新喀里多尼亚弧菌CGJ02-2中四氢嘧啶合成基因簇ectABC的克隆及其功能鉴定

doi:10.19586/j.2095-2341.2022.0049

生物技术进展 ›› 2022, Vol. 12 ›› Issue (6): 915-921.DOI: 10.19586/j.2095-2341.2022.0049

新喀里多尼亚弧菌CGJ02-2中四氢嘧啶合成基因簇ectABC的克隆及其功能鉴定

谭琳¹(), NWE Ni Win Htet¹^,², 陈偿³, PAN Zhiqiang⁴

^1.中国热带农业科学院海口实验站，海口 571101
^2.缅甸生物技术研究所微生物实验室，缅甸皎施 05151
^3.中国科学院南海海洋研究所，热带海洋生物资源和生态重点实验室，广州 510301
^4.美国农业部农业研究局天然产物利用研究中心，美国牛津 38677-1848

收稿日期:2022-04-02 接受日期:2022-06-08 出版日期:2022-11-25 发布日期:2022-11-30
通讯作者: 谭琳
作者简介:谭琳 E-mail：tanlin@catas.com
基金资助:
海南省重点研发计划项目(ZDYF2019167)

Cloning and Functional Identification of ectABC Gene Cluster from Vibrio neocaledonicus CGJ02-2

Lin TAN¹(), Ni Win Htet NWE¹^,², Chang CHEN³, Zhiqiang PAN⁴

^1.Haikou Experimental Station，Chinese Academy of Tropical Agricultural Sciences，Haikou 571101，China
^2.Microbiology Laboratory，Biotechnology Research Department，Kyaukse 05151，Myanmar
^3.Key Laboratory of Tropical Marine Bio-resources and Ecology，South China Sea Institute of Oceanology，Chinese Academy of Sciences，Guangzhou 510301，China
^4.Natural Products Utilization Research Unit，Agricultural Research Service，United States Department of Agriculture，Oxford 38677-1848，USA

Received:2022-04-02 Accepted:2022-06-08 Online:2022-11-25 Published:2022-11-30
Contact: Lin TAN

摘要/Abstract

摘要：

研究旨在克隆新的四氢嘧啶合成基因簇，并对其功能进行鉴定，为应用于四氢嘧啶的生产奠定基础。从新喀里多尼亚弧菌CGJ02-2中克隆获得四氢嘧啶合成基因簇ectABC，ectABC与表达载体pBAD连接后转化至大肠杆菌BW25113中，通过L-阿拉伯糖诱导表达。采用SDS-PAGE和液质联用鉴定重组表达蛋白，利用全细胞催化合成四氢嘧啶，通过高分辨质谱鉴定四氢嘧啶，并从天冬氨酸浓度、KCl浓度、温度和pH 4个方面优化催化条件。结果表明，来自新喀里多尼亚弧菌CGJ02-2 基因组的ectABC大小为2 235 bp。SDS-PAGE显示表达产物中有3个重组蛋白产生, 液质联用鉴定表明其分子量分别与ectA、ectB、ectC的理论分子量一致。高分辨质谱分析发现全细胞催化上清中有四氢嘧啶产生。优化后的最适全细胞催化条件为：天冬氨酸浓度100 mmol·L^-1，KCl浓度100 mmol·L^-1，温度30 ℃，pH 7.0，最优条件下产量为1.11 mg·mL^-1。研究从弧菌中克隆了四氢嘧啶合成基因簇ectABC，并在大肠杆菌BW25113中实现了异源表达和低盐环境下的四氢嘧啶合成，为大规模发酵生产四氢嘧啶奠定了基础。

关键词: 新喀里多尼亚弧菌, ectABC基因簇, 重组表达, 全细胞催化, 四氢嘧啶

Abstract:

