为研究7株克雷伯氏菌发酵棉秆水解糖液产丁二酸的关键途径及其竞争支路的代谢流分布,实现对菌株高效发酵棉秆水解糖液产丁二酸过程的有效调控。采用7株克雷伯氏菌发酵棉秆水解糖液,监测了各菌株在6、12、18、24、30和36 h的丁二酸含量、葡萄糖和木糖含量、琥珀酸脱氢酶和磷酸烯醇式丙酮酸羧化酶活性及胞内外丙酮酸和乳酸浓度,阐明了这些菌株发酵产丁二酸的代谢特性。结果表明,7株克雷伯氏菌在6~36 h内均能有效发酵棉秆水解糖液产丁二酸,且其发酵产丁二酸的时间集中于6~12 h,菌株WL1309和WL1312在发酵6 h即获得了较高的丁二酸产量(>42 g/L),其余5株细菌在发酵12 h的丁二酸含量均达峰值。至发酵结束(36 h),这7株菌的葡萄糖利用率均高于85%,木糖利用率均高于90%。各菌株琥珀酸脱氢酶活性均较低,磷酸烯醇式丙酮酸羧化酶主要在发酵6~12 h及24~36 h对丁二酸合成的影响较强。丙酮酸节点可能在发酵中后期(24~36 h)加速了丁二酸的转化,引起丁二酸合成支路代谢流分布的降低,而乳酸对其代谢流分布的影响较弱。因此,这7株克雷伯氏菌都具有高效发酵棉秆水解糖液产丁二酸的潜力,在其发酵过程中,对其关键酶和主要节点代谢物进行调控可在一定程度上实现丁二酸的高产。
To clarify the key pathways and the metabolic flux distribution of the competing branches of seven Klebsiella strains duringcotton stalk hydrolyzed sugar liquid fermentation to produce succinic acid,and to achieve high-efficiency regulation,the contents of succinic acid,glucose and xylose,the activities of succinic acid dehydrogenase and phosphoenolpyruvate carboxylase,and the concentrations of pyruvate and lactic acid of each strain at 6,12,18,24,30 and 36 h were monitored,and the metabolic characteristics were clarified.The results showed that all Klebsiella strains could effectively ferment cotton stalk to produce succinic acid in 6-36 h,and the fermentation time of succinic acid production was mostly in 6-12 h.WL1309 and WL1312 obtained higher succinic acid production (>42 g/L) at 6 h,and the succinic acid content of the other strains peaked at 12 h of fermentation.By the end of fermentation (36 h),the glucose utilization rate of all strains was higher than 85%,and the utilization rate of xylose was higher than 90%.The succinate dehydrogenase activity of each strain was low.The phosphoenolpyruvate carboxylase mainly affected the synthesis of succinic acid at 6-12 h and 24-36 h.The pyruvate node may accelerate the conversion of succinic acid in the middle and late stages of fermentation (24~36 h),causing the decrease of the metabolic flux distribution of the succinic acid synthesis branch,while the influence of lactic acid on the metabolic flux distribution was weak.During the fermentation process,the high yield of succinic acid can be achieved to a certain extent by regulating the key enzymes and major metabolites in the fermentation process.
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