Search

Bacitracin, a type of cyclic dodecapeptide antibiotic mainly produced by Bacillus , is widely used in fields of veterinary drug and feed additive. Modularization of metabolic pathways based on the concept of synthetic biology has been widely used in the efficient synthesis of target products. Here, we want to improve bacitracin production through strengthening aspartic acid (Asp) supply in B. licheniformis DW2. First, exogenous Asp addition assays implied that strengthening Asp supply benefited bacitracin production. Second, Asp synthetic pathways were strengthened via overexpressing aspartate dehydrogenase AspD and asparaginase AnsB, attaining recombinant strain DW2-ASP2, and bacitracin yield produced by DW2-ASP2 was 862.81 U/mL, increased by 14.05% compared with that of DW2 (756.49 U/mL). Then, to improve precursor oxaloacetate (OAA) accumulation for Asp synthesis, pyruvate carboxylase PycA and carbonic anhydrase EcaA were co-overexpressed in DW2-ASP2, and malic enzyme gene malS was deleted to weak overflow metabolism of tricarboxylic acid, and the attained strain DW2-ASP7 showed further increased bacitracin production from 862.81 to 989.23 U/mL. Subsequently, transporter YveA was identified as an Asp exporter, and bacitracin yield was increased to 1025.26 U/mL via deleting yveA , attaining strain DW2-ASP9. Finally, Asp ammonia-lyase gene aspA was disrupted to weaken Asp degradation, and bacitracin yield of attained strain DW2-ASP10 reached 1059.86 U/mL, increased by 40.10% compared to DW2. Taken together, this research demonstrated that metabolic engineering of Asp metabolic modules is an efficient strategy for enhancing bacitracin production, and these strategies could also be applied in the production of other peptide-related metabolites.

Bacitracin is a broad-spectrum cyclic peptide antibiotic, and mainly produced by Bacillus. Energy metabolism plays as a critical role in high-level production of target metabolites. In this study, Bacillus licheniformis DW2, an industrial strain for bacitracin production, was served as the original strain. First, our results confirmed that elimination of cytochrome bd oxidase branch via deleting gene cydB benefited bacitracin synthesis. Bacitracin titer and ATP content were increased by 10.97% and 22.96%, compared with those of original strain, respectively. Then, strengthening cytochrome aa3 oxidase branch via overexpressing gene qoxA was conducive to bacitracin production. Bacitracin titer and ATP content were increased by 18.97% and 34.00%, respectively. In addition, strengthening ADP synthesis supply is also proven as an effective strategy to promote intracellular ATP accumulation, overexpression of adenosine kinase DcK and adenylate kinase AdK could all improve bacitracin titers, among which, dck overexpression strain showed the better performance, and bacitracin titer was increased by 16.78%. Based on the above individual methods, a method of combining the deletion of gene cydB and overexpression of genes qoxA, dck were used to enhance ATP content of cells to 39.54 nmol/L, increased by 49.32% compared to original strain, and bacitracin titer produced by the final strain DW2-CQD (DW2ΔcydB::qoxA::dck) was 954.25 U/mL, increased by 21.66%. The bacitracin titer produced per cell was 2.11 U/CFU, increased by 11.05%. Collectively, this study demonstrates that improving ATP content was an efficient strategy to improve bacitracin production, and a promising strain B. licheniformis DW2-CQD was attained for industrial production of bacitracin.

