Flux Enforcement for Fermentative Production of 5-Aminovalerate and Glutarate by Corynebacterium glutamicum

Abstract

Bio-based plastics represent an increasing percentage of the plastics economy. The fermentative production of bioplastic monomer 5-aminovalerate (5AVA), which can be converted to polyamide 5 (PA 5), has been established in Corynebacterium glutamicum via two metabolic pathways. l-lysine can be converted to 5AVA by either oxidative decarboxylation and subsequent oxidative deamination or by decarboxylation to cadaverine followed by transamination and oxidation. Here, a new three-step pathway was established by using the monooxygenase putrescine oxidase (Puo), which catalyzes the oxidative deamination of cadaverine, instead of cadaverine transaminase. When the conversion of 5AVA to glutarate was eliminated and oxygen supply improved, a 5AVA titer of 3.7 ± 0.4 g/L was reached in microcultivation that was lower than when cadaverine transaminase was used. The elongation of the new pathway by 5AVA transamination by GABA/5AVA aminotransferase (GabT) and oxidation by succinate/glutarate semialdehyde dehydrogenase (GabD) allowed for glutarate production. Flux enforcement by the disruption of the l-glutamic acid dehydrogenase-encoding gene gdh rendered a single transaminase (GabT) in glutarate production via the new pathway responsible for nitrogen assimilation, which increased the glutarate titer to 7.7 ± 0.7 g/L, i.e., 40% higher than with two transaminases operating in glutarate biosynthesis. Flux enforcement was more effective with one coupling site, thus highlighting requirements regarding the modularity and stoichiometry of pathway-specific flux enforcement for microbial production.

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

“…This is unlikely due to effects on enzyme activities. C. glutamicum possesses chromosomal copies of gabT and gabD (Pérez-García et al, 2018 ; Haupka et al, 2020 ). The PucR-like regulator GabR that requires GABA as coactivator activates transcription of the gabTDP operon (Zhu et al, 2020 ).…”

“…The second pathway to 5AVA combines oxidative decarboxylation by l -lysine monooxygenase using molecular oxygen followed by desamidation by γ-aminovaleramidase from P. putida (Adkins et al, 2013 ). The third pathway is based on l -lysine decarboxylase from E. coli , putrescine oxidase PuO from Rhodococcus qingshengii and the γ-aminobutyraldehyde dehydrogenase from E. coli that catalyze decarboxylation, oxidative deamination using molecular oxygen and NAD-dependent oxidation (Haupka et al, 2020 ). The fourth pathway does not require molecular oxygen as it cascades l -lysine decarboxylase, 2-oxoglurate-dependent putrescine/cadaverine transaminase PatA, and NAD-dependent γ- aminobutyraldehyde dehydrogenase PatD from E. coli (Jorge et al, 2017 ).…”

“…The pathway combinations for LdcC-PuO-PatD-GabT-GabD and LdcC-PatA-PatD-GabT-GabD couple conversion of lysine to glutarate in one or two transaminase reactions, respectively, that generate glutamate from 2-oxoglutarate. This coupling enabled flux enforcement, i.e., growth requires production of glutarate, which was achieved by deletion of gdh , the gene for the major ammonium assimilating enzyme l -glutamic acid dehydrogenase (Pérez-García et al, 2018 ; Haupka et al, 2020 ).…”

Adaptive laboratory evolution accelerated glutarate production by Corynebacterium glutamicum

“…Bio-based production of monomeric building blocks for polyamides has been established in metabolically engineered C. glutamicum and E. coli [ 3 , 4 ]. 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.…”

“…The second pathway to 5AVA combines oxidative decarboxylation by l -lysine monooxygenase (DavA) using molecular oxygen followed by desamidation by γ-aminovaleramidase (DavB) from Pseudomonas putida [ 20 ]. The third pathway is based on l -lysine decarboxylase from E. coli , putrescine oxidase PuO from Rhodococcus qingshengii , which requires molecular oxygen, and γ-aminobutyraldehyde dehydrogenase PatD from E. coli [ 8 ]. The fourth pathway does not require molecular oxygen as it cascades l -lysine decarboxylase, 2-oxoglurate-dependent putrescine/cadaverine transaminase PatA, and NAD-dependent γ-aminobutyraldehyde dehydrogenase PatD from E. coli [ 7 ].…”