Abstract
Bacitracin is a broad-spectrum veterinary antibiotic that widely used in the fields of veterinary drug and feed additive. S-Adenosylmethionine (SAM) is a critical factor involved in many biochemical reactions, especially antibiotic production. However, whether SAM affects bacitracin synthesis is still unknown. Here, we want to analyze the relationship between SAM supply and bacitracin synthesis, and then metabolic engineering of SAM synthetic pathway for bacitracin production in Bacillus licheniformis. Firstly, our results implied that SAM exogenous addition benefited bacitracin production, which yield was increased by 12.13% under the condition of 40 mg/L SAM addition. Then, SAM synthetases and Methionine (Met) synthetases from B. licheniformis, Corynebacterium glutamicum, and Saccharomyces cerevisiae were screened and overexpressed to improve SAM accumulation, and the combination of SAM synthetase from S. cerevisiae and Met synthetase from B. licheniformis showed the best performance, and 70.12% increase of intracellular SAM concentration (31.54 mg/L) and 13.08% increase of bacitraicn yield (839.54 U/mL) were achieved in resultant strain DW2-KE. Furthermore, Met transporters MetN and MetP were, respectively, identified as Met exporter and importer, and bacitracin yield was further increased by 5.94% to 889.42 U/mL via deleting metN and overexpressing metP in DW2-KE, attaining strain DW2-KENP. Finally, SAM nucleosidase gene mtnN and SAM decarboxylase gene speD were deleted to block SAM degradation pathways, and bacitracin yield of resultant strain DW2-KENPND reached 957.53 U/mL, increased by 28.97% compared to DW2. Collectively, this study demonstrated that SAM supply served as the critical role in bacitracin synthesis, and a promising strain B. licheniformis DW2-KENPND was attained for industrial production of bacitracin.
Highlights
Bacitracin, an important peptide antibiotic that consists of 11 kinds of amino acids, is mainly produced by B. licheniformis and Bacillus subtilis
This study demonstrated that SAM supply served as the critical role in bacitracin synthesis, and a promising strain B. licheniformis DW2-KENPND was attained for industrial production of bacitracin
SAM was proven to own the critical role in the syntheses numerous kinds of antibiotic (Zhao et al, 2010), whether SAM supply affect bacitracin synthesis has not been clarified in Bacillus
Summary
Bacitracin, an important peptide antibiotic that consists of 11 kinds of amino acids, is mainly produced by B. licheniformis and Bacillus subtilis. Precursor supply served as the critical role in secondary metabolite synthesis (Wohlleben et al, 2012). Branched chain amino acid (BCAA) served as the critical role in erythromycin production, and strengthening BCAA supplies led to a 41% increase of erythromycin yield, via deleting lrp and overexpressing BCAA transporter SACE_5387 (Liu et al, 2017). Acety-CoA carboxylase was overexpressed to improve malonyl-CoA supply, which further led to a 43% increase of surfactin production, reached 13.37 g/L (Wang et al, 2019). S-adenosylmethionine (SAM) served as the critical role in antibiotic biosynthesis, and strengthening SAM supply has been proven as an efficient strategy for lincomycin, avermectin, novobiovin, cephalosporin, etc., production (Zhao et al, 2010). Whether SAM supply affects bacitracin synthesis has not been investigated in Bacillus
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