Metabolic Engineering Of Saccharomyces Cerevisiae For The Production Of Fumaric Acid

In this dissertation, we used a multi-nutrition auxotroph (leucine, tryptophan, histidine, uracil)Saccharomyces cerevisiae CEN.PK2-1C as a model to study how to achieve high yields of fumaric acidproduction. Based on the application of metabolic engineering and biochemical engineering principle andmethod, combined with genome-scale metabolic model (GSMM) of S. cerevisiae iND750. Theconstruction of pyruvate pool, the exploration of synthetic pathway for fumaric acid, and the conversion ofcarbon flow to the synthetic pathway of fumaric acid from pyruvate were investigated in detail. The mainresults were described as follows:(1) A novel strategy to improve pyruvate production via regulating thiamine synthesis was explored. Two ofthe thiamine biosynthesis regulatory genes, THI2and THI3, were disrupted in the S. cerevisiae parent strainFMME-002. The mutants FMME-002THI2and FMME-002THI3both exhibited an enhanced pyruvateyield. Moreover, the FMME-002THI2achieved a relatively higher pyruvate production (3.38±0.22g/l),and the highest concentration of pyruvate (3.82±0.14g/l) was achieved in36h when0.04mol/l thiaminewas added. To elucidate the metabolic alterations associated with THI2deletion and thiamine addition, thespecific activity of PDC was measured at various times during the fermentation process. Study showed thatit had a dramatical decrease after THI2deletion while the PDC activity of FMME-002THI2was increasedby10.5%when0.04mol/l thiamine was added. Similar fermentation profiles showed by theTHI2-complemented strain and the parent strain indicated that the observed metabolic changes representedintrinsic effects of THI2deletion on the physiology of S. cerevisiae. Then the FMME-002THI2producedpyruvate up to8.21±0.30g/l whereas ethanol titer decreased to2.21±0.24g/l when2.00g/l urea wasused and C/N ratio was set at35:2after96h cultivation.(2) A completely cytosolic fumarate biosynthetic pathway of R. oryzae was introduced in S. cerevisiae forfumarate accumulation. Firstly, comparative analysis of two species malate dehydrogenases on fumarateproduction capabilities was performed, the corresponding engineered strains, FMME-001↑MDH3SKLachieved an average of0.44±0.03g/l, while FMME-001↑RoMDH achieved an average of0.54±0.04g/l.The higher YP/Svalue associated with the FMME-001↑RoMDH strain led us to focus our subsequentstudies on the cytosolic malate dehydrogenase encoded by the RoMDH. The1H NMR spectra and13CNMR spectra showed excellent matches between the sample of the FMME-001↑RoMDH strain andfumaric acid standard, which confirmed that fumaric acid was, in fact, synthesized by our engineered S.cerevisiae strain. In order to augment the fumarate synthesis ability of FMME-001↑RoMDH, the R. oryzaeRoFUM1gene encoding cytosolic fumarase was simultaneously over-expressed, However, theco-expression strain FMME-001↑RoMDH+↑RoFUM1did not exhibit a significantly higher titer offumarate as expected; instead, the titer decreased to0.38±0.03g/l. The gene expression levels of RoMDHand RoFUM1were dramatically increased, as detected by quantitative real-time PCR, Taken together, theseresults suggest that pyruvate carboxylase represents the rate limiting factor of fumarate production. Theintegration-expression of PYC2in the engineered strain FMME-001↑RoMDH resulted in increasedfumarate titer by a remarkable488.9%(from0.54±0.04g/l to3.18±0.15g/l).(3) We report on the production of fumaric acid by direct fermentation of metabolically engineeredSaccharomyces cerevisiae based on in silico analysis of a genome-scale metabolic model. Firstly, the genesFUM1, ACR1, SDH1-4, OSM1, were selected as the candidate genes based on bibliomic survey, combinedwith metabolic pathway analysis. FBA analysis revealed that only the deletion of FUM1gene can lead tofumaric acid production and slightly lower growth of S. cerevisae. Then an oxidative production route forfumaric acid was investigated by deleting FUM1, the obtained engineered S. cerevisiae strain can producefumaric acid up to a concentration of0.61g/l (0.018mmol/mmol glucose) without any apparent change ingrowth in fed-batch culture. To further improve fumaric acid production, the addition of TCA intermediatemetabolites and amino acids were systematically simulated. Result showed that a greater production rate offumaric acid can be achieved when citric acid was added. Then0.82g/l fumaric acid was obtained when5g/l citric acid was added, which resulted in increased fumarate tier by34.6%.(4) Based on the consideration that the engineered strain can accumulate pyruvate, to obtain a higher yield of fumaric acid, simultaneous use of the reductive TCA route and the oxidative route was performed. Theoxidative production of fumaric acid in S. cerevisiae by deletion of the gene FUM1was explored, and0.25±0.01g/l of fumaric acid was achieved when FMME003was used as original strain. When the cytosolicreductive TCA route was introduced by overexpressing RoMDH and RoFUM1, the fumaric that producedby the oxidative route was almost not detected. Then we studied the effect of TCA intermediate metaboliteson fumaric acid production, results indicated that only the addition of malic acid can improve fumaric acidproduction apparently (from less than0.01g/l to0.42±0.02g/l). These results indicated that much morecarbon flow is required to channel to malate from pyruvate if our goal is high-yield production of fumaricacid.(5) The effect of pyruvate carboxylase channeling the carbon flow to fumaric acid synthetic pathway frompyruvate was explored. Firstly, the effects of endogenous pyruvate carboxylase encoded by PYC2andheterologous pyruvate carboxylase encoded by RoPYC on the production of fumaric acid was compared,results showed that pyruvate carboxylase encoded by RoPYC harbored a better effect. Then the effect ofbiotin concentration on the fumaric acid production of the engineered strain FMME-004-6was performed,and we found that fumaric acid increase from2.48±0.12g/l to3.48±0.13g/l when the fermentation brothsupplemented with32g/l biotin. Then a higher fumaric acid titer (3.92±0.15g/l) was achieved when theconcentration of MgSO4·7H2O increased to1.2g/l from0.8g/l. In addition, the effect of C/N ration onfumaric acid production was implemented. A final concentration of fumaric acid (5.64±0.16g/l) wasachieved when the urea decreased from2g/l to0.1g/l, which resulted in increased fumaric acid titer by43.9%.