Model-Guided Metabolic Rewiring for Gamma-Aminobutyric Acid and Butyrolactam Biosynthesis in Corynebacterium glutamicum ATCC13032

SIMPLE SUMMARY: The fermentative production of desired chemicals from renewable resources is one of the promising biosynthetic routes to replace the petrochemical-based process. Gamma-aminobutyric acid (GABA) can be synthesized from l-glutamic acid and used as a building block for the synthesis of butyrolactam and polyamide 4 (nylon 4). The genome-scale metabolic model can predict the growth ability and metabolic flux distribution by genetic disturbances, which provides a strategy to construct a microbial cell factory for GABA and butyrolactam biosynthesis. Here, we performed model-guided metabolic engineering of Corynebacterium glutamicum ATCC13032 for GABA and butyrolactam fermentation from glucose. The biosynthetic pathways of GABA and butyrolactam were constructed by overexpressing the heterologous genes using a bi-cistronic expression cassette. The genetic modifications of the metabolic network cooperatively forced the carbon flux toward GABA and butyrolactam synthesis. This study provides new insights into engineering industrial microorganisms to produce target chemicals from renewable carbon sources. ABSTRACT: Gamma-aminobutyric acid (GABA) can be used as a bioactive component in the pharmaceutical industry and a precursor for the synthesis of butyrolactam, which functions as a monomer for the synthesis of polyamide 4 (nylon 4) with improved thermal stability and high biodegradability. The bio-based fermentation production of chemicals using microbes as a cell factory provides an alternative to replace petrochemical-based processes. Here, we performed model-guided metabolic engineering of Corynebacterium glutamicum for GABA and butyrolactam fermentation. A GABA biosynthetic pathway was constructed using a bi-cistronic expression cassette containing mutant glutamate decarboxylase. An in silico simulation showed that the increase in the flux from acetyl-CoA to α-ketoglutarate and the decrease in the flux from α-ketoglutarate to succinate drove more flux toward GABA biosynthesis. The TCA cycle was reconstructed by increasing the expression of acn and icd genes and deleting the sucCD gene. Blocking GABA catabolism and rewiring the transport system of GABA further improved GABA production. An acetyl-CoA-dependent pathway for in vivo butyrolactam biosynthesis was constructed by overexpressing act-encoding ß-alanine CoA transferase. In fed-batch fermentation, the engineered strains produced 23.07 g/L of GABA with a yield of 0.52 mol/mol from glucose and 4.58 g/L of butyrolactam. The metabolic engineering strategies can be used for genetic modification of industrial strains to produce target chemicals from α-ketoglutarate as a precursor, and the engineered strains will be useful to synthesize the bio-based monomer of polyamide 4 from renewable resources.