State Key Laboratory of Food Science and Technology, Jiangnan University, 1800 Lihu Avenue, Wuxi 214122, China.
School of Biotechnology and Key Laboratory of Industrial Biotechnology Ministry of Education, Jiangnan University, 1800 Lihu Avenue, Wuxi 214122, China.
J Ind Microbiol Biotechnol. 2022 Jan 20;49(1). doi: 10.1093/jimb/kuab061.
A strategy for optimizing the extracellular degradation and folding environment of Brevibacillus choshinensis has been used to enhance the extracellular production of recombinant α-amylase. First, a gene (bcp) encoding an extracellular protease and another encoding an extracellular chaperone (prsC) were identified in the genome of B. choshinensis HPD31-SP3. Then, the effect of extracellular protein degradation on recombinant α-amylase production was investigated by establishing a CRISPR/Cas9n system to knock out bcp. The effect of extracellular folding capacity was investigated separately by coexpressing extracellular chaperones genes from different sources (prsA, prsC, prsL, prsQ) in B. choshinensis. The final recombinant strain (BCPPSQ), which coexpressed prsQ in a genetic background lacking bcp, produced an extracellular α-amylase activity of 6940.9 U/ml during shake-flask cultivation. This was 2.1-fold greater than that of the original strain BCWPS (3367.9 U/ml). Cultivation of BCPPSQ in a 3-l fermenter produced an extracellular α-amylase activity of 17925.6 U/ml at 72 h, which was 7.6-fold greater than that of BCWPS (2358.1 U/ml). This strategy demonstrates its great potential in enhancing extracellular α-amylase production in B. choshinensis. What's more, this study provides a strategic reference for improving the extracellular production of other recombinant proteins in B. choshinensis.
已采用优化短小芽孢杆菌胞外降解和折叠环境的策略来提高重组α-淀粉酶的胞外产量。首先，在短小芽孢杆菌 HPD31-SP3 的基因组中鉴定出编码胞外蛋白酶的基因（bcp）和编码胞外分子伴侣（prsC）的基因。然后，通过建立 CRISPR/Cas9n 系统敲除 bcp 来研究胞外蛋白降解对重组α-淀粉酶生产的影响。分别通过在短小芽孢杆菌中共表达来自不同来源的胞外折叠能力相关基因（prsA、prsC、prsL、prsQ）来研究胞外折叠能力的影响。最终的重组菌株（BCPPSQ）在缺乏 bcp 的遗传背景下共表达 prsQ，在摇瓶培养中产生的胞外α-淀粉酶活性为 6940.9 U/ml，比原始菌株 BCWPS（3367.9 U/ml）高 2.1 倍。在 3 升发酵罐中培养 BCPPSQ 时，72 h 时胞外α-淀粉酶活性达到 17925.6 U/ml，比 BCWPS（2358.1 U/ml）高 7.6 倍。该策略在提高短小芽孢杆菌胞外α-淀粉酶产量方面具有很大的潜力。此外，本研究为提高短小芽孢杆菌中其他重组蛋白的胞外生产提供了战略参考。
