Genomic Science Program
U.S. Department of Energy | Office of Science | Biological and Environmental Research Program

Exploring the Metabolic Capability of Undomesticated Thermophilic Bacillus coagulans for Biosynthesis of Designer Esters at Elevated Temperatures

Authors:

Cong T. Trinh1* (ctrinh@utk.edu), Seunghyun Ryu1, David Dooley1, Khanh Ha1, Jackson Edwards2, Richard J. Giannone3, Bruce S. Dien2

Institutions:

1University of Tennessee; 2National Center for Agricultural Utilization Research, USDA-ARS; 3Oak Ridge National Laboratory

Goals

To fundamentally understand and redirect metabolism and regulation of thermophilic Bacillus coagulans for the efficient conversion of undetoxified lignocellulosic biomass hydrolysates into designer bioesters.

Abstract

Bacillus coagulans (now recognized as Heyndrickxia coagulans), a gram-positive facultative thermophile, thrives across a broad temperature range, utilizes undetoxified biomass hydrolysates, and synthesizes valuable chemicals like acetoin, butanediol, and lactate. This project exploits the robustness of B. coagulans for converting biomass hydrolysates into designer bioesters—such as acetate and lactate esters—widely used in fragrances, flavors, pharmaceuticals, and advanced biofuels. Through comprehensive screening of a library of diverse B. coagulans strains for desirable traits, we identified B. coagulans B-768 as an optimal host for metabolic engineering, given its capability to ferment C5-C12 sugars, withstand undetoxified biomass hydrolysates, and produce significant lactate levels. Genome and proteome analyses highlighted B-768’s expanded genome, coding for enhanced sugar utilization and lactate synthesis. Successful DNA transformation in B-768 has led to the creation of production strains harboring the exchangeable ester production modules to produce acetate and lactate esters at elevated temperatures, facilitated by our engineered thermostable alcohol acetyltransferases. Notably, we uncovered B. coagulans strains capable of producing valerate esters. Current efforts focus on elucidating B. coagulans’ robust metabolism for complex hydrolysate utilization, improving synthetic biology tools (including transformation efficiency, plasmid stability, genome integration, and promoter and ribosome binding site optimization), and enabling modular cell engineering for selective biosynthesis of designer esters. Overall, B. coagulans is a promising microbial manufacturing platform that will be advanced by a fundamental understanding of its robustness, genetic engineering tool development, and the ability to harness it for production of designer bioesters from lignocellulosic hydrolysates.

References

Lee, J.-W., and C. T. Trinh. 2019. “Microbial Biosynthesis of Lactate Esters,” Biotechnology for Biofuels 12, 226. DOI:10.1186/s13068-019-1563-z.

Lee, J.-W., and C. T. Trinh. 2020. “Towards Renewable Flavors, Fragrances, and Beyond,”Current Opinion in Biotechnology 61, 168–80. DOI:10.1016/j.copbio.2019.12.017.

Lee, J.-W., and C. T. Trinh. 2022. “Controlling Selectivity of Modular Microbial Biosynthesis of Butyryl-CoA-Derived Designer Esters,” Metabolic Engineering 69, 262–74. DOI:10.1016/j.ymben.2021.12.001.

Lee, J.-W., et al. 2021. “Probing Specificities of Alcohol Acyltransferases for Designer Ester Biosynthesis with a High‐Throughput Microbial Screening Platform,” Biotechnology and Bioengineering 118(12), 4655–67. DOI:10.1002/bit.27926.

Seo, H., et al. 2021. “Engineering Promiscuity of Chloramphenicol Acetyltransferase for Microbial Designer Ester Biosynthesis,” Metabolic Engineering 66, 179–90. DOI:10.1016/j.ymben.2021.04.005.

Seo, H., et al. 2023. “Engineering a Synthetic Escherichia coli Coculture for Compartmentalized de novo Biosynthesis of Isobutyl Butyrate from Mixed Sugars,” ACS Synthetic Biology 13(1), 259–68. DOI:10.1021/acssynbio.3c00493.

Seo, H., et al. 2023. “Rewiring Metabolism of Clostridium thermocellum for Consolidated Bioprocessing of Lignocellulosic Biomass Poplar to Produce Short-Chain Esters,” Bioresource Technology 129263. DOI:10.1016/j.biortech.2023.129263.

Funding Information

This work is supported by the U.S. DOE, Office of Science, BER program, GSP under Award Number DE-SC0022226.