Metabolic Engineering Of Propionibacterium Jensenii And Process Optimiazation To Improve The Production Of Propionic Acid

In this dissertation, Propionibacterium jensenii ATCC4868was used as an initial strain，propionic acid (PA) production was improved by metabolic engineering and fermentationoptimization. An endogenous wild plasmid was screened, sequenced and analyzed. Based onthe information, a P. jensenii-E.coli shuttle expression system was constructed, and thetransformation conditions were optimized. The effect of several key enzymes on intracellularmetabolism and propionic acid production in P. jensenii ATCC4868was investigated byexpressing and knockouting corresponding genes. On the basis of the engineered stain, thecapacity of PA production was improved effectively by a series of fermentation optimization,containing nutritional and culture condition optimization. The main results were described asfollows:P. jensenii ATCC4868is able to produce PA in high yield. To further improve theproduction capacity, a P. jensenii-Escherichia coli shuttle vector was developed for themetabolic engineering of P. jensenii. Specifically, an endogenous plasmid, pZGX01, wasisolated from P. acidipropionici ATCC4875. The sequencing analysis indicated that thepZGX01could encode11proteins. Then, based on the information, a P. jensenii-Escherichiacoli shuttle vector named pZGX04was constructed using the pZGX01plasmid, the E. colipUC18plasmid, and a chloramphenicol-resistant gene. Interestingly, not only the developedshuttle vector could be transformed into P. jensenii ATCC4868and P. jensenii ATCC4870,but it could also be transformed into the freudenreichii ATCC6207subspecies (subsp.) of P.freudenreichii. Finally, the glycerol dehydrogenase gene (gldA) was overexpressed in P.jensenii ATCC4868with the constructed shuttle vector. In3-L fermentor, the PA productionby the engineered P. jensenii (pZGX04-gldA) reached28.23g L-1by optimized two stagepH-shift batch fermentation strategy, which was26.07%higher than that produced by thewild-type strain.PA production with metabolically engineered P. jensenii (pZGX04-gldA) was improved byintegrating fed-batch culture with a two-stage pH control strategy in a3-L fermentor. Atwo-stage pH control strategy was proposed by investigating the influence of different pH oncell growth and PA production. The pH was controlled at5.9during0-36h, and was shifted to6.5after36h. The titer of PA was increased to21.43g L-1. On the basis of two-stage pHcontrol, the influence of fed-batch culture mode on cell growth and PA production was furtherinvestigated, and the maximum PA production (34.62g L-1) were obtained when glycerol (300g L-1) was fed at a constant rate of3.33mL h-1from60to132h with an initial glycerolconcentration of25g L-1.A three-stage oxidoreduction potential (ORP) control strategy was developed to improvepropionic acid (PA) production using engineered P. jensenii ATCC4868(pZGX04-gldA) in a3-L bioreactor. A three-stage ORP control strategy was proposed by investigating the influence of different ORP on cell growth and PA production. ORP was controlled at-200mVfrom0to36h,-300mV from36to156h, and-400mV after156h. The PA titer increasedfrom21.38to27.31g L-1. The effects of ORP regulation on intracellular metabolism werestudied, demonstrating that ORP can both regulate NADH/NAD+ratio and the activities ofsome enzymes involved in electron transport and redistribute metabolic flux. The ORP controlstrategy was integrated with two pH-shift control strategy and fed-batch culture strategy, PAproduction was increased to39.53g L-1.Gene codes phosphoenolpyruvate carboxylase (ppc) was expressed and gene codes lactatedehydrogenase (ldh) was knockout in P. jensenii ATCC4868, respectively, and its effect oncell growth, intracellular metabolism and PA production was investigated. Compared to thewild type, two stage pH-shift control combined with fed-batch fermentation analysis revealedthat the PA production with P. jensenii-Δldh (pZGX04-ppc) increased29.61%. The effects ofthe new metabolic pathway on intracellular metabolism were also investigated. The resultsindicated that the expression of phosphoenolpyruvate carboxylase and knockout of lactatedehydrogenase affected intracellular NADH/NAD+ratio, and further influenced intracellularmetablic flux, and finally affected the cell growth and PA biosynthesis.Two rate-limiting enzymes were co-expressed to improve the production of PA.Rate-limiting metabolites for PA synthesis in P. jensenii were identified by examining theinfluence of metabolic intermediates addition on the synthesis of PA; and then furtherimproved PA production by over-expression of the identified rate-limiting enzymes, namely,glycerol dehydrogenase (GDH), malate dehydrogenase (MDH), and fumarase (FUM).Compared to the wild type, the activities of the enzymes in the engineered strain wereincreased by3.85-8.11folds. The transcription levels of corresponding enzymes in theengineered strains were2.91-8.11folds of that in the wild type. Two stage pH-shift controlcombined with fed-batch fermentation analysis revealed that the co-expression of GDH andMDH increased the PA titer to46.41g L-1.