Design And Construction Of A New Synthetic Microbial Consortium For 2-KGA Production

In recent years,artificial microbial consortium has become one of the hotspots of synthetic biology for domestic and foreign experts due to the great progress made by synthetic biology in the construction of microorganisms with clear and controllable properties.2-keto-L-gulonic acid(2-KGA)is a precursor of vitamin C.At present,domestic and foreign scholars have proposed a variety of strategies to improve the production of 2-KGA by Ketogulonicigenium vulgare strain.The accompanying bacteria are used to form microbial consortium,in which the acid-producing ability of K.vulgare could be significantly promoted.One of the disadvantages in growth and acid-producing of K.vulgare strain was derived from the oxidative stress that couldn't be tolerated by themselves.The addition of the accompanying bacteria could resist the oxidative stress of the K.vulgare strain,which can significantly improve the situation.The accompanying bacteria release metabolites for K.vulgare or induce the expression of certain antioxidant genes in K.vulgare.So the yield of 2-KGA was greatly improved.In this paper,we focused on promoting the production of 2-KGA in K.vulgare by constructing a new microbial consortium for 2-KGA fermentation.L-ascorbic acid(L-AA)was often used to protect against oxidative stress due to its potent antioxidant activity.In the present study,a strain of L-AA-producing yeast strain was first constructed and used as K.vulgare's new companion bacteria that a new 2-KGA mixed fermentation system was constructed.The biosynthesis of L-AA requires a 10-step biosynthetic reaction,in which steps 1-5(D-glucose to GDP-D-Mannose)could be spontaneously carried out in Saccharomyces cerevisiae.The genes in steps 6-10 of the L-AA synthetic pathway were introduced into S.cerevisiae CEN.PK2-1c and CEN.PK2-1d by 8 different combinations in this study,and 16 L-AA producing strains were successfully constructed.It was also found that different combinations of genes and cell mating types could affect the synthesis of L-AA.Finally,a L-AA biosynthetic strain(C-1c)with a yield of 3.7 mg/L was screened and used as a new companion for K.vulgare to construct a new microbial consortium for 2-KGA fermentation.By optimizing the inoculation ratio,fermentation medium composition and culture conditions,the yield of 2-KGA reached 14.33 g/L,and the conversion rate was 66.48%,which was significantly higher than that of mono-culturing K.vulgare(9.07 g/L).The 2-KGA production and conversion rate from sorbose to 2-KGA increased by 58% and 24.4%,respectively.The expression of genes SSDA1,SSDA2,SSDA3 and SSDB,which related to 2-KGA metabolism in K.vulgare,increased by 1.62,1.64,1.47 and 1.73 times respectively in the new synthetic microbial consortium.The expression of SNDH increased by 1.74 times.The expression of anti-oxidation related genes GPD,CAT and SOD on the genome and plasmid increased by 1.45,2.84,2.67 and 1.82 times,respectively.The up-regulation of these genes further indicated the constructed L-AAproducing yeast promotes the K.vulgare strain to produce 2-KGA.This study provides new insights for further analysis of the interaction mechanism between the companion bacteria and K.vulgare strains,and also provides a new idea for design and construction of artificial microbial consortium.