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

Carina Prell,Volker F Wendisch,Arthur Burgardt,Florian Meyer

doi:10.3389/fbioe.2020.630476

Carina Prell, Volker F Wendisch + Show 2 more

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https://doi.org/10.3389/fbioe.2020.630476

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Abstract

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.

Highlights

L-2-hydroxyglutarate (L-2HG) can be obtained by hydroxylation of the C5-dicarboxylic acid glutarate at the α-carbon position
Production of the sought after compound L-2HG by the industrial workhorse C. glutamicum was demonstrated after extension of lysine biosynthesis in a six-step cascade employing the metalloenzyme CsiD from P. putida as final step
The glucose-based process was stable in 2 L bioreactor cultivations and a L-2HG titer of 3.5 g L−1 was obtained in fed-batch fermentation

Summary

Introduction

L-2-hydroxyglutarate (L-2HG) can be obtained by hydroxylation of the C5-dicarboxylic acid glutarate at the α-carbon position. Glutarate is known to be a demanded building block for the production of biopolymers, like aliphatic polyamide 6,5 which is mainly used in the construction industry (Navarro et al, 1997). In the case of L-2HG, the additional hydroxyl group provides greater versatility for further chemical modification with the potential to alter the properties of the resulting polymer. Production of L-2HG by microbial fermentation would provide the chemical industry with an environmentally friendly building block for polyamides and polyesters. The addition of functionalities on polyester backbones (hydroxyl, carboxyl, allyl, azido, and acetylene groups) facilitates covalent post-polymerization modification. It was demonstrated that electrochromic polyamides with functional hydroxyl groups for homogeneous hybrid films can be produced. The repeating units of hydroxysuccinate in the polymer backbone provided reaction sites for organic-inorganic bonding resulting in homogeneous and transparent hybrid films (Pan et al, 2018)

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