Utilization of a Wheat Sidestream for 5-Aminovalerate Production in Corynebacterium glutamicum.

Production of plastics from petroleum-based raw materials extensively contributes to global pollution and CO2 emissions. Biotechnological production of functionalized monomers can reduce the environmental impact, in particular when using industrial sidestreams as feedstocks. Corynebacterium glutamicum, which is used in the million-ton-scale amino acid production, has been engineered for sustainable production of polyamide monomers. In this study, wheat sidestream concentrate (WSC) from industrial starch production was utilized for production of l-lysine-derived bifunctional monomers using metabolically engineered C. glutamicum strains. Growth of C. glutamicum on WSC was observed and could be improved by hydrolysis of WSC. By heterologous expression of the genes xylA Xc B Cg (xylA from Xanthomonas campestris) and araBAD Ec from E. coli, xylose, and arabinose in WSC hydrolysate (WSCH), in addition to glucose, could be consumed, and production of l-lysine could be increased. WSCH-based production of cadaverine and 5-aminovalerate (5AVA) was enabled. To this end, the lysine decarboxylase gene ldcC Ec from E. coli was expressed alone or for conversion to 5AVA cascaded either with putrescine transaminase and dehydrogenase genes patDA Ec from E. coli or with putrescine oxidase gene puo Rq from Rhodococcus qingshengii and patD Ec . Deletion of the l-glutamate dehydrogenase-encoding gene gdh reduced formation of l-glutamate as a side product for strains with either of the cascades. Since the former cascade (ldcC Ec -patDA Ec ) yields l-glutamate, 5AVA production is coupled to growth by flux enforcement resulting in the highest 5AVA titer obtained with WSCH-based media.

Adaptive laboratory evolution accelerated glutarate production by Corynebacterium glutamicum.

BACKGROUND: The demand for biobased polymers is increasing steadily worldwide. Microbial hosts for production 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 laboratory 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 processing 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.

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

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.