敲除富马酸酶基因对E.coli厌氧混合酸发酵的影响

基于基因工程菌生产丁二酸代谢途径,以E.coli BA001(△ldh,△pfl)为出发菌株,利用RED同源重组技术敲除了富马酸酶基因fumB,得到重组菌E.coli BA002(△ldh,△pfl,△fum),通过减少苹果酸生成富马酸的通量,实现苹果酸的积累.实验结果表明:对比E.coli BA001,敲除富马酸酶基因会较大程度地改变丁二酸、乙酸等的分布,在两阶段和专一性厌氧发酵中,丁二酸产率由81％、63％分别下降为76％、54％,E.coli BA002中乙酸有较大幅度的增加,而苹果酸的产量为0.25 g/L;通过外源添加1g/L的苹果酸,发现丁二酸和乙酸的产量进一步增加.实验实现了富马酸酶基因的敲除:一方面使得乙酸产量明显增加,另一方面厌氧主导酶FumB的敲除不能完全阻断厌氧发酵苹果酸到富马酸途径.

Effect of knockouting fumB on anaerobic mixed acid fermentation for E.coil

Based on the metabolic pathway of succinate,a fumB defection strain,E.coli BA002（△ldh,△pfl,△fum） was constructed,by using λ-RED homologous recombination technology with E.coli BA001（△ldh,△pfl） as the parent strain.The malate accumulated by decreasing the flux of conversion of malate to fumarate.The fermentation results showed that defection of fumB could change the yields of succinate and acetate,the yield of succinate of dual-phase fermentation and exclusively anaerobic fermentation decreased from 81% and 63% to 76% and 54%,respectively.The portion of acetate in E.coli BA002 increased largely,the final concentration of the malate was 0.25 g/L;the yields of acetate and succinate increased further when added extra malic acid.The experiments preliminarily demonstrated that the defection of fumB made the acetate increase obviously.The dominate anaerobic enzyme FumB defection could not block conversion of the malate to the fumarate compeletely.