D-丙氨酰-D-丙氨酸羧肽酶DacA表达量提高增强大肠杆菌胞外蛋白分泌水平

王浩坤; 张坤杰; 王拂祥; 官剑民; 曲金丰; 沈微; 曹钰; 夏媛媛; 杨海泉; 陈献忠

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D-丙氨酰-D-丙氨酸羧肽酶DacA表达量提高增强大肠杆菌胞外蛋白分泌水平

首发时间：2020-01-08

王浩坤 ¹
王浩坤（1994-），女，主要研究方向：微生物工程
张坤杰 ¹ 王拂祥 ¹ 官剑民 ¹ 曲金丰 ¹ 沈微 ¹ 曹钰 ¹ 夏媛媛 ¹ 杨海泉 ¹
杨海泉（1985-），男，副教授，硕导，主要研究方向：酶工程与技术、微生物工程
陈献忠 ¹

1、江南大学生物工程学院，无锡 214122

摘要：D-丙氨酰-D-丙氨酸羧肽酶(D,D-羧肽酶)DacA对大肠杆菌(Escherichia coli)细胞壁肽聚糖合成及稳定具有重要作用，研究中通过改造DacA启动子PdacA-3提高E. coli重组蛋白胞外生产水平。在基因组上启动子PdacA-3与dacA基因之间引入1个或2个SD序列后，工程菌株的胞外蛋白生产水平显著提高。工程菌株BL21-1SD::pET28a-gfp和BL21-2SD::pET28a-gfp的绿色荧光蛋白胞外生产水平分别提高至对照菌株的1.3和1.2倍。工程菌株BL21-1SD::pET28a-amyk和BL21-2SD::pET28a-amyk的胞外淀粉酶酶活力分别提高至对照菌株的2.0和1.6倍。同时，工程菌株BL21-1SD和BL21-2SD的胞外α-半乳糖苷酶酶活力分别较对照菌株提高了2.1和1.6倍。与对照菌株相比较，工程菌株BL21-1SD和BL21-2SD的外膜渗透性均增强，这为胞外蛋白生产水平增强的主要原因。工程菌株BL21-1SD和BL21-2SD的膜上D,D-羧肽酶酶活力分别提高为对照菌株2.0和1.8倍，说明SD序列引入提高了DacA的表达水平。引入SD序列后，启动子的转录水平显著提高（尤其是引入1个SD序列），这应促进了DacA表达水平的提高。通过流式细胞仪和透射电镜定量、定性分析发现，工程菌株的细胞形态发生了显著变化。启动子PdacA-3与dacA基因之间引入SD序列对E. coli细胞生长具有一定的阻碍作用。结果表明，启动子PdacA-3与dacA基因之间引入SD序列促进了D,D-羧肽酶DacA的表达，扰动了细胞壁肽聚糖的合成与结构稳定，增强了细胞膜的透性，促进了E. coli重组蛋白的胞外生产。

关键词：大肠杆菌 D-丙氨酰-D-丙氨酸羧肽酶DacA SD序列细胞壁蛋白胞外生产

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Increased expression of D-alanyl-D-alanine carboxypeptidase DacA enhances extracellular protein secretion levels

WANG Haokun ¹
王浩坤（1994-），女，主要研究方向：微生物工程
ZHANG Kunjie ¹ WANG Fuxiang ¹ GUAN Jianmin ¹ QU Jinfeng ¹ SHEN Wei ¹ CAO Yu ¹ XIA Yuanyuan ¹ YANG Haiquan ¹
杨海泉（1985-），男，副教授，硕导，主要研究方向：酶工程与技术、微生物工程
CHEN Xianzhong ¹

1、School of Biotechnology,Jiangnan University,Wuxi 214122

Abstract：D-alanyl-D-alanine carboxypeptidase(D,D-carboxypeptidase) DacAplays an important role in the synthesis and stabilization of cell wall peptidoglycan of Escherichia coli(E. coli). In this work, the DacA promoter PdacA-3 was engineered to improve extracellular recombinant protein production in E. coli. After inserting one or two SD sequences between the promoter PdacA-3 and dacA genes in the genome, the extracellular protein production level of mutants was significantly increased. The extracellular production levels of green fluorescent protein in mutants BL21-1SD::pET28a-gfp and BL21-2SD::pET28a-gfp were increased by 1.3- and 1.2-fold that of the control, respectively. Extracellular amylase activity of mutants BL21-1SD::pET28a-amyk and BL21-2SD::pET28a-amyk was increased by 2.0- and 1.6-fold that of the control, respectively. Meanwhile, the extracellular α-galactosidase activity of mutants BL21-1SD and BL21-2SD increased by 2.1- and 1.6-fold compared with that of the control. The outer membrane permeability of mutants BL21-1SD and BL21-2SD was enhanced, which should be the main reason for the increased extracellular protein production level. The D,D-carboxypeptidase activity of mutants BL21-1SD and BL21-2SD increased by 2.0- and 1.8-fold, indicating that inserting SD sequences increased the expression level of DacA. The transcription level of the promoter was significantly increased (especially inserting one SD sequence), which should enhance DacA expression level. Based on quantitative and qualitative analysis by fluorescence-activated cell sorting and transmission electron microscopy, it was found that the cell morphology of mutants was significantly changed compared with that of the control. Inserting SD sequences between the promoters PdacA-3 and dacA genes has a certain hindrance to E. coli cells growth. The results showed that inserting the SD sequence between the promoters PdacA-3 and dacA genes increased the expression of D,D-carboxypeptidase DacA, and it disturbed the synthesis and structural stability of cell wall peptidoglycans, which enhanced cell membrane permeability and improved extracellular production of recombinant proteins in E. coli.

Keywords： Escherichia coli D-alanyl-D-alanine carboxypeptidase DacA SD sequence cell wall extracellular protein production

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王浩坤，张坤杰，王拂祥，等. D-丙氨酰-D-丙氨酸羧肽酶DacA表达量提高增强大肠杆菌胞外蛋白分泌水平[EB/OL]. 北京：中国科技论文在线 [2020-01-08]. https://www.paper.edu.cn/releasepaper/content/202001-50.

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论文编号	202001-50
论文题目	D-丙氨酰-D-丙氨酸羧肽酶DacA表达量提高增强大肠杆菌胞外蛋白分泌水平
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