Expanded genome and proteome reallocation in a novel, robust Bacillus coagulans capable of utilizing pentose and hexose sugars

Authors: DOLLEY, DAVID - University Of Tennessee; RYU, SENUGHYUN - University Of Tennessee; GIANNONE, RICH - Oak Ridge National Laboratory; Edwards, Jackson; Dien, Bruce; Slininger, Patricia - Pat; TRINH, CONG - University Of Tennessee

Submitted to: mSystems
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/6/2024
Publication Date: 10/8/2024
Citation: Dolley, D., Ryu, S., Giannone, R.J., Edwards, J., Dien, B.S., Slininger, P.J., Trinh, C.T. 2024. Expanded genome and proteome reallocation in a novel, robust Bacillus coagulans capable of utilizing pentose and hexose sugars. mSystems. https://doi.org/10.1128/msystems.00952-24.
DOI: https://doi.org/10.1128/msystems.00952-24

Interpretive Summary: Development of a national industry based upon processing of agricultural and forestry residues into bio-based fuels and chemicals is a national priority. There is the potential to generate over one billion tons per year, which can significantly abate greenhouse gas emissions and be a boon for the rural economy. Achieving this dream requires domesticating hardy microbes that can be used by industry. We have identified a bacterium (named Bacillus coagulans) that has outstanding traits for industrial use: it is hardy, has a very high product yield on cellulosic sugars, it grows at 50 degrees C, requires low-cost nutrients, and grows very fast. The high temperature growth of this bacterium allows for reduced process water demand, and it impedes growth of spoilage microbes. The underlying traits that allow the microbe to be so hardy and high yielding have been determined by sequencing its genes (e.g., genome) and proteins. We also provide information that will be essential for future efforts to engineering the strain for improved properties or to make other fermentation products. While more work is required before this technology is ready to be transferred to industry, the knowledge reported in this manuscript will be of interest to the biotechnology and agricultural processing industries looking to exploit cellulosic feedstocks.

Technical Abstract: Bacillus coagulans, a Gram-positive thermophilic bacterium, is recognized for its probiotic properties and recent development as a microbial cell factory. Despite its importance for biotechnological applications, the current understanding of B. coagulans’ robustness is limited, especially for undomesticated strains. To fill this knowledge gap, we characterized the metabolic capability and performed functional genomics and systems analysis of a novel, robust strain, B. coagulans B-768. Genome sequencing revealed that B-768 has the largest B. coagulans genome known to date (3.94 Mbp), about 0.63 Mbp larger than the average genome of sequenced B. coagulans strains, with expanded carbohydrate metabolism and mobilome. Functional genomics identified a well-equipped genetic portfolio for utilizing a wide range of C5 (xylose, arabinose), C6 (glucose, mannose, galactose), and C12 (cellobiose) sugars present in biomass hydrolysates, which was validated experimentally. For growth on individual xylose and glucose, the dominant sugars in biomass hydrolysates, B-768 exhibited distinct phenotypes and proteome profiles. Faster growth and glucose uptake rates resulted in lactate overflow metabolism, which makes B. coagulans a lactate overproducer; however, slower growth and xylose uptake diminished overflow metabolism due to the high energy demand for sugar assimilation. Carbohydrate Transport and Metabolism (COG-G), Translation (COG-J), and Energy Conversion and Production (COG-C) made up 60–65% of the measured proteomes but were allocated differently when growing on xylose and glucose. The trade-off in proteome reallocation, with high investment in COG-C over COG-G, explains the xylose growth phenotype with significant upregulation of xylose metabolism, pyruvate metabolism, and TCA cycle. Strain B-768 tolerates and effectively utilizes inhibitory biomass hydrolysates containing mixed sugars and exhibits hierarchical sugar utilization with glucose as the preferential substrate.