[Enhanced production of bacitracin via energy metabolism engineering in Bacillus licheniformis DW2].

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.

Optimization of enhanced microbial production of zinc bacitracin by submerged fermentation technology.

Bacitracin is one of the most important antibiotics used in different biomedical fields. It helps to achieve sizeable amount of foreign exchange due to its use in the poultry feed. The cheap agricultural wastes are readily available for the preparation of fermentation media used for bacitracin production. The microorganisms could be mutated with different chemicals and UV radiation to improve bacitracin production. Thus, the current study was focused on the synthesis of low-cost and effective bacitracin by mutant strains of Bacillus licheniformis, employing the submerged fermentation technique. The bacteria were exposed to the UV irradiation for various time periods ranging from 5 to 40 min. These mutants were named as BLAA-5-BLAA-40. Mutant strain BLAA-25 produced maximum bacitracin, with significantly high activity (142.81 IU/mg) against Klebsiella pneumoniae but less activity against Escherichia coli (115.19 IU/mg). Several fermentation conditions were investigated to optimize bacitracin production. The highest bacitracin yield was obtained by an inoculum size of 10%, fermentation period 48 hr, pH 7, T = 37°C, using soybean meal as a substrate. Among all substrates, cucumber peel was the substrate showing the highest minimum inhibitory concentration (2.3 mg/ml and 2.7 mg/ml against K. pneumoniae and E. coli respectively). A comparison between commercial and experimentally produced Zn bacitracin showed that commercial bacitracin has a low activity (63.2 IU/mg) as compared with experimental bacitracin. Hence, the agro wastes and mutation could be used to increase the synthesis of Zn bacitracin in B. licheniformis.

Modular metabolic engineering of lysine supply for enhanced production of bacitracin in Bacillus licheniformis.

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.

Improving the utilization rate of soybean meal for efficient production of bacitracin and heterologous proteins in the aprA-deficient strain of Bacillus licheniformis.

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.

Enhancement of Bacitracin Production by NADPH Generation via Overexpressing Glucose-6-Phosphate Dehydrogenase Zwf in Bacillus licheniformis.

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.

[Enhanced production of bacitracin by knocking out of amino acid permease gene yhdG in Bacillus licheniformis DW2].

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.

Increased flux through the TCA cycle enhances bacitracin production by Bacillus licheniformis DW2.

The dodecapeptide antibiotic bacitracin, produced by several strains of Bacillus licheniformis and Bacillus subtilis, is widely used as an antibacterial animal feed additive. Several genetic strategies were explored to enhance its production. The availability of building block amino acids for bacitracin production was found to play an important role in its synthesis. In this study, the TCA cycle in the industrial strain B. licheniformis DW2 was strengthened by overexpression of the key enzymes citrate synthase and isocitrate dehydrogenase (ICDH). As the central metabolic pathway, the TCA cycle is a major source for energy supply and intermediates for anabolism. By enhancing flux through the TCA cycle, more energy and precursors were generated for amino acid biosynthesis and uptake, resulting in enlarged intracellular pool of bacitracin-containing amino acids for bacitracin production. This study unveiled the metabolic responses of the increased TCA cycle flux in B. licheniformis and provided a novel strategy for enhancing bacitracin production.

Optimization of Inexpensive Agricultural By-Products as Raw Materials for Bacitracin Production in Bacillus licheniformis DW2.

Bacitracin is a broad-spectrum antibiotic used extensively as a feed additive. In this study, inexpensive agricultural by-products were used as nitrogen sources for bacitracin production. Based on both the orthogonal tests, a combination of 7% soybean meal (SBM) +2% low protein rapeseed cake (LPRC) was optimal for bacitracin production. Compared to the original formula, the titer of bacitracin increased by 20.5% reaching 910.4 U/ml in flasks. The titer of bacitracin and the ratio of bacitracin A increased by 12.4 and 6.8% in a 50-l fermentor. Furthermore, this study also explored the effects of exogenously adding different amino acids on the yield of bacitracin. The addition of Cys and Glu enhanced bacitracin production by 5.7 and 5.0%, respectively. This study provided the inexpensive nutrient inputs into efficient bacitracin production and also the insight to further research enabling better utilization of oil cakes for economic viability of the bioprocess industry.

