Genetic Engineering Of Bacillus Sp. And Fermentation Process Optimization For Diacetyl Production

As a common C4 platform compound,diacetyl is widely used in food,chemical and other industries.Traditional methods for diacetyl production,including extraction from natural materials and chemical synthesis,are associated with high costs and cause,serious pollution.Therefore,recently microbial fermentation has become the mainstay approach for the production of diacetyl.In this study,Bacillus sp.DL01-?als D,a strain producing diacetyl,was used as the starting strain for metabolic engineering.The fermentation conditions were also optimized to maximize the yield of diacetyl in the strain.This study aims to provide a theoretical basis for the industrialized production of diacetyl by microbial fermentation.First,the temperature-sensitive plasmid p KS1-pta was constructed for the deletion of pta,the gene encoding phosphotransacetylase(PTA),a key enzyme in the acetic acid metabolic pathway.The plasmid was introduced into the host strain B.sp.DL01-?als D and the pta gene was deleted by scarless gene knockout,generating the mutant strain B.sp.DL01-?als D-?pta.The results of shake-flask fermentation showed that the yield of acetic acid in the knockout strain B.sp.DL01-?als D-?pta decreased significantly,and the yield of diacetyl increased significantly,reaching 3.67 g/L,which was 1.3-fold of that of the starting strain B.sp.DL01-?als D(2.82 g/L).The gene encoding acetolactate synthase(als)from Bacillus subtilis 168 used for the construction of the overexpression plasmid and B.sp.DL01-?als D-?pta was transformed with the plasmid to obtain the recombinant strain B.sp.DL01-?als D-?ptaals S168.The yield of diacetyl in the recombinant strain reached 5.43 g/L,which was 1.48 fold of that of the pta-mutant strain B.sp.DL01-?als D-?pta.The conditions of the fermentation were optimized to further improve the yield of diacetyl via microbial fermentation.Single-factor analysis of the fermentation conditions,including temperature,rate of ventilation and stirring speed was performed bioreactor-level fermentation.According to the results of the single-factor analysis,a BP neural network model was constructed using MATLAB software,and the optimal value of neural network was determined by genetic algorithm.The optimal conditions for the fermentation of B.sp.DL01-?als D-?pta-als S168 was predicted.The optimal conditions were 35.7°C,0.66 vvm,and 179 r/min,respectively.Under this condition,fed-batch fermentation was carried out.The concentration of glucose was set to 10 g/L,and the fermentation lasted for 60 hours.The yield of diacetyl reached 11.18 g/L,and the conversion rate was 0.16 g/g glucose,which is by far the highest value reported in the literature.In summary,this paper improved the yield of diacetyl by the over-expression of acetolactate synthase,blocking the acetic acid biosynthesis pathway,the optimization of the fermentation conditions.The yield of the final strain diacetyl was greatly improved.This study has theoretical significance and application value for the production of diacetyl by fermentation of food safety bacteria.