Systems Metabolic Engineering Of Torulopsis Glabrata For Fumarate Production

In this dissertation, a pyruvate and α-ketoglutarate producer, a multi-vitamin(thiamine, biotin, pyrodoxin and nicotinic acid) auxotroph Torulopsis glabrata CCTCC M202019 was useded as a model system to produce fumarate from pyruvate or α-ketoglutarate. This aim can be realized by combination of systems metabolic engineering and synthetic biotechnology. In this way, some challenges in metabolic engineering can be improved at least three aspects: engineering fast and efficient metabolic pathways, enhancing intermediate metabolites transmission efficiency in metabolic pathways, and reducing carbon flux loss by repressing byproducts production. The main results were described as follows:1. The yeast T. glabrata CCTCC M202019, which is used for industrial pyruvate production, was chosen to explore the suitability of engineering this multi-vitamin auxotrophic yeast for increased malate production. Various metabolic engineering strategies were used to manipulate carbon flux from pyruvate to malate:(i) overexpression of pyruvate carboxylase and malate dehydrogenase;(ii) identification of the bottleneck in malate production by model i NX804;(iii) simultaneous overexpression of genes Ro PYC, Ro MDH and Sp MAE1. Using these strategies, 8.5 g/L malate was accumulated in the engineered strain T.G-PMS, which was about 10-fold greater than that of the control strain T.G-26. The results presented here suggest T. glabrata CCTCC M202019 is a promising candidate for industrial malate production.2. Fumarate is a well-known biomass building block chemical. The substrate specificity of fumarase is one of the major factors preventing its widespread production in the engineered microorganisms. To address this issue, 159HPND162 of the fumarase from Rhizopus oryzae were selected in this study as targets for site-directed mutagenesis based on molecular docking. Twelve mutants(carrying the mutations H159 S, H159 Y, H159 V, P160 A, P160 H, P160 T, N161 R, N161 E, N161 F, D162 W, D162 K, and D162M) were generated and characterized in detail. Kinetic studies showed that the Km values of the P160 A, P160 T, P160 H, N161 E, and D162 W mutants decreased by 53.2%, 39.0%, 2.6%, 72.7%, and 62.3%, whereas that of the mutants H159 Y, H159 V, H159 S, N161 R, N161 F, D162 K, and D162 M increased by 123.4%, 120.8%, 36.4%, 39.0%, 58.4%, 89.6%, and 45.5%, respectively, compared to those of the wild-type enzyme. In addition, all mutants displayed declined catalytic efficiency except for the P160 A mutant that kcat/Km was increased by 33.2%. Mechanisms that could account for these changes were explored. Moreover, the production of fumarate was enhanced by overexpressing P160 A mutant, and the final engineered strain, T.G-PMS-P160 A, was able to produce 5.2 g/L fumarate,which was about 6.6-fold greater than that of the control strain T.G-PMS. The mutants generated in this study have potential applications in the fumarate industry.3. Microbial fumarate production from renewable feedstock is a promising and sustainable alternative to petroleum-based chemical synthesis. Here, mitochondrial engineering was used to construct the oxidative pathway for fumarate production starting from the TCA cycle intermediate α-ketoglutarate in T. glabrata. Accordingly, α-ketoglutarate dehydrogenase complex(KGD), succinyl-Co A synthetase(SUCLG), and succinate dehydrogenase(SDH) were selected to be manipulated for strengthening the oxidative pathway, and the engineered strain T.G-K-S-S exhibited increased fumarate biosynthesis(1.81 g/L). To further improve fumarate production, the oxidative route was optimized. First, three fusion proteins KGD2-SUCLG2, SUCLG2-SDH1 and KGD2-SDH1 were constructed, and KGD2-SUCLG2 led to improved fumarate production(4.24 g/L). In addition, various strengths of KGD2-SUCLG2 and SDH1 expression cassettes were designed by combinations of promoter strengths and copy numbers, resulting in a large increase in fumarate production(from 4.24 g/L to 8.24 g/L). Then, through determining intracellular amino acids and its related gene expression levels, argininosuccinate lyase in the urea cycle was identified as the key factor for restricting higher fumarate production. Correspondingly, after overexpression of it, the fumarate production was further increased to 9.96 g/L. Next, two dicarboxylic acids transporters facilitated an improvement of fumarate production, and, as a result, the final strain T.G-KS(H)-S(M)-A-2S reached fumarate titer of 15.76 g/L. This strategy described here paves the way to the development of an efficient pathway for microbial production of fumarate.4. A multi-vitamin auxotrophic T. glabrata strain, a pyruvate producer, was further engineered to produce fumaric acid. Using the genome-scale metabolic model i NX804 of T. glabrata, four fumaric acid biosynthetic pathways, involving the four cytosolic enzymes, argininosuccinate lyase(ASL), adenylosuccinate lyase(ADSL), fumarylacetoacetase(FAA) and fumarase(FUM1), were found. Athough single overexpression of each of the four enzymes in the cytosol improved fumaric acid production, the highest fumaric acid titer(5.62 g/L) was obtained with strain T.G-ASL(H)-ADSL(L) by controlling the strength of ASL at a high level and ADSL at a low level. In order to further improve the production of fumaric acid, the Sp MAE1 gene encoding the C4-dicarboxylic acids transporter was overexpressed in strain T.G-ASL(H)-ADSL(L)-Sp MAE1 and the final fumaric acid titer increased to 8.83 g/L. This study provides a novel strategy for fumaric acid biosynthesis by utilizing the urea cycle and the purine nucleotide cycle to enhance the bridge between carbon metabolism and nitrogen metabolism.5. Microbial fumarate production from renewable feedstock is a promising and sustainable alternative to petroleum-based chemical synthesis. Here, we report a modular engineering approach that systematically removed metabolic pathway bottlenecks and led to significant titre improvements in a multi-gene fumarate metabolic pathway. On the basis of central pathway architecture, fumarate biosynthesis was re-cast into three modules: reduction module, oxidation module, and byproduct module. We analyzed the module interactions between reduction module and oxidation module, and targeted to the cytoplasm and the mitochondria for enhanced metabolic flux channeling to fumarate biosynthesis, respectively. Combinatorially tuning pathway efficiency of reduction module and oxidation module by constructing protein fusions Ro MDH-P160 A and KGD2-SUCLG2 and optimizing gene Ro MDH-P160 A and KGD2-SUCLG2 expression strengths led to significantly improved fumarate production(20.46 g/L). In addition, enhancing intermediate metabolites transmission efficiency by synthetizing DNA-guided scaffolds and reducing the production of byproducts by designing s RNA switch enabled further improvement in fumarate production(33.13 g/L). These results suggest that modular pathway engineering should be optimized systematically to enable an efficient microbial production of fumarate.