【作者】 朱年青；

【导师】 赵学明；

【作者基本信息】 天津大学 ， 生物化工， 2013， 博士

【摘要】 本文围绕琥珀酸的生物合成，以野生型谷氨酸棒杆菌Corynebacteriumglutamicum ATCC13032为出发菌株进行系统代谢工程改造，构建了一系列能够在厌氧和好氧条件下高效生产琥珀酸的突变株。通过中心碳代谢的比较分析和单基因敲除分析对琥珀酸厌氧合成的途径进行了鉴定。采用干实验基元模式分析和湿实验代谢通量分析相结合的方式鉴定出基因靶点并进行改造，通过发酵和通量分析验证靶点预测的准确性。利用两阶段高密度发酵的方式来提高琥珀酸的浓度。另一方面，通过引入枯草芽孢杆菌的乙酸利用途径减少好氧条件下乙酸的积累。同时过表达柠檬酸合酶减少丙酮酸的积累，增加TCA循环的通量。最后，通过分批补料的培养方式评价好氧生产琥珀酸的可行性。得到的主要结果如下：对C. glutamicum ATCC13032进行了厌氧生产琥珀酸的途径鉴定。比较C.glutamicum ATCC13032和自然界中分离的琥珀酸生产菌株的中心代谢途径，通过理性分析得到潜在的影响琥珀酸合成的基因靶点。通过单基因敲除和厌氧发酵，证明了野生型C. glutamicum ATCC13032在厌氧条件下利用TCA还原臂合成琥珀酸。采用组合敲除策略，敲除乳酸脱氢酶、乙酸激酶、磷酸转乙酰酶、丙酮酸氧化还原酶和乙酰辅酶A转移酶后，突变株C. glutamicum SAZ1的乙酸产量显著降低，琥珀酸得率为0.99mol (mol glucose)-1。基于基因组尺度规模的谷氨酸棒杆菌模型，构建了一个精简的中心碳代谢网络模型，通过基元模式分析和突变株C. glutamicum SAZ1的代谢通量分析，鉴定出三个基因靶点：回补反应、乙醛酸循环和柠檬酸合酶。根据预测依次改造并发酵验证。进一步过表达琥珀酸输出蛋白得到突变株C. glutamicum SAZ2（pEpycgltAsucE，pXaceAB），琥珀酸得率为1.43mol (mol glucose)-1。它的通量分布与最佳通量分布更为接近。通过胞内代谢物测定分析，发现是由于胞内的琥珀酸含量下降，从而减轻了对乙醛酸循环的抑制作用。采用高密度厌氧发酵，琥珀酸产量达到930mM （110g L-1），得率为1.32mol (mol glucose)-1，生产率为9.48mM h-1。失活琥珀酸脱氢酶复合体、乙酸和乳酸生成途径，过表达丙酮酸羧化酶和磷酸烯醇式丙酮酸羧化酶，引入来自于枯草芽孢杆菌的乙酸利用途径，过表达自身的柠檬酸合酶后，突变株C. glutamicum ZX1(pEacsAgltA)在好氧条件下的琥珀酸得率为0.61mol (mol glucose)-1。在分批补料好氧培养过程中，琥珀酸浓度达到241mM （28g L-1），得率为0.63mol (mol glucose)-1。通过引入外源阿拉伯糖利用途径，突变株能够在好氧条件下利用阿拉伯糖为唯一碳源生产琥珀酸。更多还原

【Abstract】 Starting with the wild-type Corynebacterium glutamicum ATCC13032, a seriesof metabolically engineered C. glutamicum mutants were constructed that couldefficiently produce succinate under anaerobic conditions and aerobic conditions. Withthe comparative analysis of central carbon metabolism and single gene disruptionanalysis, the pathway for anaerobic biosynthesis of succinate was identified. Severalgene targets were predicted by the combination of elementary mode analysis andexperimental metabolic flux analysis, and all of them were genetically engineered stepby step. Subsequently, the designed mutants were experimentally tested via anaerobicfermentation and metabolic flux analysis to confirm the accuracy of the prediction.With the aim at increasing succinate titer, high-cell-density fermentation was carriedout. On the other hand, a pathway from Bacillus subtilis for acetate assimilation wasintroduced into succinate-producing C. glutamicum to recycle wasted carbon underaerobic conditions. The citrate synthase was further overexpressed to reduce pyruvateaccumulation and increase TCA cycle flux. Finally, the feasibility of aerobicproduction of succinate at large scale was evaluated by fed-batch culture. The mainresults were shown as follows:Through the comparison of central metabolism of C. glutamicum and naturallyisolated succinate producers and rational analysis, potential targets that involved insuccinate production were recognized. After testing these single gene disruptionstrains, the reductive TCA cycle was identified as the major pathway for anaerobicsuccinate production. Inactivation of the enzymes of pyruvate: quinoneoxidoreductase, acetyltransferase, acetyl-CoA: CoA transferase and L-lactatedehydrogenase, acetate production by strain C. glutamicum SAZ1was significantlyreduced and succinate yield of0.99mol (mol glucose)-1was achieved.A refined metabolic network was constructed based on the genome-scalenetwork. Three targets, carboxylation pathway, glyoxylate pathway and citratesynthase were identified by elementary mode analysis and metabolic flux analysis.Based on prediction, metabolic engineering and fermentation were performed. Furtheroverexpression of succinate exporter leading to strain C. glutamicum SAZ2(pEpycgltAsucE，pXaceAB), which produced succinate with a yield of1.43mol (molglucose)-1and had more identical flux distribution to the optimal EMs. The reductionof intracellular succinate alleviated its restriction to glyoxylate pathway. Usingtwo-stage high-cell-density fermentation, succinate titer up to930mM was achieved with a yield of1.32mol (mol glucose)-1and a productivity of9.48mM h-1.By inactivating the succinate dehydrogenase complex, the pathways for acetateand lactate formation, replacing the native promoters of pyruvate carboxylase andPEP carboxylase with strong promoter sod, introducing the acetate assimilationpathway from B. subtilis and overexpressing the native citrate synthase, the succinateyield of strain C. glutamicum ZX1(pEacsAgltA) increased to0.61mol (molglucose)-1. In fed-batch culture,241mM succinate with a yied of0.63mol (molglucose)-1was produced. The araBAD operon from Escherichia coli was introducedinto succinate-producing C. glutamicum, which enabled aerobic production ofsuccinate using arabinose as sole carbon source.更多还原