Unusual 1-3 peptidoglycan cross-links in Acetobacteraceae are made by L,D-transpeptidases with a catalytic domain distantly related to YkuD domains.

Peptidoglycan is an essential component of the bacterial cell envelope that contains glycan chains substituted by short peptide stems. Peptide stems are polymerized by D,D-transpeptidases, which make bonds between the amino acid in position four of a donor stem and the third residue of an acceptor stem (4-3 cross-links). Some bacterial peptidoglycans also contain 3-3 cross-links that are formed by another class of enzymes called L,D-transpeptidases which contain a YkuD catalytic domain. In this work, we investigate the formation of unusual bacterial 1-3 peptidoglycan cross-links. We describe a version of the PGFinder software that can identify 1-3 cross-links and report the high-resolution peptidoglycan structure of Gluconobacter oxydans (a model organism within the Acetobacteraceae family). We reveal that G. oxydans peptidoglycan contains peptide stems made of a single alanine as well as several dipeptide stems with unusual amino acids at their C-terminus. Using a bioinformatics approach, we identified a G. oxydans mutant from a transposon library with a drastic reduction in 1-3 cross-links. Through complementation experiments in G. oxydans and recombinant protein production in a heterologous host, we identify an L,D-transpeptidase enzyme with a domain distantly related to the YkuD domain responsible for these non-canonical reactions. This work revisits the enzymatic capabilities of L,D-transpeptidases, a versatile family of enzymes that play a key role in bacterial peptidoglycan remodelling.

Separation and purification of pyrroloquinoline quinone from Gluconobacter oxydans fermentation broth using supramolecular solvent complex extraction.

In this paper, new supramolecular extractants, which contained surfactant, alkane and alkanol, were designed and used to separate PQQ. After a series of tests, the optimal extractant composition was determined as benzalkalonium (C8-C16) chloride (BC): n-hexane:n-pentanol, and the highest extraction rate could reach 98%. The extraction equilibrium could be reached in five minutes. The mechanism of the extraction selectivity was inferred as an ion-pair and π-π complexation interaction between PQQ and BC, which was indicated by UV and fluorescence quenching experiments. To recycle the organic extractant, the extract was back-extracted with sodium chloride solution. After extraction, back extraction and crystallization, an isolated product with a purity of 97.5% was obtained from G. oxydans fermentation broth. The product was identified as PQQ by HPLC analysis and MS. Above all, the present research developed a simple and efficient method for the separation of PQQ from fermentation broth.

Improvement of pyrroloquinoline quinone-dependent d-sorbitol dehydrogenase activity from Gluconobacter oxydans via expression of Vitreoscilla hemoglobin and regulation of dissolved oxygen tension for the biosynthesis of 6-(N-hydroxyethyl)-amino-6-deoxy-α-l-sorbofuranose.

The miglitol intermediate, 6-(N-hydroxyethyl)-amino-6-deoxy-α-l-sorbofuranose (6NSL), is catalyzed from N-2-hydroxyethyl glucamine (NHEG) by resting cells of Gluconobacter oxydans. One of the key factors limiting 6NSL production was the availability of oxygen during both cell cultivation and biotransformation of NHEG to 6NSL. Based on G. oxydans/pBBR1-sldAB-pqqABCDE-tldD (G. oxydans/AB-PQQ), the Vitreoscilla hemoglobin (VHb) was heterologously expressed in G. oxydans to enhance oxygen transfer efficiency and improve 6NSL production. The recombinant G. oxydans/AB-PQQ-VHb displayed higher biomass and NHEG oxidation activity than the control stain. The transcription levels of respiratory chain-related enzyme genes in G. oxydans/AB-PQQ-VHb exhibited up-regulation, indicating that the presence of VHb promoted the respiration. The dissolved oxygen (DO) concentration for cell cultivation was optimized in a 5-L stirred bioreactor. At a DO concentration of 20%, the maximum volumetric oxidation activity of NHEG of G. oxydans/AB-PQQ-VHb in the stirred bioreactor reached 168.3 ± 3.2 U/L. Furthermore, the biotransformation of NHEG to 6NSL using G. oxydans/AB-PQQ-VHb was carried out under different oxygen tensions to investigate the effect of oxygen on 6NSL production. Finally, up to 87.5 ± 5.9 g/L 6NSL was accumulated in the reaction mixture within 16 h when the DO was controlled at 30%.

Bioconversion of recombinantly produced precursor peptide pqqA into pyrroloquinoline quinone (PQQ) using a cell-free in vitro system.

