能量代谢工程促地衣芽胞杆菌DW2高效合成杆菌肽

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

摘要:

杆菌肽是一种主要由芽胞杆菌产生的广谱性环肽类抗生素，目前广泛应用于兽药领域。能量代谢在微生物高效合成目的代谢产物中具有重要作用。文中以杆菌肽工业生产菌株地衣芽胞杆菌Bacillus licheniformis DW2为出发菌株，首先构建了呼吸链分支途径细胞色素bd泛醇氧化酶基因cydB缺失菌株，发现cydB缺失后杆菌肽效价和胞内ATP浓度相比于对照菌株分别提高了10.97%和22.96%。接着，证实了强化表达另外一条呼吸链分支途径——细胞色素aa3氧化酶基因qoxA能够提高杆菌肽合成水平，其杆菌肽效价和胞内ATP浓度相比于对照菌株分别提高了18.27%和34.00%。强化ADP合成供给也是促进胞内ATP积累的有效策略，结果表明强化表达腺苷激酶DcK和腺苷酸激酶AdK均可以提高杆菌肽效价和胞内ATP浓度，其中强化表达DcK效果较好，其杆菌肽效价相比对照提高16.78%。最后，通过组合代谢工程育种，在基因cydB缺失菌DW2ΔcydB基础上整合表达了qoxA和dck，得到工程菌株DW2-CQD (DW2ΔcydB::qoxA::dck)，发酵结果表明，DW2-CQD杆菌肽效价达到954.25 U/mL，相比于对照菌株提高了21.66%，单位菌体杆菌肽效价为2.11 U/CFU，相比对照提高了11.05%。此外，DW2-CQD胞内ATP浓度为39.54 nmol/L，相比于对照提高了49.32%。结果证实能量代谢工程是提高杆菌肽发酵水平的有效策略，提供了一株具有工业化应用前景的杆菌肽生产菌株。

Abstract:

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.