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Systematic engineering of branch chain amino acid supply modules for the enhanced production of bacitracin from Bacillus licheniformis.

ABSTRACT: Bacitracin is a broad-spectrum cyclic peptide antibiotic mainly produced by Bacillus, precursor amino acid supply served as the critical role during its synthesis. In this study, we systematically engineered branch-chain amino acid (BCAA) supply modules for bacitracin production. Firstly, we demonstrated that Ile and Leu acted as limiting precursors for bacitracin synthesis, and that BCAA synthetic pathways were strengthened via simultaneous overexpression of, feedback-resistance acetolactate synthase IlvBNfbr, 2-isopropylmalate synthetase LeuAfbr and BCAA aminotransferase YbgE. Using this approach, bacitracin yield from strain DW-BCAA2 was 892.54 U/mL, an increase of 18.32% compared with that DW2 (754.32 U/mL). Secondly, the BCAA permeases, YvbW and BraB, which have higher affinities for Leu and Ile transportation, respectively, were both identified as BCAA importers, with their overexpression improving intracellular BCAA accumulations and bacitracin yields. Finally, the leucine-responsive family regulator, lrpC was deleted to generate the final strain DW-BCAA6, with intracellular concentrations of Ile, Leu and Val increased by 2.26-, 1.90- and 0.72-fold, respectively. The bacitracin yield from DW-BCAA6 was 1029.83 U/mL, an increase of 36.52%, and is the highest bacitracin yield reported. Equally, concentrations of other byproducts including acetic acid, acetoin and 2,3-butanediol were all reduced. Taken together, we devised an efficient strategy for the enhanced production of bacitracin, and a promising B. licheniformis DW-BCAA6 strain was constructed for industrial production of bacitracin.

SUBMITTER: Cai D

PROVIDER: S-EPMC7326738 | biostudies-literature | 2020 Dec

REPOSITORIES: biostudies-literature

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Systematic engineering of branch chain amino acid supply modules for the enhanced production of bacitracin from <i>Bacillus licheniformis</i>.

Cai Dongbo D Zhu Jiang J Li Yang Y Li Lingfeng L Zhang Meng M Wang Zhi Z Yang Hanbo H Li Junhui J Yang Zhifan Z Chen Shouwen S

Metabolic engineering communications 20200611

Bacitracin is a broad-spectrum cyclic peptide antibiotic mainly produced by <i>Bacillus</i>, precursor amino acid supply served as the critical role during its synthesis. In this study, we systematically engineered branch-chain amino acid (BCAA) supply modules for bacitracin production. Firstly, we demonstrated that Ile and Leu acted as limiting precursors for bacitracin synthesis, and that BCAA synthetic pathways were strengthened via simultaneous overexpression of, feedback-resistance acetolac ...[more]

PMID: 32637317

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Project description:The cyclodipeptide pulcherriminic acid, produced by Bacillus licheniformis, is derived from cyclo(l-Leu-l-Leu) and possesses excellent antibacterial activities. In this study, we achieved the high-level production of pulcherriminic acid via multistep metabolic engineering of B. licheniformis DWc9n*. First, we increased leucine (Leu) supply by overexpressing the ilvBHC-leuABCD operon and ilvD, involved in Leu biosynthesis, to obtain strain W1, and the engineered strain W2 was further attained by the deletion of gene bkdAB, encoding a branched-chain α-keto acid dehydrogenase in W1. As a result, the intracellular Leu content and pulcherriminic acid yield of W2 reached 147.4 mg/g DCW (dry cell weight) and 189.9 mg/liter, which were 227.6% and 48.9% higher than those of DWc9n*, respectively. Second, strain W3 was constructed through overexpressing the leucyl-tRNA synthase gene leuS in W2, and it produced 367.7 mg/liter pulcherriminic acid. Third, the original promoter of the pulcherriminic acid synthetase cluster yvmC-cypX in W3 was replaced with a proven strong promoter, PbacA, to produce the strain W4, and its pulcherriminic acid yield was increased to 507.4 mg/liter. Finally, pulcherriminic acid secretion was strengthened via overexpressing the transporter gene yvmA in W4, resulting in the W4/pHY-yvmA strain, which yielded 556.1 mg/liter pulcherriminic acid, increased by 337.8% compared to DWc9n*, which is currently the highest pulcherriminic acid yield to the best of our knowledge. Taken together, we provided an efficient strategy for enhancing pulcherriminic acid production, which could apply to the high-level production of other cyclodipeptides.IMPORTANCE Pulcherriminic acid is a cyclodipeptide derived from cyclo(l-Leu-l-Leu), which shares the same iron chelation group with hydroxamate sidephores. Generally, pulcherriminic acid-producing strains could be the perfect candidates for antibacterial and anti-plant-pathogenic fungal agents. In this study, we obtained the promising W4/pHY-yvmA pulcherriminic acid-producing strain via a multistep metabolic modification. The engineered W4/pHY-yvmA strain is able to achieve 556.1 mg/liter pulcherriminic acid production, which is the highest yield so far to the best of our knowledge.

