丙氨酸转氨酶修饰对大肠杆菌L-色氨酸合成的影响

探究大肠杆菌细胞内负责L-丙氨酸合成的转氨酶对菌株代谢及L-色氨酸合成的影响。运用Red重组技术分别对编码L-丙氨酸转氨酶的基因alaA、alaC和avtA进行敲除。通过摇瓶和50 L罐中探究其对L-色氨酸积累、L-丙氨酸代谢及菌体生长变化情况。结果显示,除3种L-丙氨酸转氨酶全部缺失的工程菌L-丙氨酸合成受阻、菌体生长受到较强抑制外,其它各任意一种或两种丙氨酸转氨酶缺失菌株的生长并未有较大差异,但色氨酸的合成变化显著。其中alaA和alaC双基因缺失的E.coli FS-T4工程菌,摇瓶发酵L-色氨酸产量达6.08 g/L,L-丙氨酸含量仅0.16 g/L,较出发菌株分别提高了26.7%和降低了91.0%。在50 L罐中E.coli FS-T4工程菌L-色氨酸产量最高可达41.9 g/L,糖酸转化率达20.5%,分别较出发菌株提高了13.8%和5.1%。转氨酶AlaA和AlaC的同时缺失,既可以满足细胞整体氨基酸池的需要,而且有利于减少杂酸的积累,使得更多的碳源流向L-色氨酸的合成。

Effects of Modification of Alanine Aminotransferase on Synthesis of L-tryptophan in Escherichia coli

This work is to explore the effects of transaminase for L-alanine synthesis in Escherichia coli on the metabolism and L-tryptophan synthesis of the strain.Three genes(alaA,alaC and avtA)encoding L-alanine aminotransferase were knocked out by Red recombination technique.Fermentation experiments of shaking flask and 50 L fermenter were used to investigate the accumulation of L-tryptophan,L-alanine metabolism and cell growth.Results showed that the growth of the cells and L-tryptophan synthesis were strongly inhibited in these 3 genes deficient engineering strain E.coli FS-T0ΔalaAΔalaCΔavtA;however,the influences on the growth of single or both L-alanine aminotransferase-deficient strains were very little,but L-tryptophan synthesis varied significantly.By E.coli FS-T4ΔalaAΔalaC engineering strain,the yield of L-tryptophan was 6.08 g/L while the L-alanine production was only 0.16 g/L,which increased by 26.7%and decreased by 91.0%,respectively compared with the original strain.In the 50-L fermenter,the yield of L-tryptophan further increased to 41.9 g/L,and glucose conversion rate was 20.5%,which was 13.8%and 5.1%higher than the original strain,respectively.Deficiency of both AlaA and AlaC transaminase will not affect the needs of the whole cell amino acid pool,and is conducive to reduce the accumulation of miscellaneous acids,thus leading more carbon sources into L-tryptophan synthesis metabolic flux.