State Key Laboratory of Biocatalysis and Enzyme Engineering, Environmental Microbial Technology Center of Hubei Province, College of Life Sciences, Hubei University, 368 Youyi Avenue, Wuchang District, Wuhan, 430062, Hubei, People's Republic of China.
Chengdu Slan Biotechnology Co. Ltd, Chengdu Hi Tech Development Zone, Chengdu, 610041, People's Republic of China.
Appl Microbiol Biotechnol. 2019 Nov;103(21-22):8799-8812. doi: 10.1007/s00253-019-10110-y. Epub 2019 Sep 14.
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.
杆菌肽是由芽孢杆菌产生的一种大环十二肽，前体供应在杆菌肽生产中起着关键作用。在这里，本研究旨在通过代谢工程改造生产杆菌肽的工业菌株地衣芽孢杆菌 DW2 来提高杆菌肽的产量，从而增加赖氨酸（Lys）的供应。首先，通过外源添加 Lys 被证明有利于杆菌肽的生产，通过过表达来自地衣芽孢杆菌的二氨基庚二酸脱羧酶 LysA 和来自谷氨酸棒杆菌的二氨基庚二酸脱氢酶 DdH 来增强 Lys 合成途径，获得了菌株 LYS2，与 DW2（756.45 U/mL）相比，LYS2 产生的杆菌肽增加了 10.85%，达到 838.53 U/mL。其次，通过在 LYS2 中过表达丙酮酸羧化酶 PycA 来积累前体草酰乙酸，在 LYS3（885.53 U/mL）中获得了 17.06%的杆菌肽产量增加，与 DW2 相比。第三，通过敲除赖氨酸脱羧酶基因 yaaO 来减弱 Lys 的降解，获得的菌株 LYS4 从 885.53 增加到 923.43 U/mL，杆菌肽产量进一步增加。最后，证实转运蛋白 LysE 作为 Lys 外排蛋白；鉴定出 LysP 和 YvsH 是地衣芽孢杆菌 DW2 中的 Lys 转运蛋白，通过工程化 Lys 转运蛋白，使杆菌肽产量提高到 975.43 U/mL，增加了 28.95%。综上所述，本研究表明，Lys 供应模块的代谢工程是提高杆菌肽产量的有效策略，并为地衣芽孢杆菌的工业生产提供了一种有前途的菌株。