Untangling the transcription regulatory network of the bacitracin synthase operon in Bacillus licheniformis DW2.

The bacitracin synthetase gene cluster in Bacillus licheniformis DW2 is composed of the bacABC operon encoding a non-ribosomal peptide synthetase and bacT encoding a thioesterase. Although the bacitracin gene cluster has been well studied, little is known about how this gene cluster is regulated. This study provides insight into how the transcription factors Spo0A and AbrB regulate bacitracin biosynthesis. Deletion of spo0A resulted in drastically reduced expression of bacA and bacT, and subsequently bacitracin production. On the other hand, the expression of bacA and bacT increased significantly in B. licheniformis DW2ΔabrB and DW2Δ0AΔabrB compared to the wild-type strain DW2. The bacitracin yields on cell numbers (U/CFU) in DW2ΔabrB and DW2Δ0A/pHY300-0A-sad67 were 17.5% and 14.9% higher than that of the wild-type strain. An electrophoretic mobility shift assay (EMSA) further confirmed that AbrB could directly bind to the promoter regions of bacA and bacT. These results indicate that AbrB acts as a repressor of bacitracin biosynthesis by inhibiting bacA and bacT expression, while Spo0A indirectly promotes bacitracin biosynthesis by repressing abrB expression.

Proteomics analysis of Bacillus licheniformis in response to oligosaccharides elicitors.

The role of oligosaccharides as biotic elicitors has been recognised in the enhanced production of antibiotics from fungal and bacterial cultures. The yield of bacitracin A in cultures of Bacillus licheniformis was increased after supplementation with oligoguluronate (OG), and mannan oligosaccharides (MO) and its mechanism at transcription level been established already. However, the elicitation mechanism at post transcriptional level has not been reported so far. In this paper we investigate changes in proteomics of B. licheniformis in presence of the oligosaccharide elicitors OG and MO. Differentially expressed proteins were examined using 2D-PAGE stained with colloidal Coomassie and were further identified by LC-MS/MS. We identified 19 differentially expressed proteins including those involved in carbon metabolism, energy generation, amino acid biosynthesis, oxidative and general stress response. The novel findings of this work, together with previous reports, contribute to the unravelling of the overall mechanism of elicitation in B. licheniformis cultures and reliability of the use of these elicitors for potential industrial application.

Optimized antimicrobial peptide (Bacitracin) production by immobilized and free cells and of Bacillus Spp GU215 using Wood chips and silicon polymer beads.

The immobilization of bacillus spp. GU215 on silicon polymer beads, wood chips was performed and antibiotic peptide (bacitracin) production, optimization of parameters were investigated. The immobilized cells presented elevated levels of activity than free cells. The silicon polymer based cells showed widest zones of inhibitions (18mm) in 72 hours and 4% concentration of glucose, PH 8 and 30°C, whereas a marginal decrease in the activity (14mm) was noticed in case of wood chips based immobilization systems and least stable immobilization in 72 hours incubation time, 4% glucose concentration, PH 8 and 30°C. This study illustrates that the silicon polymer based beads facilitate a strong interactions with bacitracin producing cells and render them suitable for excessive and long time production of antibiotic.

Comparative genomic analyses of Streptococcus mutans provide insights into chromosomal shuffling and species-specific content.

BACKGROUND: Streptococcus mutans is the major pathogen of dental caries, and it occasionally causes infective endocarditis. While the pathogenicity of this species is distinct from other human pathogenic streptococci, the species-specific evolution of the genus Streptococcus and its genomic diversity are poorly understood. RESULTS: We have sequenced the complete genome of S. mutans serotype c strain NN2025, and compared it with the genome of UA159. The NN2025 genome is composed of 2,013,587 bp, and the two strains show highly conserved core-genome. However, comparison of the two S. mutans strains showed a large genomic inversion across the replication axis producing an X-shaped symmetrical DNA dot plot. This phenomenon was also observed between other streptococcal species, indicating that streptococcal genetic rearrangements across the replication axis play an important role in Streptococcus genetic shuffling. We further confirmed the genomic diversity among 95 clinical isolates using long-PCR analysis. Genomic diversity in S. mutans appears to occur frequently between insertion sequence (IS) elements and transposons, and these diversity regions consist of restriction/modification systems, antimicrobial peptide synthesis systems, and transporters. S. mutans may preferentially reject the phage infection by clustered regularly interspaced short palindromic repeats (CRISPRs). In particular, the CRISPR-2 region, which is highly divergent between strains, in NN2025 has long repeated spacer sequences corresponding to the streptococcal phage genome. CONCLUSION: These observations suggest that S. mutans strains evolve through chromosomal shuffling and that phage infection is not needed for gene acquisition. In contrast, S. pyogenes tolerates phage infection for acquisition of virulence determinants for niche adaptation.