Bacitracin is a kind of macrocyclic dodecapeptide that produced by Bacillus, precursor supply served as a critical role in bacitracin production, here, the aim of this study wants to improve bacitracin production by enhancing Lysine (Lys) supply via metabolic engineering of B. licheniformis DW2, an industrial strain for bacitracin production. Firstly, exogenous addition of Lys was proven to be favorable for bacitracin production, and the strain LYS2 was attained through strengthening Lys synthetic pathways via overexpressing diaminopimelate decarboxylase LysA from B. licheniformis and diaminopimelate dehydrogenase DdH from Corynebacterium glutamicum, and the bacitracin produced by LYS2 was increased to 838.53 U/mL by 10.85%, compared with that of DW2 (756.45 U/mL). Secondly, oxaloacetate, the precursor of Lys, was accumulated by overexpressing pyruvate carboxylase PycA in LYS2, and 17.06% increase of bacitracin yield was attained in LYS3 (885.53 U/mL), compared with DW2. Thirdly, lysine decarboxylase gene yaaO was deleted to weaken Lys degradation, and the attained strain LYS4 showed further increased bacitracin production from 885.53 to 923.43 U/mL. Lastly, the transporter LysE was confirmed to act as a Lys exporter; LysP and YvsH were identified as the Lys importers in B. licheniformis DW2, and bacitracin yield was increased to 975.43 U/mL by 28.95% in final strain LYS5 via engineering the Lys transporters. Taken together, this study implied that metabolic engineering of Lys supply modules is an efficient strategy for enhancement production of bacitracin, and provided a promising strain of B. licheniformis for industrial production of bacitracin.

Soybean meal is commonly applied as the raw material in the bio-fermentation industry, and bacitracin is a widely used feed additive in the feed industry. In this study, we investigated the influence of subtilisin enhancement on soybean meal utilization and bacitracin production in Bacillus licheniformis DW2, an industrial strain for bacitracin production. Firstly, blocking sRNA aprA expression benefited bacitracin synthesis, and the bacitracin yield produced by aprA-deficient strain DW2△PaprA reached 931.43 U/mL, 18.92% higher than that of DW2 (783.25 U/mL). The bacitracin yield was reduced by 14.27% in the aprA overexpression strain. Furthermore, our results showed that deficiency of aprA led to a 6.54-fold increase of the aprE transcriptional level and a 1.84-fold increase of subtilisin activity, respectively, which led to the increases of soybean meal utilization rate (28.86%) and precursor amino acid supplies for bacitracin synthesis. Additionally, strengthening the utilization rate of soybean meal also benefited heterologous protein production, and the α-amylase and nattokinase activities were respectively enhanced by 59.81% and 50.53% in aprA-deficient strains. Collectively, this research demonstrated that strengthening subtilisin production could improve the utilization rate of soybean meal and thereby enhance bacitracin and target protein production; also, this strategy would be useful for the improvement of protein/peptide production using soybean meal as the main nitrogen source in the fermentation process.

Primary metabolism plays a key role in the synthesis of secondary metabolite. In this study, the main transcription factors in carbon, nitrogen, and phosphorus metabolisms (CcpA, CcpC, CcpN, CodY, TnrA, GlnR, and PhoP) were engineered to improve bacitracin yield in Bacillus licheniformis DW2, an industrial strain for bacitracin production. First, our results demonstrated that deletions of ccpC and ccpN improved ATP and NADPH supplies, and the bacitracin yields were respectively increased by 14.02% and 16.06% compared with that of DW2, while it was decreased significantly in ccpA deficient strain DW2ΔccpA. Second, excessive branched chain amino acids (BCAAs) were accumulated in codY, tnrA, and glnR deletion strains DW2ΔcodY, DW2ΔtnrA, and DW2ΔglnR, which resulted in the nitrogen catabolite repressions and reductions of bacitracin yields. Moreover, overexpression of these regulators improved intracellular BCAA supplies, and further enhanced bacitracin yields by 14.17%, 12.98%, and 16.20%, respectively. Furthermore, our results confirmed that phosphate addition reduced bacitracin synthesis capability, and bacitracin yield was improved by 15.71% in gene phop deletion strain. On the contrary, overexpression of PhoP led to a 19.40% decrease of bacitracin yield. Finally, a combinatorial engineering of these above metabolic manipulations was applied, and bacitracin yield produced by the final strain DW2-CNCTGP (Simultaneously deleting ccpC, ccpN, phop and overexpressing glnR, codY, and tnrA in DW2) reached 1014.38 U/mL, increased by 35.72% compared to DW2, and this yield was the highest bacitracin yield currently reported. Taken together, this study implied that metabolic engineering of carbon, nitrogen, and phosphorus metabolism regulators is an efficient strategy to enhance bacitracin production, and provided a promising B. licheniformis strain for industrial production of bacitracin.

