Analysis of heterologous expression of phaCBA promotes the acetoin stress response mechanism in Bacillus subtilis using transcriptomics and metabolomics approaches

Abstract

Acetoin, a versatile platform chemical and popular food additive, poses a challenge to the biosafety strain Bacillus subtilis when produced in high concentrations due to its intrinsic toxicity. Incorporating the PHB synthesis pathway into Bacillus subtilis 168 has been shown to significantly enhance the strain's acetoin tolerance. This study aims to elucidate the molecular mechanisms underlying the response of B. subtilis 168-phaCBA to acetoin stress, employing transcriptomic and metabolomic analyses. Acetoin stress induces fatty acid degradation and disrupts amino acid synthesis. In response, B. subtilis 168-phaCBA down-regulates genes associated with flagellum assembly and bacterial chemotaxis, while up-regulating genes related to the ABC transport system encoding amino acid transport proteins. Notably, genes coding for cysteine and D-methionine transport proteins (tcyB, tcyC and metQ) and the biotin transporter protein bioY, are up-regulated, enhancing cellular tolerance. Our findings highlight that the expression of phaCBA significantly increases the ratio of long-chain unsaturated fatty acids and modulates intracellular concentrations of amino acids, including L-tryptophan, L-tyrosine, L-leucine, L-threonine, L-methionine, L-glutamic acid, L-proline, D-phenylalanine, L-arginine, and membrane fatty acids, thereby imparting acetoin tolerance. Furthermore, the supplementation with specific exogenous amino acids (L-alanine, L-proline, L-cysteine, L-arginine, L-glutamic acid, and L-isoleucine) alleviates acetoin's detrimental effects on the bacterium. Simultaneously, the introduction of phaCBA into the acetoin-producing strain BS03 addressed the issue of insufficient intracellular cofactors in the fermentation strain, resulting in the successful production of 70.14 g/L of acetoin through fed-batch fermentation. This study enhances our understanding of Bacillus's cellular response to acetoin-induced stress and provides valuable insights for the development of acetoin-resistant Bacillus strains.

Keywords: Acetoin; Metabolomic; Polyhydroxybutyrate (PHB); Stress tolerance; Transcriptomic.

Fig. 1

Effects of different acetoin treatments (0 g/L, 40 g/L, 60 g/L, 80 g/L) on the growth of B. subtilis 168-pMA5 (a) and B. subtilis 168-phaCBA (b)

Fig. 2

Effect of different concentrations of acetoin stress on cell growth and glucose consumption of B. subtilis 168-pMA5 (a, b) and B. subtilis 168-phaCBA (c, d)

Fig. 3

Differentially expressed genes (DEGs) GO classification analysis and KEGG classification analysis of B. subtilis 168-pMA5 and B. subtilis 168-phaCBA under acetoin stress. a Identification of enriched DEGs by KEGG database in the 60 g/L acetoin treatment group (B. subtilis 168-pMA5) and 0 g/L acetoin control group (B. subtilis 168-pMA5). b KEGG enriched pathways of DEGs after acetoin stress between B. subtilis 168-pMA5 and B. subtilis 168-phaCBA. c GO analysis of DEGs in the 60 g/L acetoin treatment group (B. subtilis 168-pMA5) and 0 g/L acetoin control group (B. subtilis 168-pMA5). d GO analysis of DEGs after acetoin stress between B. subtilis 168-pMA5 and B. subtilis 168-phaCBA

Fig. 4

Metabolic pathway analysis of significantly differential metabolites and cluster analysis of significantly differential metabolite contents after acetoin stress treatment. a Metabolic pathway analysis of significantly differential metabolites in the 60 g/L acetoin treatment group (B. subtilis 168-pMA5) and 0 g/L acetoin control group (B. subtilis 168-pMA5). b Metabolic pathway analysis of significantly differential metabolites in acetoin stress between B. subtilis 168-pMA5 and B. subtilis 168-phaCBA. c Heatmap obtained by hierarchical clustering analysis of differential metabolites between B. subtilis 168-pMA5 (0 g/L, 60 g/L acetoin stress) and B. subtilis 168-phaCBA (60 g/L acetoin stress)

Fig. 5

a Effect of exogenous addition of different amino acids (l-histidine, l-alanine, l-proline, l-valine, l-arginine, l-cysteine, l-leucine, l-glutamic acid, l-isoleucine, l-methionine) on the growth of B. subtilis 168 under acetoin stress. b Differential expression results for metabolites and related transcripts. Horizontal coordinates are associated metabolites, vertical coordinates are transcript names, red indicates positive correlation, and blue indicates negative correlation

Fig. 6

a Fermentation experiments with recombinant strains BS03 and BS03-phaCBA of acetoin **significance code: P < 0.01. b, c Effects of heterologous expression phaCBA on intracellular NADH and NAD⁺ concentrations. d Overview of the production of acetoin from glucose by B. subtilis engineered bacteria. Blue words represent genes expressed on plasmid pMA5. Green words represent genes integratively expressed. Purple words represent knockout genes

Fig. 7

Batch fermentation of recombinant strain BS03-phaCBA in a 7.5 L bioreactor for the production of acetoin