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

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 tricarboxylic acid (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.IMPORTANCEThe robustness of B. coagulans makes it a valuable microorganism for biotechnology applications; yet, this phenotype is not well understood at the cellular level. Through phenotypic characterization and systems analysis, this study elucidates the functional genomics and robustness of a novel, undomesticated strain, B. coagulans B-768, capable of utilizing inhibitory switchgrass biomass hydrolysates. The genome of B-768, enriched with carbohydrate metabolism genes, demonstrates high regulatory capacity. The coordination of proteome reallocation in Carbohydrate Transport and Metabolism (COG-G), Translation (COG-J), and Energy Conversion and Production (COG-C) is critical for effective cell growth, sugar utilization, and lactate production via overflow metabolism. Overall, B-768 is a novel, robust, and promising B. coagulans strain that can be harnessed as a microbial biomanufacturing platform to produce chemicals and fuels from biomass hydrolysates.

Keywords: B-768; Bacillus coagulans; biomass hydrolysates; carbohydrate metabolism; lactate overproducer; overflow metabolism; proteome reallocation; robustness; switchgrass; thermophile.

Fig 1

Physiological characterization of B. coagulans B-768 reveals cellular robustness and efficient utilization of various carbon sources at various temperatures. (A) Dependence of specific growth rates of B-768 on temperatures when grown on glucose, xylose, or cellobiose. The dotted lines indicate the temperature at which growth was optimal. (B) Carrying capacity and maximum specific growth rate of B-768 growing on various C5, C6, and C12 fermentable sugars at 50°C. (C) Effect of lactate concentrations on maximum specific growth rates of B-768 growing on glucose at 50°C. (D) Kinetic profiles of cell growth, glucose consumption, and lactate production in a pH-controlled bioreactor (pH = 6.5) at 50°C. Data points represent 1 mean ± 1 standard deviation of three biological replicates.

Fig 2

Circular genome map of Bacillus coagulans B-768. Tracks from outside to in are as follows: PHASTEST 3.0 prophage regions, non-coding RNAs, forward and reverse strand CDS colored by COG category, GC Skew = (G – C)/(G + C), and GC content. Abbreviations: tRNA, transfer RNA; rRNA, ribosomal RNA; ncRNA, non-coding RNA; tmRNA, transfer-messenger RNA.

Fig 3

Comparative and functional genomics of B. coagulans B-768. (A) Core genome alignment of B. coagulans pan-genome (n = 55). (B) Percent change of COG categories between strains B-768 and 36D1. Numbers above the bars indicate the excess number of proteins in B-768 relative to 36D1. Full COG Category names are listed at the end of the figure caption. (C–E) Breakdown of most abundant proteins in (C) Carbohydrate Metabolism and Transport, (D) Transcription, and (E) Mobilome COG categories. (F) Predicted CRISPR-Cas operons of B-768. COG Categories: U = Intracellular Trafficking, Secretion, and Vesicular Transport; X = Mobilome; G = Carbohydrate Transport and Metabolism; Q = Secondary Metabolite Biosynthesis, Transport and Catabolism; K = Transcription; I = Lipid Transport and Metabolism; M = Cell Wall/Membrane/Envelope Biogenesis; D = Cell Cycle Control, Cell Division, Chromosome Partitioning; H = Coenzyme Transport and Metabolism; C = Energy Production and Conversion.

Fig 4

Central carbon metabolism of B-768. Circles from left to right represent the differential expression of a given protein in (i) xylose vs glucose, (ii) xylose and glucose mixture vs glucose, and (iii) switchgrass hydrolysate vs glucose conditions. Red = upregulated with log₂-fold change ≥1 and q < 0.05; blue = downregulated with log₂-fold change ≤ −1 and q < 0.05; Gray = not significant. Paralogs of the same enzyme were plotted as half circles with the most abundant paralog on top.

Fig 5

B. coagulans B-768 exhibited distinct proteomes for growing on glucose and xylose. (A–B) Kinetic profiles of (A) cell growth and (B) glucose/xylose consumption and lactate production. Proteomics samples were collected from parallel cultures at the time points indicated by black arrows. (C) Principal component analysis of B. coagulans B-768 proteomes for growth on glucose and xylose. (D) Number of differentially expressed proteins between glucose and xylose conditions for most perturbed COG categories. (E) Mass fraction of proteomes in top COG categories (f_COG) for glucose and xylose conditions. (F–K) Proteome allocation by pathway for glucose and xylose conditions. (L) Percent change in amino acid allocation of proteomes between glucose and xylose conditions. COG Categories: G = Carbohydrate Transport and Metabolism; C = Energy Production and Conversion; I = Lipid Transport and Metabolism; E = Amino Acid Transport and Metabolism; F = Nucleotide Transport and Metabolism; NA = No COG annotation; J = Translation; M = Cell Wall/Membrane/Envelope Biogenesis; S = Function Unknown; T = Signal Transduction Mechanisms; H = Coenzyme Transport and Metabolism; L = Replication, Recombination, and Repair; R = General Function Prediction Only; V = Defense Mechanisms; K = Transcription; O = Post-translation Modification, Protein Turnover, Chaperones; P = Inorganic Ion Transport and Metabolism; D = Cell Cycle Control, Cell Division, Chromosome Partitioning. Statistics for F–K were performed using an unpaired t-test with default settings in GraphPad Prism v10.2.3. Error bars represent the standard deviation of three biological replicates. ****P-value <0.0001; ***P-value <0.001; **P-value <0.01.

Fig 6

Growth of B-768 in refined sugars and raw hydrolysate reveals mechanisms of cellular robustness and carbon catabolite repression. (A) Growth profiles of B-768 grown on glucose + xylose (G + X) mixture and switchgrass hydrolysate (SGH). (B) Glucose and xylose consumption and lactate production profiles of B-768 grown on G + X and SGH. (C) Principal component analysis plot of B-768 proteomes grown on different sugars. (D) Schematic of CCR in Bacillus species. (E) Levels of CCR proteins in B-768 proteomes grown on different sugars. (F) Levels of xyl proteins in B-768 proteomes grown on different sugars. (G) Volcano plot of differentially expressed proteins between SGH and G + X conditions. (H) Number of significantly perturbed proteins by COG category. COG Categories: G = Carbohydrate Transport and Metabolism; E = Amino Acid Transport and Metabolism; T = Signal Transduction Mechanisms; C = Energy Production and Conversion; H = Coenzyme Transport and Metabolism; NA = No COG annotation; K = Transcription; J = Translation; I = Lipid Transport and Metabolism; P = Inorganic Ion Transport and Metabolism; S = Function Unknown; M = Cell Wall/Membrane/Envelope Biogenesis; R = General Function Prediction Only.