通过同源介导a-淀粉酶基因扩增改良地衣芽孢杆菌a-淀粉酶生产菌株

Genetic improvement of α-amylase producing Bacillus licheniformis by homolog-mediated α-amylase gene amplification

摘要:

地衣芽孢杆菌高温a-淀粉酶(BLA)是淀粉水解与生物加工过程中重要关键酶制剂之一。为了进一步提高地衣芽孢杆菌高温a-淀粉酶生产菌株的生产性能, 本研究构建了一种含有地衣芽孢杆菌高温a-淀粉酶编码基因amyL的整合性重组质粒pBL-amyL。将重组质粒pBL-amyL转化入BLA工业生产菌株Bacillus licheniformis B0204, 再在卡那霉素存在下介导其B. licheniformis B0204染色体中的同源整合与高温a-淀粉酶编码基因amyL的扩增, 由此获得了携带多个amyL拷贝的转化子。对转化子的amyL拷贝数及其BLA发酵水平分别用荧光实时定量PCR及摇瓶发酵试验进行评价与鉴定。与出发菌株B0204相比, 含2~5倍amyL拷贝数的重组菌的BLA的合成水平显著提高。其中, 重组菌REBL18生产BLA的水平提高了89.2%。

Abstract:

Bacillus licheniformis a-amylase (BLA) is one of the most important enzymes involved in starch hydrolysis and many biotechnological processes. To improve the BLA productivity, an integrative plasmid pBL-amyL carrying amyL gene encoding a thermophilic a-amylase of B. licheniformis was constructed and transformed into B. licheniformis B0204, an industrial α-amylase producer. The transformants harboring different copies of amyL were developed on kanamycin by using homolog-mediated chromosomal amplification of a-amylase gene. The recombinants with different multiple copies of amyL integrated in the chromosome were identified by real-time PCR and evaluated by shake-flask fermentation. Recombinants harboring 2?5 multiple copies of amyL produced more a-amylase comparison to the parental strain B0204.