Effect of oligosaccharide elicitors on bacitracin A production and evidence of transcriptional level control.

The role of oligosaccharides as biotic elicitors for the enhanced production of antibiotics and enzymes has been established in recent years. These findings could have significant promise for the pharmaceutical and biotechnology industries. Before the elicitors could be used at large-scale, reproducible production and high levels of enhancement are essential. In order to ensure the robustness of elicitation and exploit the process an understanding of this phenomenon at the molecular level is required. Bacitracin produced by Bacillus licheniformis is widely used as an animal feed additive and antibiotic against Gram-positive bacteria. In this work we report, the effect of single and multiple additions of different elicitors to B. licheniformis cultures for the production of bacitracin A. The results obtained showed that single addition of oligoguluronate to the cultures enhanced the levels of bacitracin A by 23.3%. Moreover, multiple elicitor addition of oligoguluronate and mannan oligosaccharide further enhanced the production of bacitracin A by 13.2% and 36.5% compared to single elicitor addition and control cultures, respectively. In addition, for the first time, it was found that elicitor supplementation of cultures increased the transcription level of the bacitracin biosynthetic genes bacABC, indicating transcriptional level control of the elicitor.

Effect of partial replacement of different defatted oil seed cakes as substrate in biosynthesis of bacitracin in solid-state fermentation by Bacillus licheniformis.

The present study was conducted to ascertain the effect of partial replacement of different defatted oil seed cakes as substrate i.e. sunflower meal, rice hulls and soybean meal, in biosynthesis of Bacitracin in Solid-State Fermentation by Bacillus licheniformis on laboratory scale. In solid-state fermentation, wheat bran, soybean meal, sunflower meal, rice hulls and their different combinations were used. The antibiotic activity was determined at various intervals and recorded 48 hours gave maximum yield, 4375 i.u/gm when only soybean was used. However, maximum titre 4820 i.u / gm of antibiotic were obtained when wheat bran and soybean meal was in ratio of 1:3. The raw material for its production is readily available and cheap such as soybean meal, sunflower meal and wheat bran. Thus development of this technology in our country would result in utilizing our own resources in Pakistan.

Isolation of bacillus subtilis MH-4 from soil and its potential of polypeptidic antibiotic production.

The genus Bacillus produces mainly polypeptide antibiotics such as bacitracin and polymyxin. Bacillus species were isolated from soil by soil sprinkle technique. And all were screened for the production of antibiotic. Bacillus subtilis MH-4 gave the maximum antimicrobial activity so finally selected for optimization. During optimization of culture conditions for Bacillus subtilis MH-4 best antibacterial activity was obtained at 96 hours of incubation period, at pH-8 and by using glycerol as carbon and L-glutamic acid as nitrogen source. Optimum temperature for antibiotic production was 37 degrees C. The antibiotic was confirmed to be bacitracin by paper chromatography. Antibiotic was further extracted successfully with 1-Butanol, and aqueous concentrate showed activity of 0.8 mg/ml. The antibiotic so produced was found to be narrow spectrum active against only Gram-positive bacteria.

Optimization of bacitracin production by Bacillus licheniformis B5.

Production of the polypeptide antibiotic bacitracin by a newly isolated Bacillus licheniformis B5 strain, using a laboratory medium was optimized. A medium of the following conditions was obtained: 1.5% glycerol as carbon source, 0.05% glutamic acid as nitrogen source, 1.43% inorganic phosphate, 0.0025% magnesium sulfate, with an initial pH of 7.5. Culture was incubated at 37 degrees C for 48 h. The bacitracin productivity was about 174.2 units mL(-1). Bacitracin antibiotic was also produced using immobilized cells of the B5 isolate in different immobilization supports (7.5% polyacrylamide, 4% sodium alginate beads and 2% agar). It was found that the highest bacitracin production rate (192 units mL(-1)) was obtained when using 7.5% polyacrylamide gel as an immobilization material.

