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首页> 外文期刊>Applied and Environmental Microbiology >Comparative Metabolic Flux Analysis of Lysine-Producing Corynebacterium glutamicum Cultured on Glucose or Fructose
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Comparative Metabolic Flux Analysis of Lysine-Producing Corynebacterium glutamicum Cultured on Glucose or Fructose

机译：在葡萄糖或果糖上培养的赖氨酸生产谷氨酸棒杆菌的比较代谢通量分析

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摘要

A comprehensive approach to 13C tracer studies, labeling measurements by gas chromatography-mass spectrometry, metabolite balancing, and isotopomer modeling, was applied for comparative metabolic network analysis of lysine-producing Corynebacterium glutamicum on glucose or fructose. Significantly reduced yields of lysine and biomass and enhanced formation of dihydroxyacetone, glycerol, and lactate in comparison to those for glucose resulted on fructose. Metabolic flux analysis revealed drastic differences in intracellular flux depending on the carbon source applied. On fructose, flux through the pentose phosphate pathway (PPP) was only 14.4% of the total substrate uptake flux and therefore markedly decreased compared to that for glucose (62.0%). This result is due mainly to (i) the predominance of phosphoenolpyruvate-dependent phosphotransferase systems for fructose uptake (PTSFructose) (92.3%), resulting in a major entry of fructose via fructose 1,6-bisphosphate, and (ii) the inactivity of fructose 1,6-bisphosphatase (0.0%). The uptake of fructose during flux via PTSMannose was only 7.7%. In glucose-grown cells, the flux through pyruvate dehydrogenase (70.9%) was much less than that in fructose-grown cells (95.2%). Accordingly, flux through the tricarboxylic acid cycle was decreased on glucose. Normalized to that for glucose uptake, the supply of NADPH during flux was only 112.4% on fructose compared to 176.9% on glucose, which might explain the substantially lower lysine yield of C. glutamicum on fructose. Balancing NADPH levels even revealed an apparent deficiency of NADPH on fructose, which is probably overcome by in vivo activity of malic enzyme. Based on these results, potential targets could be identified for optimization of lysine production by C. glutamicum on fructose, involving (i) modification of flux through the two PTS for fructose uptake, (ii) amplification of fructose 1,6-bisphosphatase to increase flux through the PPP, and (iii) knockout of a not-yet-annotated gene encoding dihydroxyacetone phosphatase or kinase activity to suppress overflow metabolism. Statistical evaluation revealed high precision of the estimates of flux, so the observed differences for metabolic flux are clearly substrate specific.
机译：采用13C示踪剂研究的综合方法，通过气相色谱-质谱法进行标记测量，代谢物平衡和同位素异构体建模，可用于在葡萄糖或果糖上比较赖氨酸生产谷氨酸棒杆菌的代谢网络。与果糖相比，与葡萄糖相比，赖氨酸和生物质的产量显着降低，二羟基丙酮，甘油和乳酸的形成增加。代谢通量分析表明，取决于所应用的碳源，细胞内通量存在巨大差异。在果糖上，通过戊糖磷酸途径（PPP）的通量仅占总底物吸收通量的14.4％，因此与葡萄糖相比（62.0％）显着降低。该结果主要是由于（i）磷酸烯醇丙酮酸依赖性磷酸转移酶系统在果糖摄取（PTSF果糖）（92.3％）方面占主导地位，导致果糖主要通过果糖1,6-双磷酸酯进入，以及（ii）无活性果糖1，6-双磷酸酶（0.0％）。通过PTSM葡萄糖通量期间果糖的吸收仅为7.7％。在葡萄糖生长的细胞中，通过丙酮酸脱氢酶的通量（70.9％）远小于在果糖生长的细胞中（95.2％）。因此，通过三羧酸循环的通量在葡萄糖上降低。归一化为葡萄糖摄取，果糖中NADPH的供应量仅为果糖的112.4％，而葡萄糖为176.9％，这可能解释了谷氨酸棒状杆菌在果糖上赖氨酸的产量要低得多。平衡NADPH水平甚至显示出果糖上NADPH的明显缺乏，这可能是苹果酸酶的体内活性所克服的。根据这些结果，可以确定潜在的靶点，以优化谷氨酸棒杆菌对果糖的赖氨酸生产，包括（i）修改通过两个PTS的通量以吸收果糖，（ii）扩增果糖1,6-双磷酸酶以增加通过PPP的流量，以及（iii）敲除尚未注释的编码二羟基丙酮磷酸酶或激酶活性的基因，以抑制溢出代谢。统计评估显示通量估算值的高精度，因此观察到的代谢通量差异显然是底物特异性的。

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