肌酐酶在枯草芽孢杆菌中的异源表达及分泌机制

doi:10.16085/j.issn.1000-6613.2019-1115

摘要/Abstract

摘要：

肌酐酶是用于临床检测肾小球滤过功能的关键酶之一，但目前国内肌酐酶的生产量较低无法满足市场需求，多依赖于国外进口。为了解决这一问题，本研究将恶臭假单胞杆菌肌酐酶基因克隆至原核表达载体pMA5实现肌酐酶在枯草芽孢杆菌1A751中的异源表达。随后通过启动子优化，使肌酐酶的蛋白表达量显著提高到1.08mg/mL，并且胞外肌酐酶的比酶活力达到238U/mg。同时发现肌酐酶可不依赖于信号肽即可实现胞外分泌，因此对肌酐酶在枯草芽孢杆菌中的分泌机制进行研究。通过对经典分泌途径和Holin途径的分析排除、利用Calcein-AM/PI双染色法鉴定表达菌株1AGC的细胞膜不完整，最后通过电子显微镜观察结果表明表达菌株表面存在潜在的泄漏位点，因此证实肌酐酶在枯草芽孢杆菌中通过细胞泄漏的方式，释放到胞外培养基中。本文构建了一株基于细胞泄漏的肌酐酶高产菌株，为肌酐酶的表达和潜在工业化应用提供理论基础，同时也为枯草芽孢杆菌泄漏表达系统提供了一定的研究基础。

关键词: 枯草芽孢杆菌, 肌酐酶, 电子显微镜观察, 泄漏表达

Abstract:

Creatininase is one of the key enzymes used to evaluate glomerular filtration function. At present, the domestic creatininase production cannot meet the market demand, and it is mostly dependent on import. To solve this problem, our study employed the prokaryotic expression vector pMA5 to achieve the initial expression of creatininase from Pseudomonas putida in Bacillus subtilis 1A751. Subsequently, the promoter optimization successfully increased the creatininase expression level to 1.08 mg/mL, and the extracellular specific enzyme activity was improved to 238 U/mg. Meanwhile, we found that the creatininase without any signal peptides could be secreted into the extracellular medium directly, so the potential secretion mechanism of creatininase in B. subtilis was investigated. The classical secretion pathway and Holin pathway were excluded by experiments, while the membrane damage of strain 1AGC was detected by the Calcein-AM/PI double staining assay. Also, the scanning and transmission electron microscopy analysis revealed that the potential leakage sites were present on the surface of the expressing bacteria. Therefore, we speculated that the creatininase was secreted into extracellular medium by cell leakage in B. subtilis. Overall, our study constructed a strain with high-level creatininase extracellular production based on cell leakage strategy, which provided the theoretical basis for the expression and potential industrial application of creatininase. Moreover, it could help us in establishing a leakage-based expression system in B. subtilis.

Key words: Bacillus subtilis, creatininase, electron microscopy observation, leakage expression

中图分类号:

TQ464.7

陶政宇,付刚,闻建平,张大伟. 肌酐酶在枯草芽孢杆菌中的异源表达及分泌机制[J]. 化工进展, 2020, 39(4): 1458-1468.

Zhengyu TAO,Gang FU,Jianping WEN,Dawei ZHANG. Secretion exploration and heterologous expression of creatininase in Bacillus subtilis[J]. Chemical Industry and Engineering Progress, 2020, 39(4): 1458-1468.