Pyrroloquinoline quinone (PQQ) has been recognized as the third class of redox cofactors in addition to the well-known nicotinamides (NAD(P)+) and flavins (FAD, FMN). It plays important physiological roles in various organisms and has strong antioxidant properties. The biosynthetic pathway of PQQ involves a gene cluster composed of 4-7 genes, named pqqA-G, among which pqqA is a key gene for PQQ synthesis, encoding the precursor peptide PqqA. To produce recombinant PqqA in E. coli, fusion tags were used to increase the stability and solubility of the peptide, as well simplify the scale-up of the fermentation process. In this paper, pqqA from Gluconobacter oxydans 621H was expressed in E. coli BL21 (DE3) as a fusion protein with SUMO and purified using a hexahistidine (His6) tag. The SUMO fusion protein and His6 tag were specifically recognized and cleaved by the SUMO specific ULP protease, and immobilized-metal affinity chromatography was used to obtain high-purity precursor peptide PqqA. Expression and purification of target proteins was confirmed by Tricine-SDS-PAGE. Finally, the synthesis of PQQ in a cell-free enzymatic reaction in vitro was confirmed by LC-MS.

The Auxiliary NADH Dehydrogenase Plays a Crucial Role in Redox Homeostasis of Nicotinamide Cofactors in the Absence of the Periplasmic Oxidation System in Gluconobacter oxydans NBRC3293.

Gluconobacter oxydans has the unique property of a glucose oxidation system in the periplasmic space, where glucose is oxidized incompletely to ketogluconic acids in a nicotinamide cofactor-independent manner. Elimination of the gdhM gene for membrane-bound glucose dehydrogenase, the first enzyme for the periplasmic glucose oxidation system, induces a metabolic change whereby glucose is oxidized in the cytoplasm to acetic acid. G. oxydans strain NBRC3293 possesses two molecular species of type II NADH dehydrogenase (NDH), the primary and auxiliary NDHs that oxidize NAD(P)H by reducing ubiquinone in the cell membrane. The substrate specificities of the two NDHs are different from each other: primary NDH (p-NDH) oxidizes NADH specifically but auxiliary NDH (a-NDH) oxidizes both NADH and NADPH. We constructed G. oxydans NBRC3293 derivatives defective in the ndhA gene for a-NDH, in the gdhM gene, and in both. Our ΔgdhM derivative yielded higher cell biomass on glucose, as reported previously, but grew at a lower rate than the wild-type strain. The ΔndhA derivative showed growth behavior on glucose similar to that of the wild type. The ΔgdhM ΔndhA double mutant showed greatly delayed growth on glucose, but its cell biomass was similar to that of the ΔgdhM strain. The double mutant accumulated intracellular levels of NAD(P)H and thus shifted the redox balance to reduction. Accumulated NAD(P)H levels might repress growth on glucose by limiting oxidative metabolisms in the cytoplasm. We suggest that a-NDH plays a crucial role in redox homeostasis of nicotinamide cofactors in the absence of the periplasmic oxidation system in G. oxydansIMPORTANCE Nicotinamide cofactors NAD+ and NADP+ mediate redox reactions in metabolism. Gluconobacter oxydans, a member of the acetic acid bacteria, oxidizes glucose incompletely in the periplasmic space-outside the cell. This incomplete oxidation of glucose is independent of nicotinamide cofactors. However, if the periplasmic oxidation of glucose is abolished, the cells oxidize glucose in the cytoplasm by reducing nicotinamide cofactors. Reduced forms of nicotinamide cofactors are reoxidized by NADH dehydrogenase (NDH) on the cell membrane. We found that two kinds of NDH in G. oxydans have different substrate specificities: the primary enzyme is NADH specific, and the auxiliary one oxidizes both NADH and NADPH. Inactivation of the latter enzyme in G. oxydans cells in which we had induced cytoplasmic glucose oxidation resulted in elevated intracellular levels of NAD(P)H, limiting cell growth on glucose. We suggest that the auxiliary enzyme is important if G. oxydans grows independently of the periplasmic oxidation system.

Investigating the Product Profiles and Structural Relationships of New Levansucrases with Conventional and Non-Conventional Substrates.

The synthesis of complex oligosaccharides is desired for their potential as prebiotics, and their role in the pharmaceutical and food industry. Levansucrase (LS, EC 2.4.1.10), a fructosyl-transferase, can catalyze the synthesis of these compounds. LS acquires a fructosyl residue from a donor molecule and performs a non-Lenoir transfer to an acceptor molecule, via ß-(2→6)-glycosidic linkages. Genome mining was used to uncover new LS enzymes with increased transfructosylating activity and wider acceptor promiscuity, with an initial screening revealing five LS enzymes. The product profiles and activities of these enzymes were examined after their incubation with sucrose. Alternate acceptor molecules were also incubated with the enzymes to study their consumption. LSs from Gluconobacter oxydans and Novosphingobium aromaticivorans synthesized fructooligosaccharides (FOSs) with up to 13 units in length. Alignment of their amino acid sequences and substrate docking with homology models identified structural elements causing differences in their product spectra. Raffinose, over sucrose, was the preferred donor molecule for the LS from Vibrio natriegens, N. aromaticivorans, and Paraburkolderia graminis. The LSs examined were found to have wide acceptor promiscuity, utilizing monosaccharides, disaccharides, and two alcohols to a high degree.

Learning about Enzyme Stability against Organic Cosolvents from Structural Insights by Ion Mobility Mass Spectrometry.

