| 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-â…¡ 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. |
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