Design And Construction Of The Microbial Consortium For Vitamin C One-step Fermentation

As synthetic biology begins to address the problems involved in the programing novel biological systems,design and construction of the microbial consortia is emerging as a key tool for building synthetic systems that robustly perform complex behaviors.In these consortia,the microbes communicate with others through the exchange of materials,energy and information to perform as a group.And this will be more diverse in the industrial environment.In this dissertation,the conventional twostep fermentation system for vitamin C production is taken as an example.Different artificial microbial consortia were designed and constructed to realize the one-step fermentation of 2-keto-L-gulonic acid(2-KGA),the precursor of vitamin C,directly from D-sorbitol.Furthermore,the consortia were then optimized with the engineered Gluconobacter oxydans to improve the interaction between the microbes,which provided us potential strategies for improving the synthetic consortium.In order to solve the issues about the long fermentation cycle and the secondary sterilization process,a synthetic consortium of G.oxydans and Ketogulonicigenium vulgare producing 2-KGA directly from D-sorbitol was constructed based on the conventional two-step fermentation process.Taken the conditions of the laboratory and the production cost into consideration,the fermentation condition was optimized.Besides,five relevant genes involved in the L-sorbose consumption pathway in G.oxydans were deleted,respectively,to alleviate the metabolic competition of G.oxydans with K.vulgare for sorbose,which was helpful for reconstruction of a better homeostasis between microbes and making them work better together.With the engineered G.oxydans H6 and K.vulgare,the molar yield of 2-KGA reached to 89.7% within 36 h,which was 29.6% higher than that of the primary consortium.On the basis of the G.oxydans-K.vulgare consortium,Bacillus megaterium was introduced to reconstruct the consortium for further optimizing the one-step fermentation of 2-KGA from D-sorbitol.A series of quorum sensing system modules,tufB-luxI-Ter-tufB-luxR-Ter-(N×)lux pR-RBS-ccdB-Ter(N=1,2,3,5),were constructed and optimized in G.oxydans to realize the programmed cell death.With K.vulgare,B.megaterium and the engineered G.oxydans IR3 C,the molar yield of 2-KGA reached to 80.7% within 36 h,which was 21.4% higher than that of the primary consortium.Furthermore,metabolomic analysis was used to verify the enhancement of the symbiotic relationship of both consortia constructed in this dissertation.It was found that simplifying the sorbose metabolic pathway will affect not only itself alone,but also other related metabolic characteristics,such as the metabolism of amino acids,nucleotides,unsaturated fatty acids and TCA cycle.It was suggested that the relationship between the two microbes changed from commensalism and competition to mutualism.In addition,it was found that the quorum sensing system modules for the population control in G.oxydans did have a certain effect on the intracellular amino acids,TCA cycle and saturated fatty acids in G.oxydans-K.vulgare-B.megaterium consortium.On the basis of the G.oxydans-K.vulgare consortium,B.megaterium showed the synergistic effect with the engineered G.oxydans to improve the growth and the productivity of K.vulgare.