ascorbic-acid and sorbosone

Membrane-bound sorbosone dehydrogenase (SNDH) of Gluconacetobacter liquefaciens oxidizes l-sorbosone to 2-keto-l-gulonic acid (2KGLA), a key intermediate in vitamin C production. We constructed recombinant Escherichia coli and Gluconobacter strains harboring plasmids carrying the sndh gene from Ga. liquefaciens strain RCTMR10 to identify the prosthetic group of SNDH. The membranes of the recombinant E. coli showed l-sorbosone oxidation activity, only after the holo-enzyme formation with pyrroloquinoline quinone (PQQ), indicating that SNDH is a PQQ-dependent enzyme. The sorbosone-oxidizing respiratory chain was thus heterologously reconstituted in the E. coli membranes. The membranes that contained SNDH showed the activity of sorbosone:ubiquinone analogue oxidoreductase. These results suggest that the natural electron acceptor for SNDH is membranous ubiquinone, and it functions as the primary dehydrogenase in the sorbosone oxidation respiratory chain in Ga. liquefaciens. A biotransformation experiment showed l-sorbosone oxidation to 2KGLA in a nearly quantitative manner. Phylogenetic analysis for prokaryotic SNDH homologues revealed that they are found only in the Proteobacteria phylum and those of the Acetobacteraceae family are clustered in a group where all members possess a transmembrane segment. A three-dimensional structure model of the SNDH constructed with an in silico fold recognition method was similar to the crystal structure of the PQQ-dependent pyranose dehydrogenase from Coprinopsis cinerea. The structural similarity suggests a reaction mechanism under which PQQ participates in l-sorbosone oxidation.

Topics: Ascorbic Acid; Bacterial Proteins; Cell Membrane; Computer Simulation; Crystallization; Escherichia coli; Gluconacetobacter; Metabolic Engineering; Oxidoreductases; Phylogeny; Sorbose; Sugar Acids

2-Keto-L-gulonic acid (2-KLG) is the direct precursor of vitamin C in industrial synthesis. 2-KLG is mainly produced via the classical two-step fermentation route. In the two-step fermentation process, 2-KLG can be synthesized from L-sorbose by Ketogulonicigenium vulgare aided by Bacillus megaterium. There are five sorbose/sorbosone dehydrogenases (SSDHs), SSDA1, SSDA1-P, SSDA2, SSDA3 and SSDB, and two sorbosone dehydrogenases (SNDHs), glucose/sorbosone dehydrogenase (GSNDH) and sorbosone dehydrogenase (SNDH), in K. vulgare, which could play crucial roles in transforming L-sorbose or L-sorbosone to 2-KLG. However, confusion about the catalytic characteristics of the individual SSDHs and SNDHs makes construction of a recombinational strain for the purpose of enhancing 2-KLG production difficult. In this study, the five SSDHs and two SNDHs from K. vulgare WSH-001 were purified, and their optimal pH values and reaction temperatures, kinetic properties, thermostabilities, substrate spectra and effects of electron acceptors on their performances were systematically determined. Among these dehydrogenases, only SSDA1 and SSDA3 have high activity for catalyzing L-sorbose to 2-KLG directly. These data provide more clues for ways to achieve enhanced conversion of L-sorbose in K. vulgare, which could facilitate both the construction of a more efficient one-step fermentation 2-KLG producer and the reconstruction of a one-step fermentation process.

Topics: Ascorbic Acid; Bacterial Proteins; Carbohydrate Dehydrogenases; Enzyme Stability; Metabolic Engineering; Rhodobacteraceae; Sorbose; Sugar Acids

The expression levels of sorbose/sorbosone dehydrogenase genes (sdh and sndh) and the synthesis genes (pqqABCDEN) of the adjoint cofactor pyrroloquinoline quinone (PQQ) were genetically manipulated in Ketogulonigenium vulgare to increase the production of 2-keto-l-gulonic acid (2-KLG), the precursor of vitamin C, in the consortium of K. vulgare and Bacillus cereus. We found that overexpression of sdh-sndh alone in K. vulgare could not significantly enhance the production of 2-KLG, revealing the cofactor PQQ was required for the biosynthesis of 2-KLG. Various expression levels of PQQ were achieved by differential expression of pqqA, pqqABCDE and pqqABCDEN, respectively. The combinatorial expression of sdh/sndh and pqqABCDEN in K. vulgare enabled a 20% increase in the production of 2-KLG (79.1±0.6gl(-1)) than that of the parental K. vulgare (65.9±0.4gl(-1)) in shaking flasks. Our results demonstrated the balanced co-expression of both the key enzymes and the related cofactors was an efficient strategy to increase chemicals' biosynthesis.

Topics: Ascorbic Acid; Bacillus cereus; Bacterial Proteins; Carbohydrate Dehydrogenases; Gene Expression Regulation, Bacterial; Gene Expression Regulation, Enzymologic; Metabolic Engineering; PQQ Cofactor; Sorbose; Sugar Acids

A novel enzyme, L-sorbosone dehydrogenase 1 (SNDH1), which directly converts L-sorbosone to L-ascorbic acid (L-AA), was isolated from Ketogulonicigenium vulgare DSM 4025 and characterized. This enzyme was a homooligomer of 75-kDa subunits containing pyrroloquinoline quinone (PQQ) and heme c as the prosthetic groups. Two isozymes of SNDH, SNDH2 consisting of 75-kDa and 55-kDa subunits and SNDH3 consisting of 55-kDa subunits, were also purified from the bacterium. All of the SNDHs produced L-AA, as well as 2-keto-L-gulonic acid (2KGA), from L-sorbosone, suggesting that tautomerization of L-sorbosone causes the dual conversion by SNDHs. The sndH gene coding for SNDH1 was isolated and analyzed. The N-terminal four-fifths of the SNDH amino acid sequence exhibited 40% identity to the sequence of a soluble quinoprotein glucose dehydrogenase from Acinetobacter calcoaceticus. The C-terminal one-fifth of the sequence exhibited similarity to a c-type cytochrome with a heme-binding motif. A lysate of Escherichia coli cells expressing sndH exhibited SNDH activity in the presence of PQQ and CaCl2. Gene disruption analysis of K. vulgare indicated that all of the SNDH proteins are encoded by the sndH gene. The 55-kDa subunit was derived from the 75-kDa subunit, as indicated by cleavage of the C-terminal domain in the bacterial cells.

Topics: Aldehyde Oxidoreductases; Amino Acid Sequence; Ascorbic Acid; Base Sequence; DNA, Bacterial; Gene Expression; Genes, Bacterial; Heme; Isoenzymes; Models, Biological; Molecular Sequence Data; Molecular Weight; Mutagenesis, Insertional; PQQ Cofactor; Protein Subunits; Rhodobacteraceae; Sorbose

Ketogulonicigenium vulgare DSM 4025, known as a 2-keto-L-gulonic acid producing strain from L-sorbose via L-sorbosone, surprisingly produced L-ascorbic acid from D-sorbitol, L-sorbose, L-gulose, and L-sorbosone as the substrate under a growing or resting condition. As the best result, K. vulgare DSM 4025 produced 1.37 g per liter of L-AA from 5.00 g per liter of L-sorbosone during 4 h incubation time at 30 degrees C under the resting cell condition having 5.70 g per liter of wet cells. The precursor of L-AA formation from D-sorbitol and L-sorbose, except for L-gulose, was thought to be the putative furanose form of L-sorbosone. This is the first time it is reported that bacteria can produce vitamin C via L-sorbosone.

Topics: Ascorbic Acid; Gluconobacter oxydans; Hexoses; Sorbitol; Sorbose