Metabolic engineering of Corynebacterium glutamicum for the sustainable production of functional C5-monomers for polyamides

Prell C. Metabolic engineering of Corynebacterium glutamicum for the sustainable production of functional C5-monomers for polyamides. Bielefeld: Universität Bielefeld; 2022

Utilization of a wheat sidestream for 5-aminovalerate production by Corynebacterium glutamicum

Burgardt A, Prell C, Wendisch VF. Utilization of a wheat sidestream for 5-aminovalerate production by Corynebacterium glutamicum. Frontiers in Bioengineering and Biotechnology. 2021;9: 732271

Fermentative production of ʟ-2-hydroxyglutarate by engineered Corynebacterium glutamicum via pathway extension of ʟ-lysine biosynthesis

Prell C, Burgardt A, Meyer F, Wendisch VF. Fermentative production of ʟ-2-hydroxyglutarate by engineered Corynebacterium glutamicum via pathway extension of ʟ-lysine biosynthesis. Frontiers in Bioengineering and Biotechnology. 2021;8: 630476.L-2-hydroxyglutarate (L-2HG) is a trifunctional building block and highly attractive for the chemical and pharmaceutical industries. The natural L-lysine biosynthesis pathway of the amino acid producer Corynebacterium glutamicum was extended for the fermentative production of L-2HG. Since L-2HG is not native to the metabolism of C. glutamicum metabolic engineering of a genome-streamlined L-lysine overproducing strain was required to enable the conversion of L-lysine to L-2HG in a six-step synthetic pathway. To this end, L-lysine decarboxylase was cascaded with two transamination reactions, two NAD(P)-dependent oxidation reactions and the terminal 2-oxoglutarate-dependent glutarate hydroxylase. Of three sources for glutarate hydroxylase the metalloenzyme CsiD from Pseudomonas putida supported L-2HG production to the highest titers. Genetic experiments suggested a role of succinate exporter SucE for export of L-2HG and improving expression of its gene by chromosomal exchange of its native promoter improved L-2HG production. The availability of Fe2+ as cofactor of CsiD was identified as a major bottleneck in the conversion of glutarate to L-2HG. As consequence of strain engineering and media adaptation product titers of 34 ± 0 mM were obtained in a microcultivation system. The glucose-based process was stable in 2 L bioreactor cultivations and a L-2HG titer of 3.5 g L−1 was obtained at the higher of two tested aeration levels. Production of L-2HG from a sidestream of the starch industry as renewable substrate was demonstrated. To the best of our knowledge, this study is the first description of fermentative production of L-2HG, a monomeric precursor used in electrochromic polyamides, to cross-link polyamides or to increase their biodegradability

Metabolic engineering of Corynebacterium glutamicum for de novo production of 3-hydroxycadaverine

Prell C, Vonderbank S, Meyer F, Perez F, Wendisch VF. Metabolic engineering of Corynebacterium glutamicum for de novo production of 3-hydroxycadaverine. Current Research in Biotechnology. 2022;4:32-46.Functionalization of amino acids and their derivatives opens up the possibility to produce novel compounds with additional functional groups, which can expand their application spectra. Hydroxylation of polyamide building blocks might allow crosslinking between the molecular chains by esterification. Consequently, this can alter the functional properties of the resulting polymers. C. glutamicum represents a well-known industrial workhorse and has been used extensively to produce lysine and lysine derivatives. These are used as building blocks for chemical and pharmaceutical applications. In this study, it was shown for the first time that C3-hydroxylated cadaverine can be produced de novo by a lysine overproducing C. glutamicum strain. The lysine hydroxylase from Flavobacterium johnsoniae is highly specific for its natural substrate lysine and, therefore, hydroxylation of lysine precedes decarboxylation of 4-hydroxylysine (4-HL) to 3-hydroxycadaverine (3-HC). For optimal precursor supply, various cultivation parameters were investigated identifying the iron concentration and pH as major effectors on 4-HL production, whereas the supply with the cosubstrate 2-oxoglutarate (2-OG) was sufficient. Deletion of the gene coding for the lysine exporter LysE suggested that the exporter may also be involved in the export of the structurally similar 4-HL. With the optimised setting for 4-HL production, the pathway was extended towards 3-HC by decarboxylation. Three different genes coding for lysine/4-HL decarboxylases, LdcC and CadA from E. coli and DCFj from F. johnsoniae, were expressed in the 4-HL producing strain and compared regarding 3-HC production. It was shown in a semi-preparative biocatalysis that all three decarboxylases can accept 4-HL as substrate with varying efficiencies. In vivo, LdcC supported 3-HC production best with a final titer of 11 mM. To improve titers a fed-batch cultivation in 1 L bioreactor scale was performed and the plasmid-based overexpression of ldcC was induced after 24 h resulting in the highest titer of 8.6 g L-1 (74 mM) of 3-hydroxycadaverine reported up to now

Adaptive Laboratory Evolution accelerated glutarate production by Corynebacterium glutamicum

Prell C, Busche T, Rückert C, Nolte L, Brandenbusch C, Wendisch VF. Adaptive Laboratory Evolution accelerated glutarate production by Corynebacterium glutamicum. Microbial Cell Factories. 2021;20:97.Background: The demand for biobased polymers is increasing steadily worldwide. Microbial hosts for produc-tion of their monomeric precursors such as glutarate are developed. To meet the market demand, production hosts have to be improved constantly with respect to product titers and yields, but also shortening bioprocess duration is important. Results: In this study, adaptive laboratory evolution was used to improve a C. glutamicum strain engineered for production of the C5-dicarboxylic acid glutarate by flux enforcement. Deletion of the l-glutamic acid dehydrogenase gene gdh coupled growth to glutarate production since two transaminases in the glutarate pathway are crucial for nitrogen assimilation. The hypothesis that strains selected for faster glutarate-coupled growth by adaptive labora-tory evolution show improved glutarate production was tested. A serial dilution growth experiment allowed isolating faster growing mutants with growth rates increasing from 0.10 h−1 by the parental strain to 0.17 h−1 by the fastest mutant. Indeed, the fastest growing mutant produced glutarate with a twofold higher volumetric productivity of 0.18 g L−1 h−1 than the parental strain. Genome sequencing of the evolved strain revealed candidate mutations for improved production. Reverse genetic engineering revealed that an amino acid exchange in the large subunit of l-glutamic acid-2-oxoglutarate aminotransferase was causal for accelerated glutarate production and its beneficial effect was dependent on flux enforcement due to deletion of gdh. Performance of the evolved mutant was stable at the 2 L bioreactor-scale operated in batch and fed-batch mode in a mineral salts medium and reached a titer of 22.7 g L−1, a yield of 0.23 g g−1 and a volumetric productivity of 0.35 g L−1 h−1. Reactive extraction of glutarate directly from the fermentation broth was optimized leading to yields of 58% and 99% in the reactive extraction and reactive re-extraction step, respectively. The fermentation medium was adapted according to the downstream pro-cessing results. Conclusion: Flux enforcement to couple growth to operation of a product biosynthesis pathway provides a basis to select strains growing and producing faster by adaptive laboratory evolution. After identifying candidate mutations by genome sequencing causal mutations can be identified by reverse genetics. As exemplified here for glutarate production by C. glutamicum, this approach allowed deducing rational metabolic engineering strategies