Glutaric acid production by systems metabolic engineering of an l -lysine–overproducing Corynebacterium glutamicum

Abstract

There is increasing industrial demand for five-carbon platform chemicals, particularly glutaric acid, a widely used building block chemical for the synthesis of polyesters and polyamides. Here we report the development of an efficient glutaric acid microbial producer by systems metabolic engineering of anl-lysine–overproducingCorynebacterium glutamicumBE strain. Based on our previous study, an optimal synthetic metabolic pathway comprisingPseudomonas putidal-lysine monooxygenase (davB) and 5-aminovaleramide amidohydrolase (davA) genes andC. glutamicum4-aminobutyrate aminotransferase (gabT) and succinate-semialdehyde dehydrogenase (gabD) genes, was introduced into theC. glutamicumBE strain. Through system-wide analyses including genome-scale metabolic simulation, comparative transcriptome analysis, and flux response analysis, 11 target genes to be manipulated were identified and expressed at desired levels to increase the supply of direct precursorl-lysine and reduce precursor loss. A glutaric acid exporter encoded byynfMwas discovered and overexpressed to further enhance glutaric acid production. Fermentation conditions, including oxygen transfer rate, batch-phase glucose level, and nutrient feeding strategy, were optimized for the efficient production of glutaric acid. Fed-batch culture of the final engineered strain produced 105.3 g/L of glutaric acid in 69 h without any byproduct. The strategies of metabolic engineering and fermentation optimization described here will be useful for developing engineered microorganisms for the high-level bio-based production of other chemicals of interest to industry.

Fermentative Production of l-2-Hydroxyglutarate by Engineered Corynebacterium glutamicum via Pathway Extension of l-Lysine Biosynthesis

“…Unlike l -2HG, which likely is exported by SucE (s. above), glutarate is exported by YnfM (Fukui et al, 2019 ). Overexpression of ynfM improved production of glutarate, succinate, and 2-OG (Fukui et al, 2019 ; Han et al, 2020 ). Thus, deletion of ynfM is a suitable strategy to abolish export of glutarate as by-product of l -2HG production.…”

Adaptive laboratory evolution accelerated glutarate production by Corynebacterium glutamicum

“…This report is not the first on metabolic engineering of C. glutamicum for glutarate production [ 8 , 16 , 17 , 26 ], but the first example of improving volumetric productivity by flux enforcement and ALE. Notably, engineering of the GS/GOGAT system, which was identified here as crucial to accelerate glutarate production, has not been reported previously as metabolic engineering target for glutarate production by C. glutamicum .…”

“…Notably, engineering of the GS/GOGAT system, which was identified here as crucial to accelerate glutarate production, has not been reported previously as metabolic engineering target for glutarate production by C. glutamicum . Strain engineering, e.g., by overexpressing ynfM encoding the recently discovered glutarate exporter, media optimization, e.g., by using mixtures of glucose and sucrose, and process intensification, e.g., by using a pH–stat feeding strategy in fed-batch cultures, have been described to boost glutarate production to titers of more than 100 g L −1 [ 17 ]. Thus, the mutation pair identified here (GltB E686Q and deletion of gdh ) complements the previously described metabolic engineering strategies for glutarate production by C. glutamicum .…”

“…Fermentative production of the C4-ω-amino acid γ-aminobutyrate (GABA) [ 5 , 6 ] and the C5-ω-amino acid 5-aminovalerate (5AVA) has been established [ 7 , 8 ] and, e.g., ring-opening polymerization of 5AVA can be used to produce the polyamide 5 (PA 5) [ 9 , 10 ]. Moreover, diamines like putrescine [ 11 , 12 ] and cadaverine [ 13 , 14 ] as well as the dicarboxylic acids succinate and glutarate [ 15 – 17 ] were successfully produced in high titers. Glutarate, e.g., is used as a building block for polyamides such as PA 4.5 [ 18 ], PA 6.5, PA 12.5 [ 19 ] or PA 5.5 the latter of which is synthesized by polycondensation of the C5-dicarboxylic acid glutarate with C5-diamine cadaverine [ 20 ].…”

“…Systems metabolic engineering proved successful to achieve high titer glutarate production by metabolically engineered C. glutamicum [ 16 , 17 , 26 ]. In this study, we aimed to accelerate glutarate production by evolutionary engineering.…”