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B12的影响

摘要

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 methyltransferase (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.%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B12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RCcobA1,RCcobA2和脱氮假单胞菌来源的PDcobA,并在VB12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B12发酵结果分析可知,SUMT(PDcobA),SUMT1(RCco-bA1)和SUMT2(RCcobA2)的表达有利于维生素B12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5L发酵罐上进行了放大实验,pBBR122-Pbla-RCcobA2的产量为144.5 mg/L,相比对照菌pBBR122-Pb1a(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B12合成途径中的瓶颈,提高维生素B12产量.