腺苷钴胺素合成酶基因cobs的克隆及其在产1,3-丙二醇菌中的应用

甘油脱水酶是催化由甘油到1,3-丙二醇过程中的关键酶,它需要在辅酶B_(12)存在的情况下才能有效的进行催化;而在此催化过程中甘油脱水酶会出现失活现象,研究表明辅酶B_(12)可以有效的促使甘油脱水酶复活。因此,辅酶B_(12)在由甘油生物催化生产1,3-丙二醇过程中起到非常重要的作用。本研究利用PCR扩增技术,从Escherichia K-12菌株中扩增出产VB_(12)关键酶—腺苷钴胺素合成酶基因cobs,其序列与NCBI上已经公布的序列比对,同源性为99.6%,将基因cobs与产1,3-丙二醇关键酶基因dhaB、yqhD在Klebsiella pneumoniae中共表达,发酵结果显示重组菌所需额外添加的VB_(12)由原始菌株的0.01 g/L下降到0.004 g/L。

Cloning of cobalamin-5-phosphate synthase gene cobs and its expression in Klebsiella pneumoniae

The coenzyme B12-dependent glycerol dehydratase is the key enzyme in the transformation of glycerol to 1,3- propanediol, and it was subject to suicide by the natural substrate glycerol during catalysis. Studies showed that coenzyme B12 could effectively promote revival of glycerol dehydratase. Therefore, cocnzyme B12 played a very important role in the process of glycerol to 1,3-propanediol catalyzed by microorganisms. In this study the cobalamin-5-phosphate synthase gene cobs was amplified from Escherichia coli K-12 by using PCR method, which was 99.6% similarity with cobs sequence from NCBI. The gene cobs with genes dhaB and yqhD, key enzymes for producing 1,3-PD, were co-expressed in K. pneumoniae. Compared with the original strain, the recombinant bacteria required less additional VB12, reduced from 0.01 g/L to 0. 004 g/L by preliminary shake-flask fermentation.