图/表 13

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菌种	基因型或相关特性	来源
大肠杆菌商业化感受态DH5α	F-φ80lacZ ΔM15 Δ(lacZYA-argF) U169 endA1 recA1 gyrA96 endA1 thi-1 relA1	Invitrogen
B. subtilis1A751	eglSΔ102 bglT/bglS ΔEV aprE nprE his	实验室保存
1AGC	菌株1A751转入质粒pMGC	本次实验构建
1A751H	菌株1A751转入质粒pMA5	本次实验构建
1AHC	菌株1A751转入质粒pMA5-CR	本次实验构建
1DGC	菌株1A751转入质粒pDL-DnaK和质粒pMGC	本次实验构建
1PGC	菌株1A751转入质粒pDL-PrsA和质粒pMGC	本次实验构建
1GGC	菌株1A751转入质粒pDL-GroEsL和质粒pMGC	本次实验构建
ΔTat-GC	菌株1A751敲除Tat分泌途径的编码基因，转入质粒pMGC	实验室保存
ΔPbsX-GC	菌株1A751敲除Holin蛋白编码基因pbsx，转入质粒pMGC	本次实验构建
ΔBhlAB-GC	菌株1A751敲除Holin蛋白编码基因bhlA、bhlB，转入质粒pMGC	本次实验构建
SecY-GC	菌株1A751转入整合型质粒pDL-SecY和表达质粒pMGC	本次实验构建
SecE-GC	菌株1A751转入整合型质粒pDL-SecE和表达质粒pMGC	本次实验构建
SecYEG-GC	菌株1A751转入整合型质粒pDL-SecYEG和表达质粒pMGC	本次实验构建
SecY-GC	菌株1A751转入整合型质粒pDL-SecY和表达质粒pMGC	本次实验构建
SecE-GC	菌株1A751转入整合型质粒pDL-SecE和表达质粒pMGC	本次实验构建

菌种	基因型或相关特性	来源
大肠杆菌商业化感受态DH5α	F-φ80lacZ ΔM15 Δ(lacZYA-argF) U169 endA1 recA1 gyrA96 endA1 thi-1 relA1	Invitrogen
B. subtilis1A751	eglSΔ102 bglT/bglS ΔEV aprE nprE his	实验室保存
1AGC	菌株1A751转入质粒pMGC	本次实验构建
1A751H	菌株1A751转入质粒pMA5	本次实验构建
1AHC	菌株1A751转入质粒pMA5-CR	本次实验构建
1DGC	菌株1A751转入质粒pDL-DnaK和质粒pMGC	本次实验构建
1PGC	菌株1A751转入质粒pDL-PrsA和质粒pMGC	本次实验构建
1GGC	菌株1A751转入质粒pDL-GroEsL和质粒pMGC	本次实验构建
ΔTat-GC	菌株1A751敲除Tat分泌途径的编码基因，转入质粒pMGC	实验室保存
ΔPbsX-GC	菌株1A751敲除Holin蛋白编码基因pbsx，转入质粒pMGC	本次实验构建
ΔBhlAB-GC	菌株1A751敲除Holin蛋白编码基因bhlA、bhlB，转入质粒pMGC	本次实验构建
SecY-GC	菌株1A751转入整合型质粒pDL-SecY和表达质粒pMGC	本次实验构建
SecE-GC	菌株1A751转入整合型质粒pDL-SecE和表达质粒pMGC	本次实验构建
SecYEG-GC	菌株1A751转入整合型质粒pDL-SecYEG和表达质粒pMGC	本次实验构建
SecY-GC	菌株1A751转入整合型质粒pDL-SecY和表达质粒pMGC	本次实验构建
SecE-GC	菌株1A751转入整合型质粒pDL-SecE和表达质粒pMGC	本次实验构建

质粒	相关特性	来源
pMA5	穿梭型质粒，启动子P_hpaⅡ；氨苄西林抗性，卡那霉素抗性	实验室保存
pMA5-CR	肌酐酶重组质粒，启动子为P_hpaⅡ；氨苄西林抗性，卡那霉素抗性	本次实验构建
pMGC	肌酐酶重组质粒，麦芽糖诱导型启动子P_glv；氨苄西林抗性，卡那霉素抗性	本次实验构建
pDL-GroEsL	pDL整合型质粒衍生物，groESL操纵子；氯霉素抗性	实验室保存
pDL-PrsA	pDL整合型质粒衍生物，prsA操纵子；氯霉素抗性	实验室保存
pDL-DnaK	pDL整合型质粒衍生物，dnaK操纵子；氯霉素抗性	实验室保存
pDL-SecY	pDL整合型质粒衍生物，元件SecY编码基因；氯霉素抗性	实验室保存
pDL-SecE	pDL整合型质粒衍生物，元件SecE编码基因；氯霉素抗性	实验室保存
pDL-SecYEG	pDL整合型质粒衍生物，元件SecYEG编码基因；氯霉素抗性	实验室保存

