(325b) Metabolic Engineering of Corynebacterium Glutamicum for Production of a Four-Carbon Platform Chemical ?-Hydroxybutyrate

Within sustainable industrial frameworks, the biosynthesis of four-carbon (C4) platform chemicals is increasingly recognized for its environmental advantages and superior efficiency over conventional production techniques. γ-hydroxybutyrate (GHB) serves as a vital precursor for synthesizing key compounds like γ-butyrolactone (GBL) and 1,4-butanediol (1,4-BDO). This study details the development of an efficient GHB microbial producer by metabolic engineering of a L-glutamate-overproducing Corynebacterium glutamicum S9114 strain. A synthetic pathway consisting of mutated L-glutamate decarboxylase (gadB^{E89Q Δ465-466}), γ-aminobutyric transaminase (gabT), and alcohol dehydrogenase (yqhD) genes from Escherichia coli was introduced into the C. glutamicum S9114 strain. Suppression of by-products such as succinate, lactate, and acetate through gene knockouts further enhanced the GHB production. System-wide analyses, including genome-scale metabolic simulation and comparative transcriptome analysis, were conducted to better understand the metabolic characteristics of our biosynthetic pathway. A new aldehyde dehydrogenase mediating the oxidation of succinate semialdehyde was also identified by transcriptome analysis and confirmed in the knock-out experiment. Additionally, incorporating a phosphoketolase-mediated non-oxidative glycolysis pathway enhanced the theoretical pathway yields of GHB, and was successfully applied in further improving its production. The optimized strain produced 38.1 g/L of GHB from batch culture in a 1.3L fermenter, with a yield of 0.615 mol/mol glucose, without byproduct accumulation. Furthermore, GBL can be easily produced from the fermentation broth by acid treatment. This study provides valuable insights and methodologies for biotechnological production of GHB and other C4 chemicals.