不同来源的尿卟啉原Ⅲ转甲基酶在脱氮假单胞菌中的表达及其对生产维生素B_(12)的影响

Over-expression uroporphyrinogen Ⅲ methyltransferase from different strains in Pseudomonas denitrificans and their effects on vitamin B_(12) production

S-腺苷-L-甲硫氨酸依赖型尿卟啉原Ⅲ转甲基酶(S-adenosy-L-methionine uroprophyrinogen Ⅲ methyltransferase,SUMT)催化尿卟啉原Ⅲ(uroprophyrinogen Ⅲ,urogen Ⅲ)中心碳原子C-2和C-7甲基化生成前咕啉-2,是维生素B_(12)生物合成途径中的一步关键酶。本文分别克隆了荚膜红细菌来源的RCcob A1,RCcob A2和脱氮假单胞菌来源的PDcob A,并在VB_(12)生产菌株脱氮假单胞菌中过表达和发酵。通过对三株重组菌维生素B_(12)发酵结果分析可知,SUMT(PDcob A),SUMT1(RCcob A1)和SUMT2(RCcob A2)的表达有利于维生素B_(12)产量的提高,与对照菌株相比分别提高了16.48%,10.2%和31.86%。根据摇瓶发酵的结果在5 L发酵罐上进行了放大实验,p BBR122-PblaRCcob A2的产量为144.5 mg/L,相比对照菌p BBR122-Pbla(111.3 mg/L)产量提高了29.83%左右。结论:SUMT的表达可以在一定程度上解除维生素B_(12)合成途径中的瓶颈,提高维生素B_(12)产量。

The carbon atoms in the center position of C-2 and C-7 of uroprophyrinogen III （ urogen III） can be methylated to produce precorrin-2 by S-adenosy-L-methionine uroprophyrinogen III methyhransferase （SUMT）, which is the key enzyme of vitamin B12 （ VB12 ） biosynthetic pathway. In this study, RCcobA1 and RCcobA2 from Rhodobacter capsulatus and PDcobA from Pseudomonas denitrificans were cloned and over-expressed in VB12-producing strains P. denitrificans. The resulting strains PD1/2/3/4 were fermented in shake flasks. The results showed that the VB12 production of PD2 （overexpressing PDcobA ） increased 16. 48%, the VB12 production of PD3 （overexpressing RCcobA1 ） and PD4 （overexpressing RCcobA2） increased 10.2% and 31.86% when compared with the control strain （PD1） , respectively. According to the results of shaking flasks fermentation, the fermentation of the strains PD1 and PD4 in 5 L fermentor was conducted. The output of VB12 of PD4 was 144.5 mg/L, which increased by 29.83% comparing to that of PD1 （ 111.3 mg/L）. It revealed that the overexpression of cobA2, to a certain extent, could release the bottlenecks of VB12 synthetic pathway and increase the production of VB12.