Project description:Bacterial resistance against antibiotics often involves multiple mechanisms that are interconnected to ensure robust protection. So far, the knowledge about underlying regulatory features of those resistance networks is sparse, since they can hardly be determined by experimentation alone. Here, we present the first computational approach to elucidate the interplay between multiple resistance modules against a single antibiotic and how regulatory network structure allows the cell to respond to and compensate for perturbations of resistance. Based on the response of Bacillus subtilis toward the cell wall synthesis-inhibiting antibiotic bacitracin, we developed a mathematical model that comprehensively describes the protective effect of two well-studied resistance modules (BceAB and BcrC) on the progression of the lipid II cycle. By integrating experimental measurements of expression levels, the model accurately predicts the efficacy of bacitracin against the B. subtilis wild type as well as mutant strains lacking one or both of the resistance modules. Our study reveals that bacitracin-induced changes in the properties of the lipid II cycle itself control the interplay between the two resistance modules. In particular, variations in the concentrations of UPP, the lipid II cycle intermediate that is targeted by bacitracin, connect the effect of the BceAB transporter and the homeostatic response via BcrC to an overall resistance response. We propose that monitoring changes in pathway properties caused by a stressor allows the cell to fine-tune deployment of multiple resistance systems and may serve as a cost-beneficial strategy to control the overall response toward this stressor.IMPORTANCE Antibiotic resistance poses a major threat to global health, and systematic studies to understand the underlying resistance mechanisms are urgently needed. Although significant progress has been made in deciphering the mechanistic basis of individual resistance determinants, many bacterial species rely on the induction of a whole battery of resistance modules, and the complex regulatory networks controlling these modules in response to antibiotic stress are often poorly understood. In this work we combined experiments and theoretical modeling to decipher the resistance network of Bacillus subtilis against bacitracin, which inhibits cell wall biosynthesis in Gram-positive bacteria. We found a high level of cross-regulation between the two major resistance modules in response to bacitracin stress and quantified their effects on bacterial resistance. To rationalize our experimental data, we expanded a previously established computational model for the lipid II cycle through incorporating the quantitative action of the resistance modules. This led us to a systems-level description of the bacitracin stress response network that captures the complex interplay between resistance modules and the essential lipid II cycle of cell wall biosynthesis and accurately predicts the minimal inhibitory bacitracin concentration in all the studied mutants. With this, our study highlights how bacterial resistance emerges from an interlaced network of redundant homeostasis and stress response modules.

Project description:BACKGROUND:Bacillus licheniformis 2709 is extensively applied as a host for the high-level production of heterologous proteins, but Bacillus cells often possess unfavorable wild-type properties, such as production of viscous materials and foam during fermentation, which seriously influenced the application in industrial fermentation. How to develop it from a soil bacterium to a super-secreting cell factory harboring less undomesticated properties always plays vital role in industrial production. Besides, the optimal expression pattern of the inducible enzymes like alkaline protease has not been optimized by comparing the transcriptional efficiency of different plasmids and genomic integration sites in B. licheniformis. RESULT:Bacillus licheniformis 2709 was genetically modified by disrupting the native lchAC genes related to foaming and the eps cluster encoding the extracellular mucopolysaccharide via a markerless genome-editing method. We further optimized the expression of the alkaline protease gene (aprE) by screening the most efficient expression system among different modular plasmids and genomic loci. The results indicated that genomic expression of aprE was superior to plasmid expression and finally the transcriptional level of aprE greatly increased 1.67-fold through host optimization and chromosomal integration in the vicinity of the origin of replication, while the enzyme activity significantly improved 62.19% compared with the wild-type alkaline protease-producing strain B. licheniformis. CONCLUSION:We successfully engineered an AprE high-yielding strain free of undesirable properties and its fermentation traits could be applied to bulk-production by host genetic modification and expression optimization. In summary, host optimization is an enabling technology for improving enzyme production by eliminating the harmful traits of the host and optimizing expression patterns. We believe that these strategies can be applied to improve heterologous protein expression in other Bacillus species.

Dataset Information

Systematic engineering of branch chain amino acid supply modules for the enhanced production of bacitracin from Bacillus licheniformis.

Publications

Systematic engineering of branch chain amino acid supply modules for the enhanced production of bacitracin from <i>Bacillus licheniformis</i>.

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