrolysis
Background/Objective
Products, such as footwear, carpet, automotive interiors, and multi-layer packaging
that are manufactured from plastic blends or laminates are extremely difficult to
recycle due to their different chemistries and various separation-purification
methods.
Approach
Chemical circularity in mixed-polymer recycling becomes possible by controlling
the rates of depolymerization of circular plastics (called polydiketoenamines or
PDKs) through molecular design.
Results
Stepwise deconstruction of mixed-PDK (blend or laminate) or multi-material
PDKs (including glass or metal) is chemospecific, allowing a prescribed sub-set of
monomers, fillers, and additives to be recovered in pristine condition at each stage
of the recycling process.
Significance/Impacts
Circularity in mixed PDK plastics has the potential to pair sustainable
manufacturing with chemical recycling, a top priority of our current society.
J. Demarteau, A. R. Epstein, P. R. Christensen, M. Abubekerov, H. Wang, S. J. Teat, T. J. Seguin, C. W. Chan, C. D. Scown,
T. P. Russell, J. D. Keasling, K. A. Persson, B. A. Helms, Circularity in mixed-plastic chemical recycling enabled by variable
rates of polydiketoenamine hydrolysis. Sci. Adv. 8, eabp8823 (2022).

6. Office of Biological and Environmental Research
Use of Chemical Catalysis to Help Prioritize Biological Targets
Background/Objective
• Selection of biological targets needs to consider their
potential further chemical conversion to desirable products
• Evaluating two scenarios
- Diversification of products from limonene
- Discovering target monomers for flame retardant nylon
Approach
• Develop “star” diagram for limonene with further experimental work on
the epoxidation of limonene
• Grafted different nitrogen-containing moieties onto the muconic acid
derivative 3-hexenedioc acid
Results
• Found improved catalytic system for limonene epoxidation
• All of the grafted aromatics gave improved flame retardance properties
with 2-mercaptopyrazine being particularly effective
Significance/Impacts
• Limonene would be an attractive biological target as it can be readily
converted to both fuel molecules and a range of valuable products.
• Muconic acid is an excellent target for synthesizing flame retardant
polymers, which was a key polymer target from our Product Council.
H-H Lin, Y. Cheng, J. Huo, B.H. Shanks. Selective Ammonolysis of Bioderived Esters for Biobased Amide Synthesis

7. Office of Biological and Environmental Research
JBEI Enabled Publications

8. Office of Biological and Environmental Research
Sirirungruang S., Ad O., Privalsky T. M., Ramesh S., Sax J. L., Dong H., Baidoo E., Amer B., Khosla C., Chang M. (2022) “Engineering
site-selective incorporation of fluorine into polyketides.” Nature chemical biology. doi: 10.1038/s41589-022-01070-y
Background/Objective
• To expand the molecular diversity available for drug discovery by blending
the effective attributes of synthetic and natural molecules
Approach
• Creating a method to site-selectively incorporate fluorine into complex
structures to produce regioselectively fluorinated full-length polyketides
Results
• Engineered a fluorine-selective trans-acyltransferase to produce site-
selectively fluorinated erythromycin precursors in vitro
Significance/Impacts
• Demonstrated that these analogs could be produced in vivo in Escherichia
coli on engineering of the fluorinated extender unit pool
• Using engineered microbes, elaborate fluorinated compounds can be
produced by fermentation, offering the potential for expanding the
identification and development of bioactive fluorinated small molecules
Engineering site-selective incorporation of fluorine into polyketides
Figure: By merging the ability of living systems to produce
complex molecular architecture with design elements from
synthetic chemistry we can expand the accessible scope of
molecular space for discovery of new function.

9. Office of Biological and Environmental Research
Enhancing the interaction between cellulose and dilute aqueous ionic
liquid solutions (ILs) and its implication to ionic liquid recycling and reuse
Background/Objective
• Cellulose solubility in the ILs is strongly inhibited by water, which has
negative impacts on IL pretreatment and reuse of the recycled ILs
• To develop an effective biomass pretreatment using dilute aqueous IL
solutions under mild pretreatment conditions
Approach
A distillation and aeration apparatus was used as the reactor for biomass
pretreatment in dilute aqueous IL solutions and in recycled IL liquor without drying
or purification.. The biomass samples were subjected to a dynamic IL environment
during pretreatment as water was constantly removed from the reactor.
Results
Enhanced interactions between biomass and the aqueous IL solutions were achieved,
indicated by the transformation of cellulose I crystalline structure to amorphous
cellulose or cellulose II, which were easily digestible by enzymes.
Significance/Impacts
An efficient biomass pretreatment process using dilute aqueous IL solutions was
demonstrated on a laboratory scale. It provided a basis for further development of IL
recycle and reuse in biomass pretreatment
Jinxu Zhang, Dongzhe Zou , Siyu Zhai , Yin Yan , Hua Yang , Chunyong He , Yubin Ke , Seema Singh , Gang
Cheng* Carbohydrate Polymers 277 (2022) 118848
Figure 2. Enzymatic hydrolysis data of pretreated
samples using dilute IL solutions.
Figure 1. A distillation and aeration apparatus
was used as the reactor for biomass pretreatment.

10. Office of Biological and Environmental Research
MACAW: An Accessible Tool for Molecular Embedding
and Inverse Molecular Design
Background/Objective
• Synthetic biologists and organic chemists are continuously expanding
the universe of synthesizable small molecules.
• Identifying molecules useful for an application amongst the myriad
that could be synthesized is a lengthy and costly process.
Approach
• We present MACAW, a tool to recommend molecules that fit a
desired specification (inverse molecular design).
Results
• We demonstrate this capability by recommending molecules with
predicted octane numbers of 40, 80, and 120, and by identifying
molecules with high predicted binding affinity to the histamine H1
receptor and limited affinity to the muscarinic M2 receptor.
Significance/Impacts
• MACAW augments classical retrosynthesis tools by providing
recommendations for molecules on specification on top of the pathways
to synthesize them.
Blay et al. . Chem. Inf. Model. 2022, 62, 15, 3551–3564