Bacitracin, a kind of cyclic peptide antibiotic mainly produced by Bacillus, has wide ranges of applications. NADPH generation plays an important role in amino acid synthesis, which might influence precursor amino acid supply for bacitracin production. In this study, we want to improve bacitracin yield by enhancing intracellular precursor amino acids via strengthening NAPDH generation pathways in the bacitracin industrial production strain Bacillus licheniformis DW2. Based on our results, strengthening of NADPH pathway genes (zwf, gnd, ppnk, pntAB, and udhA) could all improve bacitracin yields in DW2, and the glucose-6-phosphate dehydrogenase Zwf overexpression strain DW2::Zwf displayed the best performance, the yield of which (886.43 U/mL) was increased by 12.43% compared to DW2 (788.40 U/mL). Then, the zwf transcriptional level and Zwf activity of DW2::Zwf were increased by 12.24-fold and 1.57-fold; NADPH and NADPH/NADH were enhanced by 61.24% and 90.63%, compared with those of DW2, respectively. Moreover, the concentrations of intracellular precursor amino acids (isoleucine, leucine, cysteine, ornithine, lysine, glutamic acid) were all enhanced obviously for bacitracin production in DW2::Zwf. Collectively, this research constructed a promising B. licheniformis strain for industrial production of bacitracin, more importantly, which revealed that strengthening of NADPH generation is an efficient strategy to improve precursor amino acid supplies for bacitracin production.

Bacitracin is a broad-spectrum polypeptide antibiotic, which is formed by 11 amino acids residues. Precursor amino acids supply might be the limit factor during bacitracin fermentation. First, our results demonstrated that increasing Ile and Leu supplies were regarded as the efficient strategies for the enhanced titer of bacitracin. Then, the amino acid permease YhdG, which was identified as the BCAA permease, was deleted and overexpressed in DW2, respectively. Our results showed that knocking out of permease YhdG could improve bacitracin production remarkablely. The bacitracin titer of the yhdG deficient strain DW2ΔyhdG reached 917.35 U/mL by flask fermentation, increased by 11% compared with that of DW2. In addition, the bacitracin titer was decreased by 25% in the YhdG overexpressed strain. Meanwhile, the intracellular concentrations of BCAA were higher than DW2 during the biosynthesis of bacitracin. The above results suggested that the permease YhdG might act as an exporter for branched chain amino acids in B. licheniformis DW2. Taken together, the increasing intracellular concentrations of branched chain amino acids by deleting amino acid permease YhdG could improve bacitracin titer. This study provided a new strategy for high-level production of bacitracin.

[Display omitted] • Achieving cost-effective bioproduction from starch by engineered B. licheniformis. • Modular metabolic engineering to improve starch utilization in B. licheniformis. • Starch conversion rate was increased by over 22% in multiple products production. • The highest bacitracin titer of 1252.2 U/mL was achieved in batch culture. Starch is an attractive feedstock in biorefinery processes, while the low natural conversion rate of most microorganisms limits its applications. Herein, starch metabolic pathway was systematically investigated using Bacillus licheniformis DW2 as the host organism. Initially, the effects of overexpressing amylolytic enzymes on starch hydrolysis were evaluated. Subsequently, the transmembrane transport system and intracellular degradation module were modified to accelerate the uptake of hydrolysates and their further conversion to glucose-6-phosphate. The DW2-derived strains exhibited robust growth in starch medium, and productivity of bacitracin and subtilisin were improved by 38.5% and 32.6%, with an 32.3% and 22.9% increase of starch conversion rate, respectively. Lastly, the employment of engineering strategies enabled another B. licheniformis WX-02 to produce poly-γ-glutamic acid from starch with a 2.1-fold increase of starch conversion rate. This study not only provided excellent B. licheniformis chassis for sustainable bioproduction from starch, but shed light on researches of substrate utilization. [ABSTRACT FROM AUTHOR]