Anbu Rajan Lawrance, Joseph Toms C, Thampuran Nirmala, James Roswin, Ashok Kumar Kesavan, Viswanathan Chinnusamy, Bansal Kailash C
Microbiology, Fermentation and Biotechnology Division, Central Institute of Fisheries Technology, Cochin 682 029, Kerala, India.
Biotechnol Lett. 2008 Aug;30(8):1403-7. doi: 10.1007/s10529-008-9688-3. Epub 2008 May 17.
The genes involved in the biosynthetic pathway of ectoine (2-methyl-1,4,5,6-tetrahydropyrimidine-4-carboxylic acid) from Bacillus halodurans were cloned as an operon and expressed in E. coli. Analysis of the deduced ectoine biosynthesis cluster amino acid sequence revealed that the ectoine operon contain 2,389 bp, encoded by three genes; ectA, ectB and ectC that encode proteins of 189, 427 and 129 amino acids with deduced molecular masses of 21,048, 47,120 and 14,797 Da respectively. Extracts of induced cells showed two bands at 41 kDa and 17 kDa, possibly corresponding to the products of the later two genes. However the expression of ectA gene could not be ascertained by SDS-PAGE. The activity of the ectA protein was confirmed by an acylation assay. The transgenic E. coli accumulated upto 4.6 mg ectoine/l culture. This is the first report of an engineered E. coli strain carrying the ectoine genes of the alkaliphilic bacterium, B. halodurans.
将嗜碱芽孢杆菌中与依克多因（2-甲基-1,4,5,6-四氢嘧啶-4-羧酸）生物合成途径相关的基因作为一个操纵子进行克隆，并在大肠杆菌中表达。对推导的依克多因生物合成簇氨基酸序列的分析表明，依克多因操纵子包含2389 bp，由三个基因编码；ectA、ectB和ectC，分别编码189、427和129个氨基酸的蛋白质，推导的分子量分别为21,048、47,120和14,797 Da。诱导细胞提取物在41 kDa和17 kDa处显示两条带，可能对应后两个基因的产物。然而，通过SDS-PAGE无法确定ectA基因的表达。通过酰化试验证实了ectA蛋白的活性。转基因大肠杆菌每升培养物中积累高达4.6 mg依克多因。这是关于携带嗜碱细菌嗜碱芽孢杆菌依克多因基因的工程大肠杆菌菌株的首次报道。