Biosynthesis of bacitracin in solid-state fermentation by Bacillus licheniformis using defatted oil seed cakes as substrate.

Bacitracin is being imported in Pakistan involving substantial amount of foreign exchange for its incorporation in poultry feed. The cheap raw material for its production is readily available and cheap such as soybean meal, sunflower meal, wheat bran etc. Thus development of this technology in our country would result in saving a reasonable amount of foreign exchange by exploiting indigenous resources. The present study is concerned with the biosynthesis of antibiotic bacitracin in solid-state fermentation by Bacillus licheniformis on laboratory scale using defatted oil seed cakes of agricultural by-products as starting material for maximum production of the antibiotic Bacitracin. In solid-state fermentation, wheat bran, soybean meal, sunflower meal, rice hulls and their different combinations were used. The antibiotic activity, 48 hours after inoculation was 4375 i.u / gm when only soybean was used. However, maximum titre 4820 i.u / gm of antibiotic was obtained using wheat bran and soybean meal in ratio of 1:3.

Engineered biosynthesis of the peptide antibiotic bacitracin in the surrogate host Bacillus subtilis.

Nonribosomal peptides are processed on multifunctional enzymes called nonribosomal peptide synthetases (NRPSs), whose modular multidomain arrangement allowed the rational design of new peptide products. However, the lack of natural competence and efficient transformation methods for most of nonribosomal peptide producer strains prevented the in vivo manipulation of these biosynthetic gene clusters. In this study, we present methods for the construction of a genetically engineered Bacillus subtilis surrogate host for the integration and heterologous expression of foreign NRPS genes. In the B. subtilis surrogate host, we deleted the resident 26-kilobase srfA gene cluster encoding the surfactin synthetases and subsequently used the same chromosomal location for integration of the entire 49-kilobase bacitracin biosynthetic gene cluster from Bacillus licheniformis by a stepwise homologous recombination method. Synthesis of the branched cyclic peptide antibiotic bacitracin in the engineered B. subtilis strain was achieved at high level, indicating a functional production and proper posttranslational modification of the bacitracin synthetases BacABC, as well as the expression of the associated bacitracin self-resistance genes. This engineered and genetically amenable B. subtilis strain will facilitate the rational design of new bacitracin derivatives.

The two-component regulatory system BacRS is associated with bacitracin 'self-resistance' of Bacillus licheniformis ATCC 10716.

Bacitracin is a peptide antibiotic produced by several Bacillus licheniformis strains that is most active against other Gram-positive microorganisms, but not against the producer strain itself. Recently, heterologous expression of the bacitracin resistance mediating BcrABC transporter in Bacillus subtilis and Escherichia coli was described. In this study we could determine that the transporter encoding bcrABC genes are localized about 3 kb downstream of the 44-kb bacitracin biosynthetic operon bacABC. Between the bac operon and the bcrABC genes two orfs, designated bacR and bacS, were identified. They code for proteins with high homology to regulator and sensor proteins of two-component systems. A disruption mutant of the bacRS genes was constructed. While the mutant displayed no effects on the bacitracin production it exhibited highly increased bacitracin sensitivity compared to the wild-type strain. Western blot analysis of the expression of BcrA, the ATP-binding cassette of the transporter, showed in the wild-type a moderate BcrA induction in late stationary cells that accumulate bacitracin, whereas in the bacRS mutant cells the BcrA expression was constitutive. A comparison of bacitracin stressed and nonstressed wild-type cells in Western blot analysis revealed increasing amounts of BcrA and a decrease in BacR in the stressed cells. From these findings we infer that BacR acts as a negative regulator for controlling the expression of the bcrABC transporter genes.

Amplification of bacitracin transporter genes in the bacitracin producing Bacillus licheniformis.

We have amplified the previously cloned and sequenced genes of the bacitracin exporter (bcr), a member of the ATP-binding transport protein family, within the chromosome of the bacitracin producing Bacillus licheniformis. Amplification of the transporter genes was followed by greatly increased bacitracin resistance. Antibiotic production was enhanced at a low level of bcr genes amplification. An enlarged increase in the copy number of the bcr genes negatively affects the overall growth of bacteria.