一种谷氨酸棒杆菌4-异丙基苯甲酸诱导型启动子的设计与应用

doi:10.13560/j.cnki.biotech.bull.1985.2024-0174

摘要/Abstract

摘要：

【目的】 谷氨酸棒杆菌是一种重要的工业微生物，挖掘可调控性元件可有效拓展谷氨酸棒杆菌研究和应用的广度和深度。【方法】 在谷氨酸棒杆菌组成型强启动子P_H10上嵌入阻遏蛋白CymR结合的操纵序列CuO，构建了4-异丙基苯甲酸（Cumate）为诱导剂的启动子P_H10-CuO。【结果】 绿色荧光蛋白作为报告基因的实验结果显示，无诱导剂时，谷氨酸棒杆菌工程菌相对荧光强度较低；以25 μg/mL 4-异丙基苯甲酸诱导12 h时，谷氨酸棒杆菌工程菌荧光强度达62 000，证明P_H10-CuO具有很好的严谨性和诱导表达强度。构建了以启动子P_H10-CuO调控recET和Cas12a表达的谷氨酸棒杆菌基因编辑质粒，实现谷氨酸棒杆菌染色体上靶标基因的精准编辑和外源基因插入。【结论】 诱导型启动子P_H10-CuO具有表达强度高且渗漏表达低的特点，可用于谷氨酸棒杆菌中被其调控基因的时序性表达。

关键词: 谷氨酸棒杆菌, 诱导型启动子, 阻遏蛋白CymR, 4-异丙基苯甲酸

Abstract:

【Objective】 Corynebacterium glutamicum is an important industrial microorganism, and its metabolic engineering modification by gene editing may effectively broaden the diversity of its fermentation products. The lack of high-intensity, low-leakage, and low-cost inducible promoters limits the metabolic engineering modification, thereby the design and application of novel inducible promoters are necessary. 【Method】 An cumate-induced promoter P_H10-CuO was constructed by embedding the operator sequence CuO on the constitutive promoter P_H10. 【Result】 Using green fluorescent protein as a quantitative reporter, the relative fluorescence intensity was low due to the basal leakage of P_H10-CuO when without the inducer 4-isopropylbenzoic acid; and gfp expression significantly increased when the fluorescence intensity was up to 62 000 at presence of 25 μg/mL 4-isopropylbenzoic acid for 12 h. This proved that PH 10-CuO has very good rigor and induced expression intensity. Meanwhile, in Constructing the gene editing plasmid for C. glutamicum with P_H10-CuO regulating the expression of recET and cas12a, The accurate editing of target gene and insertion of foreign genes in C. glutamicum chromosome. 【Conclusion】 The inducible promoter PH_10-CuO presented the advantages with high intensity and low leakage, which enables it as an useful element to regulate temporal expression of specific genes in C. glutamicum.

Key words: Corynebacterium glutamicum, inducible promoter, repressor protein CymR, 4-isopropylbenzoic acid

毋舒宁, 苏永平, 李冬雪, 柏映国, 刘波, 张志伟. 一种谷氨酸棒杆菌4-异丙基苯甲酸诱导型启动子的设计与应用[J]. 生物技术通报, 2024, 40(7): 108-116.

WU Shu-ning, SU Yong-ping, LI Dong-xue, BAI Ying-guo, LIU Bo, ZHANG Zhi-wei. Design and Application of a Cumate-inducible Promoter for Corynebacterium glutamicum[J]. Biotechnology Bulletin, 2024, 40(7): 108-116.