质粒	相关特性	来源
pMA5	穿梭型质粒，启动子P_hpaⅡ；氨苄西林抗性，卡那霉素抗性	实验室保存
pMA5-CR	肌酐酶重组质粒，启动子为P_hpaⅡ；氨苄西林抗性，卡那霉素抗性	本次实验构建
pMGC	肌酐酶重组质粒，麦芽糖诱导型启动子P_glv；氨苄西林抗性，卡那霉素抗性	本次实验构建
pDL-GroEsL	pDL整合型质粒衍生物，groESL操纵子；氯霉素抗性	实验室保存
pDL-PrsA	pDL整合型质粒衍生物，prsA操纵子；氯霉素抗性	实验室保存
pDL-DnaK	pDL整合型质粒衍生物，dnaK操纵子；氯霉素抗性	实验室保存
pDL-SecY	pDL整合型质粒衍生物，元件SecY编码基因；氯霉素抗性	实验室保存
pDL-SecE	pDL整合型质粒衍生物，元件SecE编码基因；氯霉素抗性	实验室保存
pDL-SecYEG	pDL整合型质粒衍生物，元件SecYEG编码基因；氯霉素抗性	实验室保存

引物	序列（5’→3’）
pMA5-CR-Vector-F	GAAAAGGCAGGGGACAAAAGGCCTGGGGTGCCTA
pMA5-CR-Vector-R	ACTCATTAGGCACCCCAGGCCTTTTGTCCCCTGCCTT
Creatininase-F	ATTAGCTAGGGGGAATAATTATGGGCCATCACCATC
Creatininase-R	GTCGGAACGAGACTTCTTCTCGAGTTAGGTAGGCG
P_glv-F	AGAGCACACGGATCCCTTTTGTCCCCTGCC
P_glv-R	AATTATTCCCCCTAGCTAATTTTCG
P_glv-Vector-F	CTCGAGAAGAAGTCTCGTTCCGACAGTTGGCAT
P_glv-Vector-R	AATTATTCCCCCTAGCTAATTTTCGTTTAATTATAAA
PG-Vali-F	TCTTCTCAAAAAATACTACC
PG-Vali-R	GCAATTCCAAGCTCTGCCTCGCG
PbsX-1-F	TTACTTCAAGAGAGCTTCAAGTTT
PbsX-1-R	CTTTAGTTGAAGAGCCAGTGGCTTTTTTTATCTTACTG
PbsX-2-F	GCCACTGGCTCTTCAACTAAAGCACCCATTAGT
PbsX-2-R	GGAGGTGATTAGATTCAACTAACGGGGCAGGTTAGT
PbsX-3-F	CCGTTAGTTGAATCTAATCACCTCCTCCGAGGCAA
PbsX-3-R	ATGGCATCATATAGTTTTCAATTTTCAAC
PbsX-4-F	CCGGATGGTAGCGCATATGACGT
PbsX-4-R	GCTGACGAACGCAGTTTT
PbsX-CR-R	AAGGCTTGAATCTTAGCTCATTAGACAC
BhlAB-1-F	ATGTCAGTTTTCACTAATAGCTACATTC
BhlAB-1-R	GCTTTAGTTGAAGAAAAATGTTCACCACCTTAGTACCA
BhlAB-2-F	TGGTGAACATTTTTCTTCAACTAAAGCACCCATTAGT
BhlAB-2-R	ATCCGAAAAAAGTTCAACTAACGGGGCAGGTTAGT
BhlAB-3-F	CCGTTAGTTGAACTTTTTTCGGATTACCTGTGTAAC
BhlAB-3-R	CATGAAGAACCTATGAATTATAATCATAGTCTAC
BhlAB-4-F	GTAGATAAGGCTGAAGCAATTTGTA
BhlAB-4-R	GAAGAAATGTTAAGATTGGAGCC
BhlAB-CR-R	CTTTCATCAATTGGATTGATTATTGCGT
CR-F	TTTCTATTATTCCATGGACTTCATTTACTGGG