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

ABSTRACT: 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.

SUBMITTER: Valk LC

PROVIDER: S-EPMC6978723 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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A Novel D-Galacturonate Fermentation Pathway in <i>Lactobacillus suebicus</i> Links Initial Reactions of the Galacturonate-Isomerase Route With the Phosphoketolase Pathway.

Valk Laura C LC Luttik Marijke A H MAH de Ram C C Pabst Martin M van den Broek Marcel M van Loosdrecht Mark C M MCM Pronk Jack T JT

Frontiers in microbiology 20200117

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 pos ...[more]

PMID: 32010092

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Project description:Lactobacillus plantarum is the lactic acid bacterial species most frequently found in plant-food fermentations where hydroxycinnamic acids are abundant. L. plantarum efficiently decarboxylates these compounds and also reduces them, yielding substituted phenylpropionic acids. Although the reduction step is known to be induced by a hydroxycinnamic acid, the enzymatic machinery responsible for this reduction pathway has not been yet identified and characterized. A previous study on the transcriptomic response of L. plantarum to p-coumaric acid revealed a marked induction of two contiguous genes, lp_1424 and lp_1425, encoding putative reductases. In this work, the disruption of these genes abolished the hydroxycinnamate reductase activity of L. plantarum, supporting their involvement in such chemical activity. Functional in vitro studies revealed that Lp_1425 (HcrB) exhibits hydroxycinnamate reductase activity but was unstable in solution. In contrast, Lp_1424 (HcrA) was inactive but showed high stability. When the hcrAB genes were co-overexpressed, the formation of an active heterodimer (HcrAB) was observed. Since L. plantarum reductase activity was only observed on hydroxycinnamic acids (o-coumaric, m-coumaric, p-coumaric, caffeic, ferulic, and sinapic acids), the presence of a hydroxyl group substituent on the benzene ring appears to be required for activity. In addition, hydroxycinnamate reductase activity was not widely present among lactic acid bacteria, and it was associated with the presence of hcrAB genes. This study revealed that L. plantarum hydroxycinnamate reductase is a heterodimeric NADH-dependent coumarate reductase acting on a carbon-carbon double bond.IMPORTANCELactobacillus plantarum is a bacterial species frequently found in the fermentation of vegetables where hydroxycinnamic acids are present. The bacterial metabolism on these compounds during fermentation plays a fundamental role in the biological activity of hydroxycinnamates. L. plantarum strains exhibit an as yet unknown reducing activity, transforming hydroxycinnamates to substituted phenylpropionic acids, which possess higher antioxidant activity than their precursors. The protein machinery involved in hydroxycinnamate reduction, HcrAB, was genetically identified and characterized. The heterodimeric NADH-dependent coumarate reductase HcrAB described in this work provides new insights on the L. plantarum metabolic response to counteract the stressful conditions generated by food phenolics.

Project description:BackgroundSynthetic L-ascorbic acid (vitamin C) is widely used as a preservative and nutrient in food and pharmaceutical industries. In the current production method, D-glucose is converted to L-ascorbic acid via several biochemical and chemical steps. The main source of L-ascorbic acid in human nutrition is plants. Several alternative metabolic pathways for L-ascorbic acid biosynthesis are known in plants. In one of them, D-galacturonic acid is the precursor. D-Galacturonic acid is also the main monomer in pectin, a plant cell wall polysaccharide. Pectin is abundant in biomass and is readily available from several waste streams from fruit and sugar processing industries.ResultsIn the present work, we engineered the filamentous fungus Aspergillus niger for the conversion of D-galacturonic acid to L-ascorbic acid. In the generated pathway, the native D-galacturonate reductase activity was utilized while the gene coding for the second enzyme in the fungal D-galacturonic acid pathway, an L-galactonate consuming dehydratase, was deleted. Two heterologous genes coding for enzymes from the plant L-ascorbic acid pathway--L-galactono-1,4-lactone lactonase from Euglena gracilis (EgALase) and L-galactono-1,4-lactone dehydrogenase from Malpighia glabra (MgGALDH)--were introduced into the A. niger strain. Alternatively, an unspecific L-gulono-1,4-lactone lactonase (smp30) from the animal L-ascorbic acid pathway was introduced in the fungal strain instead of the plant L-galactono-1,4-lactone lactonase. In addition, a strain with the production pathway inducible with D-galacturonic acid was generated by using a bidirectional and D-galacturonic acid inducible promoter from the fungus. Even though, the lactonase enzyme activity was not observed in the resulting strains, they were capable of producing L-ascorbic acid from pure D-galacturonic acid or pectin-rich biomass in a consolidated bioprocess. Product titers up to 170 mg/l were achieved.ConclusionsIn the current study, an L-ascorbic acid pathway using D-galacturonic acid as a precursor was introduced to a microorganism for the first time. This is also the first report on an engineered filamentous fungus for L-ascorbic acid production and a proof-of-concept of consolidated bioprocess for the production.

