【作者】 李宜奎；

【导师】 祁庆生；

【作者基本信息】 山东大学 ， 微生物学， 2014， 博士

【摘要】 琥珀酸是一种重要的平台化合物,位于美国能源部选出的12种(组)具有高附加值的平台化合物之首。作为兼性厌氧菌,现有的利用大肠杆菌工程菌株生产琥珀酸的策略有三种：好氧生产、厌氧发酵和好氧生长-厌氧发酵两阶段法等。鉴于这三个琥珀酸发酵策略的优势与瓶颈,我们构建了一株在好氧、微好氧、及厌氧条件下均生产琥珀酸的大肠杆菌工程菌株,并开发了好氧-微好氧-厌氧全阶段发酵策略。解除低溶氧对琥珀酸好氧产生途径的抑制。通过在好氧产琥珀酸菌株QZ1111中敲除氧气感应系统ArcAB中ArcA组分的编码基因arcA,获得工程菌株QMJ03。QMJ03的琥珀酸产量提高81.65%,副产物乙酸的分泌量下降60%。QMJ03在对数生长期保持一个几乎恒定的琥珀酸产生速率。在溶氧较低的对数生长后期,QMJ03中TCA循环和乙醛酸支路等途径中相关基因较QZ1111均上调。arcA基因的敲除解除了低溶氧对好氧琥珀酸产生途径中相关基因的抑制,有利于微好氧条件下琥珀酸的积累。提高工程菌株厌氧产琥珀酸能力。通过敲除大肠杆菌厌氧混合酸发酵中NADH利用的两条代谢支路一乙醇脱氢酶和乳酸脱氢酶,得到工程菌株QMJ09。QMJ09的厌氧琥珀酸产量较QMJ03提高了2.5倍,而二者好氧琥珀酸产量相当。这样,我们成功构建了一株好氧-厌氧产琥珀酸的大肠杆菌工程菌株。再分配代谢流提高厌氧琥珀酸得率。通过在QMJ09基因组上对磷酸烯醇式丙酮酸羧激酶编码基因(pckA)的启动子进行了点突变(YL102)和在质粒上过量表达NAD+依赖的苹果酸酶(pSCsfcA),以增加磷酸烯醇式丙酮酸和丙酮酸的羧化流量。厌氧阶段的琥珀酸得率由0.95mol/mol Glucose (QMJ09)提高到1.35mol/mol Glucose (YL102/pSCsfcA)。加快葡萄糖的跨膜运输提高琥珀酸产量和生产强度。组成型表达具有高速率、低能耗的运动假单胞杆菌来源的葡萄糖运输载体GlfZm的编码基因(trc-rbs-glfZm),并整合到QMJ09和YL102的基因组上,分别获得工程菌株YL104和YL106,进而获得重组菌株YL104/pSCsfcA和YL106/pSCsfcA。操纵子trc-rbs-glfZm的基因组整合插入不仅提高了全阶段葡萄糖的总利用量,而且提高了全阶段琥珀酸的产量。这说明,加快碳源的跨膜转运对于提高琥珀酸产量和生产强度是关键。其中,YL106/pSCsfcA的琥珀酸产量提高到34.40g/l,厌氧阶段的琥珀酸得率达到1.57mol/mol Glucose,全阶段的琥珀酸得率为1.23mol/mol Glucose。开发好氧-微好氧-厌氧全阶段琥珀酸发酵策略。在51发酵罐中通过控制通气量对YL106/pSCsfcA进行全阶段发酵。在起始的好氧阶段,细胞快速生长,发酵液中琥珀酸的积累基本上与菌株生长相偶联。在微好氧阶段,YL106/pSCsfcA以最大的生产强度积累了最多的琥珀酸。最后,通过停止通气使发酵进入自然厌氧阶段。经过40h全阶段发酵,琥珀酸产量达到85.30g/l,其总得率和总生产强度分别为0.99mol/mol和2.13g/l/h。该总生产强度是利用大肠杆菌生产琥珀酸中最高的。一致化NADH脱氢酶提高工程菌株厌氧琥珀酸生产性能。通过敲除大肠杆菌好氧电子传递链中主要的两个NADH脱氢酶(NDH-II和WrbA),使好氧琥珀酸生产和厌氧琥珀酸发酵中NADH电子传递链中NADH脱氢酶一致化。NADH脱氢酶一致化提高了厌氧阶段葡萄糖消耗和琥珀酸的产量及得率,使厌氧阶段与好氧阶段琥珀酸产量比由0.74逐步提高到1.03和2.28,获得具有提高的厌氧琥珀酸生产能力和稳健的全阶段琥珀酸生产性能的工程菌株YL104NW。本论文还研究了失活细胞色素氧化酶Cyt bo、组成型过表达琥珀酸产生途径中与NADH利用相关的酶(苹果酸酶SfcA和延胡索酸还原酶FrdABCD)、及组成型过表达厌氧琥珀酸发酵途径中的关键基因,对工程菌株好氧-厌氧全阶段发酵过程中的厌氧琥珀酸发酵性能影响。综上所述,本论文构建的在好氧、微好氧及厌氧条件下均能够生产琥珀酸的大肠杆菌和开发的好氧-微好氧-厌氧全阶段琥珀酸发酵策略,很好地解决了好氧琥珀酸生产策略的产物得率低、厌氧琥珀酸发酵策略的发酵周期长、和好氧生长-厌氧发酵两阶段法中琥珀酸生产能力不稳定和总生产强度低的缺点。本研究中获得的总生产强度(2.13g/l/h)是目前利用大肠杆菌生产琥珀酸中最高的。这一全阶段琥珀酸生产过程,不仅能实现高产量、高生产强度和高设备周转率,而且积累的副产物较少,降低了琥珀酸的分离纯化成本,具有可观的工业化应用价值和前景。更多还原

