Department of Bioprocess Technology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia.
Mol Biotechnol. 2013 Jul;54(3):961-8. doi: 10.1007/s12033-013-9647-7.
Regulation of RNA transcription in controlling the expression of genes at promoter and terminator regions is crucial as the interaction of RNA polymerase occurred at both sites. Gene encoding cyclodextrin glycosyltransferase (CGTase) from Bacillus sp. NR5 UPM isolated in the previous study was used for further construction of pTZCGT-SS, pTZCGT-BS and pTZCGT-BT expression systems for enhancement of CGTase production. The putative promoter regions, -35 and -10 sequences were found in the upstream of the mature gene start codon. Whereas, long inverted repeats sequences which can form a stable stem and loop structure was found downstream of the open reading frame (ORF) of Bacillus sp. NR5 UPM CGTase. The construction of E. coli strain harbouring pTZCGT-BS showed increment of 3.2-fold in CGTase activity compared to the wild type producer. However, insertion of terminator downstream of CGTase gene in E. coli strain harbouring pTZCGT-BT only resulted in 4.42 % increment of CGTase production compared to E. coli strain containing pTZCGT-BS, perhaps due to low intrinsic termination efficiency. Thus, it is suggested that the insertion of the putative promoter regions upstream of the coding sequence for the construction of CGTase expression system will further enhance in the recombinant enzyme production.
调控 RNA 转录在控制启动子和终止子区域基因的表达方面至关重要，因为 RNA 聚合酶在这两个位点都发生相互作用。从之前的研究中分离出的芽孢杆菌 NR5 UPM 中的环糊精葡萄糖基转移酶 (CGTase) 基因被用于进一步构建 pTZCGT-SS、pTZCGT-BS 和 pTZCGT-BT 表达系统，以提高 CGTase 的产量。在成熟基因起始密码子的上游发现了假定的启动子区域 -35 和 -10 序列。而在 Bacillus sp. NR5 UPM CGTase 的开放阅读框 (ORF) 下游发现了长的反向重复序列，这些序列可以形成稳定的茎环结构。与野生型产酶菌相比，携带 pTZCGT-BS 的大肠杆菌菌株的构建显示 CGTase 活性增加了 3.2 倍。然而，在携带 pTZCGT-BT 的大肠杆菌菌株中，终止子插入 CGTASE 基因的下游仅导致 CGTase 产量增加了 4.42%，与携带 pTZCGT-BS 的大肠杆菌菌株相比，这可能是由于内在终止效率低所致。因此，建议在构建 CGTase 表达系统时，在上游插入编码序列的假定启动子区域，将进一步提高重组酶的产量。