图/表 7

表1 本研究所用引物

Table 1 Primers used in this study

引物名称 Primer name	引物序列 Primer sequence（5' -3'）	引物用途 Primer usage
cymR-F	CCAGGGTGGTTTTTCTTTCTATCGCTTGAACTTAGCGTAGC	扩增CymR
cymR-R	AGAGTCAATTCAGGGTGGTGAATATGAGCCCGAAGCGCCGCAC	扩增CymR
LacIq-F	ATTCACCACCCTGAATTGACTCT	扩增P_lacIq
LacIq-R	GGATCAGCTTGCAATTCGCGCGCGAAGGCGAAGCGGCATTTACG	扩增P_lacIq
Mazf-F	CACGGCGAAAGGATACTCATGGTAAGCCGATACGTACCCG	扩增mazf
Mazf-R	GTCGACCTGCAGGCATGCCTACCCAATCAGTACGTTAATTTTGG	扩增mazf
gfp-F gfp-R	GCATGCCTGCAGGTCGACATGGTGAGCAAGGGCGAGGA ACCCGGGGATCCTCTAGATTACTTGTACAGCTCGTCCATG	扩增gfp
cymR/H10CuO-F cymR/H10CuO-R	ATGGGTCTTGTTGTTGGCAGACCGTATCCAAAGCATCCG AGGAAGAGTGGTTTTGTGCTCATGAGTATCCTTTCGCCGTGCT	扩增cymR/H10CuO
recET-F	ATGAGCACAAAACCACTCTTCCT	扩增recET
recET-R	CTTCCTGGCATCTTCCAGCAAAAAAAGCACCGACTCGGTGCCACTTTTTCAAGTTGATAATTATTCCTCTGAATTATCGATTACACTG	扩增recET
H10CuO/cpf1-F	GTTCGCGGAATCATGACCGCTCAACCCTTACCGGTCG	P_H10-CuO扩增
H10CuO/cpf1-R	TCACAAACTCTTGGTAGATGGACATGAGTATCCTTTCGCCGTGCTC	P_H10-CuO扩增
LysE-LF	TCAGTGGAACGAAAACTCACTGCGCGAGCAAGGAGAGTAC	扩增lysE左同源臂
LysE-LR	CGCCATGACAAACAAGGTGG	扩增lysE左同源臂
LysE-RF	GTGGCACCGAGTCGGTGCTTTTTTTGAGGTAAGCGATGCCACCCCA	扩增lysE右同源臂
LysE-RR	TCTCATCCGCCAAAACAGCC GTTATGGTTTGCCGTCATGGCAGCATCTACAACAGTAGAAATTCGGATCCAT- TATACCTAGGACTGAGCTAGCTGTCAACCAAGAAGCTACCTCGTTGAACA	扩增lysE右同源臂
asd/H10CuO-F	CCACCTTGTTTGTCATGGCGGCTCAACCCTTACCGGTCGGCTCTAAGCCGGCGGCGTATGGTAAGCTCTGTTATGTATAGTCCGAGCACGGCGAAAGGATACTC ATGACCACCATCGCAGTTGTT	扩增asd
asd/H10CuO-R	AAAGCACCGACTCGGTGCCACTTTTTCAAGTTGATAATTACTTAACCAGCAGCTCAGCG	扩增asd

图1 启动子PH10-CuO序列

Fig. 1 Sequence of promoter PH10-CuO

图2 不同质粒示意图 A：质粒pXJM19/cymR；B：质粒pRHCuO

Fig. 2 Profiles of various plasmids A: Plasmid pXJM19/cymR. B: Plasmid pRHCuO

图3 启动子PH10-CuO性能鉴定 A：质粒pRHCuO/gfp；B：pRHCuO/gfp阳性转化子表征（左图：点接于含有4-异丙基苯甲酸的氯霉素抗性LB培养基，右图：点接于氯霉素抗性LB培养基）

Fig. 3 Characterization of PH10-CuO A: Plasmid pRHCuO/gfp. B: Profiles of positive transformants harboring pRHCuO/gfp on different media (Left: chloramphenicol-contained LB medium with 4-isopropylbenzoic acid; right: chloramphenicol-contained LB medium)

图4 启动子PH10-CuO性能鉴定 13032-01在不同4-异丙基苯甲酸浓度下诱导12 h时的相对荧光强度

Fig. 4 Characterization of PH10-CuO Fluorescence intensity at different 4-isopropylbenzoic acid concentrations at 12 h of 13032-01 induction

图5 基因编辑质粒 A：PH10-CuO调控recET表达的质粒pYJR01；B：PH10-CuO调控recET和cas12a表达的质粒pYJRC01

Fig. 5 Plasmids via gene editing A: PH10-CuO regulating the plasmid pYJR01 expressed with recET. B: PH10-CuO regulating the plasmid pYJR01 expressed with recET and cas12a

图6 基因编辑及质粒消除 A：用于敲除lysE和过表达asd的质粒pYJRC/lysE-asd；B：转化子验证（泳道M：DNA marker；泳道1-8：在含25 μg/mL 卡那霉素和12.5 μg/mL 4-异丙基苯甲酸的BHISG固体培养基上转化子染色体为模板时PCR扩增产物）；C：转化子验证（泳道M：DNA marker；泳道1-8：在含25 μg/mL 卡那霉素和12.5 μg/mL 4-异丙基苯甲酸的液体培养基BHISG中再次培养24 h的菌体为模板时PCR扩增产物）；D：质粒pYJRC/lysE-asd消除菌株筛选

Fig. 6 Gene editing and plasmid elimination A: Plasmid pYJRC/lysE-asd for deleting lysE and overexpressing asd. B: Transformants verification（Lane M: DNA marker; lane 1-8: PCR products with the chromosomal DNA of transformants on solid medium BHISG containing 25 μg/mL kanamycin and 12.5 μg/mL 4-isopropylbenzoic acid as templates. C: Transformants verification（Lane M: DNA marker; lane 1-8: PCR products with the chromosomal DNA of transformants cultured in liquid medium BHISG containing 25 μg/mL kanamycin and 12.5 μg/mL 4-isopropylbenzoic acid for 24 h as templates）. D: Screen of transformants without plasmid pYJRC/lysE-asd

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