Synthesis Of Diacetyl By Corynebacterium Glutamicum Through Metabolic Engineering

Diacetyl is an important natural flavor compound with high commercial value,widely used in food,cosmetics,chemical and pharmaceutical industries.In our study,?-acetolactate synthase of diacetyl synthesis pathway,as well as by-products synthesis pathways of lactate,acetoin,2,3-butanediol and acetate were metabolically modified in Corynebacterium glutamicum ATCC 13032 to realize the goal of high production of diacetyl,combining with modification the NADH oxidase or the cytochrome bd oxidase of the respiratory chain.Integrating the?-acetolactate synthase encoding gene als S from Bacillus subtilis168 into the pta-ack A site of the C.glutamicum ATCC 13032 genome and introducing a constitutive als S expression vector,the resulting strain showed diacetyl production increased from 0.01 g/L to 0.23 g/L.Besides the NADH/NAD~+ratio increased to from0.53 to 0.75.The biosynthetic pathways of lactate,acetoin and acetate were disrupted to improve diacetyl accumulation to 0.58 g/L.These modifications of C.glutamicum resulted in a sharp increase of the NADH/NAD~+ratio from 0.53 to 1.10,which limiting the synthesis of diacetyl.Three NADH oxidases were selected to compare the sequences and evolutionary trees.The codon preference of three NADH oxidases was analyzed,and it was speculated that the NADH oxidase from B.subtilis 168 had the best heterologous expression effect.Comparing the effects of overexpression of three NADH oxidase on diacetyl synthesis,overexpression the NADH oxidase from B.subtilis 168 was the optimal strategy for diacetyl synthesis.This result is consistent with the speculation of codon preference analysis.The production of diacetyl increased to 0.79 g/L while NADH/NAD~+ratio decreased from 1.09 to 0.75.Overexpression of cytochrome bd oxidase doubled the diacetyl production to 1.29g/L,and NADH/NAD~+ratio decreased from 1.09 to 0.45,which was close to the level of the starting strain.To further analyze the mechanism of efficient biosynthesis of diacetyl,the real-time quantitative PCR was used to determine genes expression level of diacetyl synthesis pathway and the respiratory chain.By modifying the diacetyl synthesis pathway and balancing the intracellular reducing power,diacetyl production became the highest in the overexpressed cyd ABDC strain.Determine NADH/NAD~+ratio of this strain and genes expression level of diacetyl synthesis pathway and the respiratory chain,which can deepen the the understanding of the relationship between the respiratory chain and reducing power of C.glutamicum and in-depth analyze the mechanism of efficient biosynthesis of diacetyl.It is of great significance in modifying the strain by metabolic engineering to efficiently produce diacetyl and improving the synthesis of other bio-based products with imbalance of NADH.