This study aimed to clone and functionally characterize ectABC gene cluster so as to lay a foundation for the production of ectoine. The ectABC gene cluster was cloned from Vibrio neocaledonicus CGJ02-2 by PCR and ligated to expression vector pBAD， which was transformed into Escherichia coli BW25113 and expressed with L-arabinose induction. The recombinant protein was detected by SDS-PAGE and authenticated by LC-MS. The whole cell biocatalysis was used to synthesize ectoine， and high resolution mass spectrum was employed to identify the production of ectoine， the catalysis conditions were optimized regarding to aspartate concentration， KCl concentration， temperature and pH. Results showed the size of ectABC gene cluster from the genome of Vibrio neocaledonicus CGJ02-2 was 2 235 bp. Three recombinant proteins were detected in the expression product by SDS-PAGE， and LC-MS analysis indicated that the molecular weight of the three recombinant proteins is consistent with the theoretical molecular weight of ectA， ectB， ectC， respectively. High resolution tandem mass spectrum identification found that ectoine was produced in the supernatant of whole cell catalysis. The optimal catalysis conditions obtained as follows： aspartate concentration 100 mmol·L^-1， KCl concentration 100 mmol·L^-1， temperature 30 ℃， pH 7 with a yield of 1.11 mg·mL^-1. In this study， the gene cluster ectABC for ectoine biosynthesis was cloned from genus Vibrio， and it was heterologously expressed with biosynthesis of ectoine in low saline environment， which has laid foundation for the scale production of ectione.

Key words: Vibrio neocaledonicus, ectABC gene cluster, recombinant expression, whole-cell biocatalysis, ectoine

中图分类号:

谭琳, NWE Ni Win Htet, 陈偿, PAN Zhiqiang. 新喀里多尼亚弧菌CGJ02-2中四氢嘧啶合成基因簇ectABC的克隆及其功能鉴定[J]. 生物技术进展, 2022, 12(6): 915-921.

Lin TAN, Ni Win Htet NWE, Chang CHEN, Zhiqiang PAN. Cloning and Functional Identification of ectABC Gene Cluster from Vibrio neocaledonicus CGJ02-2[J]. Current Biotechnology, 2022, 12(6): 915-921.

图/表 8

图1 四氢嘧啶合成基因簇etcABC PCR扩增注：M—1 kb DNA ladder；1—PCR扩增产物。

Fig. 1 PCR of etcABC gene cluster for ectoine

图2 重组质粒pBADetcABC的双酶切鉴定注：M—1 kb DNA ladder；1—重组质粒pBAD ettABC双酶切产物。

Fig. 2 Identification of recombinant plasmid pBADetcABC by synthesis double restriction enzyme digestion

图3 SDS-PAGE分析ectA、ectB、ectC的重组表达注：M—蛋白质Marker；1—含有pBAD表达载体的BW25113诱导后细胞裂解液上清；2—含有pBAD-ectABCBW25113未诱导培养后细胞裂解液上清；3—诱导BW25113pBAD-ectABC培养后细胞裂解液上清。

Fig. 3 SDS-PAGE analysis for the recombinant expression of ectA, ectB, ectC

图4 四氢嘧啶的高分辨质谱鉴定A：四氢嘧啶标准品高分辨质谱图；B：未诱导重组菌株pBAD-ectABC全细胞催化上清高分辨质谱图；C：诱导重组菌株pBAD-ectABC全细胞催化上清高分辨质谱图

Fig. 4 Identification of ectoine by high resolution mass spectrometry

图5 天冬氨酸钠浓度对四氢嘧啶产量的影响注：不同字母表示不同处理间差异具有统计学意义（P<0.05）。

Fig. 5 The effect of sodiumasparpate concentration on the yield of ectione

图6 KCl浓度对四氢嘧啶产量的影响注：不同字母表示不同处理间差异具有统计学意义（P<0.05）。

Fig. 6 The effect of KCl concentration on the yield of ectione

图7 温度对四氢嘧啶产量的影响

Fig. 7 The effect of temperature on the yield of ectione

图8 pH对四氢嘧啶产量的影响

Fig. 8 The effect of pH on the yield of ectione

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新喀里多尼亚弧菌CGJ02-2中四氢嘧啶合成基因簇ectABC的克隆及其功能鉴定

Cloning and Functional Identification of ectABC Gene Cluster from Vibrio neocaledonicus CGJ02-2

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