【Abstract】 Succinate (butanedioic acid) is one of the12value-added platform chemicals identified by the US Department of Energy (DOE). Currently, metabolically engineered Escherichia coli has been used for succinate production via aerobic culture, anaerobic fermentation, or the dual-phase strategy (aerobic growth first and then anaerobic succinate production). But these strategies have both advantages and drawbacks. In this project, E. coli strains were engineered for being able to produce succinate from glucose under aerobic, microaerobic and anaerobic conditions, and corresponding processes were developed.Derepressing the inhibition of low dissolved oxygen on aerobic succinate-producing pathways. The gene arc A, encoding the Arc A component of the oxygen sensor ArcAB two-component signal transduction system, was knocked out in an aerobic succinate-producing strain QZ1111, and the engineered strain was named as E. coli QMJ03. Succinate production of QMJ03was increased by81.65%. The acetate secretion was also reduced by60%. Importantly, QMJ03maintained an almost constant succinate production rate during the exponential growth phase. In the late exponential phase, the genes in TCA cycle and glyoxylate shunt were all up-regulated in QMJ03. The genes in glyoxylate shunt and the oxidative branch of TCA cycle were partially or even completely derepressed in QMJ03during the fermentation process. Deletion of arcA gene was beneficial to microaerobic succinate production.Improving the strain capability for anaerobic succinate production. QMJ03was modified genetically to an anaerobic succinate producer by blocking two NADH-utilization pathways, ethanol dehydrogenase (adhE) and lactate dehydrogenase (ldhA), generating E. coli QMJ09. Anaerobic succinate production of QMJ09was improved2.5-fold when compared to that of QMJ03. Aerobic succinate production of QMJ09was comparable to that of QMJ03. So QMJ09was capable to produce succinate under both aerobic and anaerobic conditions. Modulation of PEP/Pyruvate carboxylation flux to enhance the anaerobic succinate yield. A single point mutation in pckA promoter (G to A transition at position-64relative to the ATG start codon of pckA denoted pckA*) was introduced into QMJ09, generating E. coli YL102. Meanwhile, the NAD+-linked malic enzyme SfcA was overexpressed under a constitutive trc promoter with a strong RBS in a low copy-number plasmid pSCsfcA. The strain YL102/pSCsfcA showed the highest anaerobic succinate yield of1.35mol/mol Glucose.Accelerating glucose uptake to increase succinate production and productivity. The glfZm gene, encoding a high velocity and low-energy cost glucose facilitator GlfZm from Zymomonas mobilis ATCC10988, was integrated into the chromosome (single copy) of QMJ09and YL102, generating E. coli YL104and YL106, respectively. SfcA was also overexpressed in YL104and YL106. In both cases, integration of glfZm increased glucose consumption and succinate production greatly. Succinate production of YL106/pSCsfcA was34.40g/l. The results indicate that accelerating glucose transport is critical to the improvement of succinate production and productivity. It is worth to note that anaerobic succinate yield of YL106/pSCsfcA reached1.57mol/mol Glucose, while the overall yiled was1.23mol/mol Glucose.Developing a novel aerobic-microaerobic-anaerobic whole-phase succinate fermentation strategy. In the initial aerobic phase, the cells grew very fast and succinate production was growth associated as expected. During the microaerobic phase, the strain YL106/pSCsfcA accumulated the most succinate with maximum productivity. Finally, the culture was shifted to anaerobic phase by dropping the air flow rate to zero. After40h cultivation, the final succinate concentration reached85.30g/1. For whole-phase fed-batch fermentation, the overall volumetric productivity and molar yield of succinate were2.13g/l/h and0.99mol per mol glucose, respectively. The overall volumetric productivity was the highest to date.Uniforming NADH dehydrogenase to improve the anaerobic succinate-producing capacity. The two main NADH dehydrogenases (NDH-Ⅱ and WrbA) of aerobic electron transfer chain were inactivated to drive NDH-I to be the main participant in aerobic succinate production in order to uniform aerobic and anaerobic NADH dehydrogenase (YL104NW). The process of NADH dehydrogenase uniformity enhanced the anaerobic glucose consumption and succinate production and yield, and gradually improved the ratio of anaerobic succinate production to aerobic succinate production from0.74to1.03and2.28. The engineered strain YL104NW had a robust ability of succinate production in whole-phase fermentation.The effect of inactivating the main cytochrome oxidase Cyt bo in aerobic electron transport chain, overexpressing the NADH-utilization enzymes in succinate fermentation pathways (SfcA and FrdABCD), and the key genes in the reductive arm of TCA cycle (pckA, mdh, fumB, frdABCD) on anaerobic succinate fermentation were also investigated.In the present study, an engineered E. coli was constructed for succinate production from glucose under aerobic, microaerobic and anaerobic conditions, and a novel aerobic-microaerobic-anaerobic whole-phase fermentation strategy was developed for effecient succinate production. The engineered strain and the novel whole-phase fermentation strategy solve the problems well, which are that the carbon conversion ratio in aerobic succinate production is low, the duration of anaerobic succinate fermentation is long, and the succinate production capacity of the dual-phase strategy is not stable and the overall productivity is low. The overall productivity (2.13g/l/h) achieved in the present study is highest in E. coli reported to date. The whole-phase succinate production process not only can achieve high production, high productivity and high turnover of the equipment, but also accumulates less by-products and reduces the cost of the separation and purification of succinate, and so has a significant application value and prospect in industrialization.更多还原