A Novel D-Galacturonate Fermentation Pathway in Lactobacillus suebicus Links Initial Reactions of the Galacturonate-Isomerase Route With the Phosphoketolase Pathway

A Novel D-Galacturonate Fermentation Pathway in Lactobacillus suebicus Links Initial Reactions of the Galacturonate-Isomerase Route With the Phosphoketolase Pathway

Valk, L.C. (TU Delft BT/Industriele Microbiologie)
Luttik, M.A.H. (TU Delft BT/Industriele Microbiologie)
de Ram, C. (TU Delft OLD BT/Cell Systems Engineering)
Pabst, Martin (TU Delft OLD BT/Cell Systems Engineering)
van den Broek, M.A. (TU Delft BT/Industriele Microbiologie)
van Loosdrecht, Mark C.M. (TU Delft BT/Environmental Biotechnology)
Pronk, J.T. (TU Delft BT/Biotechnologie)

BT/Biotechnologie

2020

D-galacturonate, a key constituent of pectin, is a ubiquitous monomer in plant biomass. Anaerobic, fermentative conversion of D-galacturonate is therefore relevant in natural environments as well as in microbial processes for microbial conversion of pectin-containing agricultural residues. In currently known microorganisms that anaerobically ferment D-galacturonate, its catabolism occurs via the galacturonate-isomerase pathway. Redox-cofactor balancing in this pathway strongly constrains the possible range of products generated from anaerobic D-galacturonate fermentation, resulting in acetate as the predominant organic fermentation product. To explore metabolic diversity of microbial D-galacturonate fermentation, anaerobic enrichment cultures were performed at pH 4. Anaerobic batch and chemostat cultures of a dominant Lactobacillus suebicus strain isolated from these enrichment cultures produced near-equimolar amounts of lactate and acetate from D-galacturonate. A combination of whole-genome sequence analysis, quantitative proteomics, enzyme activity assays in cell extracts, and in vitro product identification demonstrated that D-galacturonate metabolism in L. suebicus occurs via a novel pathway. In this pathway, mannonate generated by the initial reactions of the canonical isomerase pathway is converted to 6-phosphogluconate by two novel biochemical reactions, catalyzed by a mannonate kinase and a 6-phosphomannonate 2-epimerase. Further catabolism of 6-phosphogluconate then proceeds via known reactions of the phosphoketolase pathway. In contrast to the classical isomerase pathway for D-galacturonate catabolism, the novel pathway enables redox-cofactor-neutral conversion of D-galacturonate to ribulose-5-phosphate. While further research is required to identify the structural genes encoding the key enzymes for the novel pathway, its redox-cofactor coupling is highly interesting for metabolic engineering of microbial cell factories for conversion of pectin-containing feedstocks into added-value fermentation products such as ethanol or lactate. This study illustrates the potential of microbial enrichment cultivation to identify novel pathways for the conversion of environmentally and industrially relevant compounds.

anaerobic metabolism
enrichment cultivation
Entner–Doudoroff pathway
galacturonic acid
heterolactic fermentation
pectin degradation

http://resolver.tudelft.nl/uuid:cf430d52-0db1-4cb5-98cd-f7686501b2cd

https://doi.org/10.3389/fmicb.2019.03027

1664-302X

Frontiers in Microbiology, 10

Institutional Repository

journal article

© 2020 L.C. Valk, M.A.H. Luttik, C. de Ram, Martin Pabst, M.A. van den Broek, Mark C.M. van Loosdrecht, J.T. Pronk