青海湖盐单胞菌Ectoine合成基因簇ectABC的重组共表达

盐单胞菌属(Halomonas)通过胞内积聚有机相容溶质(Compatible solutes)来抵抗胞外的高盐渗透压。为了探究相容溶质Ectoine合成代谢相关基因的结构特征和异源共表达的可能性, 以青海湖盐单胞菌Halomonas sp. QHL1为材料, 通过高效液相色谱(HPLC)分析不同盐梯度下QHL1胞内Ectoine的积聚量, 并借助于染色体步移技术(Genome walking)捕获QHL1菌株的Ectoine生物合成基因簇ectABC, 利用分子克隆技术分析ectABC基因簇的异源重组表达(E.coli BL21)。研究结果表明: 胞内Ectoine的积聚量随着培养基中Na+浓度的增加而增加, 最大积聚量为167.1 mg/g细胞干重(1.0 mol/L Na+), 但菌体生长却受到高浓度Na+的强烈抑制作用。QHL1的ectABC操纵子全长序列为3580 bp, 结构基因ectA(579 bp)、ectB(1269 bp)与ectC (390 bp)串联排列。基于生物信息学预测分析, 两个启动子(70与38因子控制)和若干未知功能的保守模序(Motifs)存在于QHL1的ect操纵子上游。构建重组表达载体pET-28-ectABC, 并在E.coli BL21中异源表达ectABC基因簇(2438 bp)。SDS-PAGE结果显示EctA、EctB和EctC分别为27.2、52.5 和 20.8 kD, 与预测结果一致, 表明ectA、ectB和ectC基因能在E. coli BL21中实现异源共表达, 为构建Ectoine合成代谢基因整合的系统代谢工程, 并实现低盐发酵控制和过量化生产提供了重要的理论基础。  

RECOMBINANT CO-EXPRESSION OF THE ECTOINE BIOSYNTHESIS GENE CLUSTER ectABC IN HALOMONAS FROM QINGHAI LAKE

Halomonas is capable of synthesizing organic compatible solutes ectoine in response to high osmotic pressure. To reveal the possibility of heterologous co-expression of ectoine biosynthesis genes, intracellular ectoine in Halomonas sp. QHL1 strain was determined by HPLC under different salt gradients. The entire ectABC gene cluster for ectoine synthesis was cloned using genome walking and expressed in the heterologous recombinant E. coli BL21. The results showed that the concentration of ectoine accumulated in the cells had a positive correlation with the extracellular Na+ concentration and reached a maximum value (167.1 mg/g cell dry weight) at 1.0 mol/L Na+, and high concentration of Na+ strongly inhibited the bacteria growth. The entire ectABC gene cluster in QHL1 strain was 3580 bp, containing structural gene ectA (579 bp), ectB (1269 bp) and ectC (390 bp). Based on bioinformatics prediction analysis, two putative promoters (70 and 38-controlled promoter) and several conserved motifs with unknown function were identified in the upstream of ect-operon. The recombinant plasmid pET-28a (+)-ectABC was successfully constructed, and the results of heterologous expression indicated that these three genes could be simultaneously translated to protein EctA (27.2 kD), EctB (52.5 kD) and EctC (20.8 kD). These results contribute further improvements in ectoine high yield and hypohaline biotechnological process optimization, and also provided a framework for future genetic manipulation of systems metabolic engineering.