Project description:Agricultural residues such as sugar beet pulp and citrus peel are rich in pectin, which contains galacturonic acid as a main monomer. Pectin-rich residues are underexploited as feedstocks for production of bulk chemicals or biofuels. The anaerobic, fermentative conversion of d-galacturonate in anaerobic chemostat enrichment cultures provides valuable information toward valorization of these pectin-rich feedstocks. Replicate anaerobic chemostat enrichments, with d-galacturonate as the sole limiting carbon source and inoculum from cow rumen content and rotting orange peels, yielded stable microbial communities, which were dominated by a novel Lachnospiraceae species, for which the name "Candidatus Galacturonibacter soehngenii" was proposed. Acetate was the dominant catabolic product, with formate and H2 as coproducts. The observed molar ratio of acetate and the combined amounts of H2 and formate deviated significantly from 1, which suggested that some of the hydrogen and CO2 formed during d-galacturonate fermentation was converted into acetate via the Wood-Ljungdahl acetogenesis pathway. Indeed, metagenomic analysis of the enrichment cultures indicated that the genome of "Candidatus G. soehngenii" encoded enzymes of the adapted Entner-Doudoroff pathway for d-galacturonate metabolism as well as enzymes of the Wood-Ljungdahl pathway. The simultaneous operation of these pathways may provide a selective advantage under d-galacturonate-limited conditions by enabling a higher specific ATP production rate and lower residual d-galacturonate concentration than would be possible with a strictly fermentative metabolism of this carbon and energy source.IMPORTANCE This study on d-galacturonate metabolism by open, mixed-culture enrichments under anaerobic, d-galacturonate-limited chemostat conditions shows a stable and efficient fermentation of d-galacturonate into acetate as the dominant organic fermentation product. This fermentation stoichiometry and population analyses provide a valuable baseline for interpretation of the conversion of pectin-rich agricultural feedstocks by mixed microbial cultures. Moreover, the results of this study provide a reference for studies on the microbial metabolism of d-galacturonate under different cultivation regimes.

Project description:YEAST STRAIN: A genetically modified Saccharomyces cerevisiae yeast strain with PMI40 knockout (MATa URA3 TRP1 LEU2 his3- 1 MAL2-8C SUC2 PMI40::HIS3). The parent strain was CEN.PK113-7A (MATa URA3 TRP1 LEU2 his3- 1 MAL2-8C SUC2) obtained from Euroscarf (9). CULTIVATION MEDIUM: The mineral medium used for the bioreactor cultivations was based on the composition described by Verduyn and co-workers (11). The medium consisted of 5 g/l (NH4)2SO4 (Sigma-Aldrich, USA), 3 g/l KH2PO4 (Fluka, Swizerland) and 0.5 g/l MgSO4 × 7 H2O (Sigma-Aldrich, USA), as well as 1 ml/l trace element and 1 ml/l vitamin solution. The composition of the trace element solution was 15 g/l EDTA, 4.5 g/l ZnSO4 × 7 H2O, 0.84 g/l MnCl2 × H2O, 0.3 g/l CuSO4 × 5 H2O, 3 g/l FeSO4 × 7 H2O (Riedel-de-Haën AG, Germany), 0.1 g/l KI, 0.3 g/l CoCl3 × 6 H2O, 1 g/l H3BO3, 4.5 g/l CaCl2 × 2 H2O (Merck, Germany), and 0.4 g/l Na2MoO4 × 2 H2O (BDH Laboratory Supplies, UK). The composition of the vitamin solution was 0.05 g/l D-Biotin, 1 g/l Ca-pantothenate, 1 g/l Nicotinic acid, 25 g/l myo-inositol, 1 g/l Thiamine-HCl, 1 g/l Pyridoxine HCl, and 0.2 g/l p-aminobenzoic acid (Sigma-Aldrich, USA). The media in each cultivation contained initially 15 g/L D-glucose and a varying amount of D-mannose (0.75 g/L, 1.0 g/L, 1.5 g/L, 3.0 g/L, or 5.0 g/L). CULTIVATIONS AND SAMPLING: Bioreactor cultivations were performed in 3.2 l Braun Biostat CT2-DCU 3 instruments (B. Braun Biotech International GmbH, Germany). The cultivation temperature was +30 °C, agitation speed 1000 rpm, initial cultivation volume 2.0 l, airflow 1.0 l/min, and pH 5.0. In each cultivation, samples for the measurement of gene expression levels were taken 6 hours after inoculation. MEASUREMENT OF GENE EXPRESSION LEVELS: 0.9 mg CDW of cells were quenched in cold (-40 °C) methanol (45%) and centrifuged (1 min, +4 °C, 16100 G). The pellets were suspended in 400 µl of breaking buffer (20 mM Tris-HCl pH 7.4, 100 mM KCl, 2 mM MgCl2, 2mM DTT), 400 µl 1:1 phenol-chloroform, and 5 µl 20% SDS. Equal volume of glass beads (diameter 0.40-0.60 mm, B.Braun Biotech International, Germany) were added and samples were shaken in a bead beater (Mini-BeadBeater-8, BioSpec Products, USA) three times 1 minute at +4 °C. The mixtures were centrifuged (15 min, +4 °C, 16100 G in 5415 R, Eppendorf AG, Germany) and supernatants were recovered. The total RNA was purified from the supernatants using the QIAGEN RNeasy Mini kit (Qiagen, USA) according to manufacturers instructions. After this step the double-stranded cDNA was synthesized from 15 µg of total RNA with SuperScript Double-Stranded cDNA synthesis kit (Invitrogen, USA) using a T7-oligo(dT) primer. Biotin-labeled cRNA was then synthesized employing an Enzo High Yield RNA Transcript Labeling Kit (Enzo Life Science, USA). The biotin-labeled cRNA was heat-fragmented and biotinylated cRNAs were hybridized to Affymetrix YG-S98 microarrays in Affymetrix Hybridization Oven 640. Washing and staining of arrays were performed using the GeneChip Fluidics Station 450 and scanning with the Affymetrix GeneChip Scanner 3000. Acquisition and quantification of array images were performed using the Affymetrix GeneChip Operating System 1.0. The signals were scaled into target intensity of 100. All samples were assayed in triplicates. Keywords: dose response

Dataset Information

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

Publications

A Novel D-Galacturonate Fermentation Pathway in <i>Lactobacillus suebicus</i> Links Initial Reactions of the Galacturonate-Isomerase Route With the Phosphoketolase Pathway.

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