Ion mobility spectrometry (IMS) coupled with mass spectrometry (MS) enables the investigation of protein folding in solution. Herein, a proof-of-concept for obtaining structural information about the folding of a protein in dependency of the amount of an organic cosolvent in the aqueous medium by means of this IMS-MS method is presented. By analyzing the protein with native nano-electrospray ionization IMS-MS, the impact of acetonitrile as a representative organic cosolvent and/or pH values on the folding of an enzyme was successfully evaluated in a fast and straightforward fashion, as exemplified for an ene reductase from Gluconobacter oxydans. The IMS-MS results are in agreement with findings from the nicotinamide adenine dinucleotide phosphate (NADPH)-based spectrophotometric enzyme activity tests under analogous conditions, and thus, also rationalizing these "wet" analytical data. For this ene reductase, a higher tolerance against CH3 CN in the presence of a buffer was observed by both analytical methods. The results suggest that this IMS-MS methodology could be a useful complementary tool to existing methods in process optimization and fine-tuning of solvent conditions for biotransformations.

Synergistic improvement of PQQ-dependent D-sorbitol dehydrogenase activity from Gluconobacter oxydans for the biosynthesis of miglitol precursor 6-(N-hydroxyethyl)-amino-6-deoxy-α-L-sorbofuranose.

6-(N-hydroxyethyl) amino-6-deoxy-l-sorbofuranose (6NSL) is the direct precursor of miglitol for diabetes therapy. The regio- and stereo-selective dehydrogenation offered by the membrane-bound d-sorbitol dehydrogenase (mSLDH) from Gluconobacter oxydans provides an elegant enzymatic method for 6NSL production. In this study, two subunits sldA and sldB of mSLDH were introduced into G. oxydans ZJB-605, and the specific enzyme activity of mSLDH towards NHEG was enhanced by 2.15-fold. However, the endogenous PQQ level was dramatically reduced in the recombinant strain and became a bottleneck to support the holo-enzyme activity. A combined supplementation of four amino acids (Glu, Ile, Ser, Arg) involved in biosynthesis of PQQ in conventional media effectively increased extracellular accumulation of PQQ by 1.49-fold, which further enhanced mSLDH activity by 1.33-fold. The synergic improvement of mSLDH activity provided in this study supports the superior high dehydrogenate activity towards substrate N-2-hydroxyethyl-glucamine, 184.28 g·L-1 of 6NSL was produced after a repeated bioconversion process catalyzed by the resting cells of G. oxydans/pBB-sldAB, all of which presenting a great potential of their industrial application in 6NSL biosynthesis.

L-Erythrulose production with a multideletion strain of Gluconobacter oxydans.

Many ketoses or organic acids can be produced by membrane-associated oxidation with Gluconobacter oxydans. In this study, the oxidation of meso-erythritol to L-erythrulose was investigated with the strain G. oxydans 621HΔupp BP.8, a multideletion strain lacking the genes for eight membrane-bound dehydrogenases. First batch biotransformations with growing cells showed re-consumption of L-erythrulose by G. oxydans 621HΔupp BP.8 in contrast to resting cells. The batch biotransformation with 2.8 g L-1 resting cells of G. oxydans 621HΔupp BP.8 in a DO-controlled stirred-tank bioreactor resulted in 242 g L-1 L-erythrulose with a product yield of 99% (w/w) and a space-time yield of 10 g L-1 h-1. Reaction engineering studies showed substrate excess inhibition as well as product inhibition of G. oxydans 621HΔupp BP.8 in batch biotransformations. In order to overcome substrate inhibition, a continuous membrane bioreactor with full cell retention was applied for meso-erythritol oxidation with resting cells of G. oxydans 621HΔupp BP.8. At a mean hydraulic residence time of 2 h, a space-time yield of 27 g L-1 h-1 L-erythrulose was achieved without changing the product yield of 99% (w/w) resulting in a cell-specific product yield of up to 4.4 gP gX-1 in the steady state. The product concentration (54 g L-1 L-erythrulose) was reduced in the continuous biotransformation process compared with the batch process to avoid product inhibition.

Overcoming NADPH product inhibition improves D-sorbitol conversion to L-sorbose.

Gluconobacter oxydans sorbitol dehydrogenase (GoSLDH) exhibits a higher catalytic efficiency than other L-sorbose producing enzymes. During the reaction catalysed by GoSLDH, NADP+ is reduced to NADPH and D-sorbitol is oxidized to L-sorbose. However, GoSLDH activity is inhibited by the NADPH (Ki = 100 µM) formed during the enzymatic reaction. Therefore, Escherichia coligosldh-lrenox producing both GoSLDH for D-sorbitol oxidation and LreNOX (NAD(P)H oxidase from Lactobacillus reuteri) for NADP+ regeneration was generated and used for L-sorbose production. Whole cell biocatalysts with the LreNOX cofactor recycling system showed a high conversion rate (92%) of D-sorbitol to L-sorbose in the presence of low concentration of NADP+ (0.5 mM). By alleviating NADPH accumulation during the catalytic reactions, E. coligosldh-lrenox exhibited 23-fold higher conversion rate of D-sorbitol than E. coligosldh. L-Sorbose production by E. coligosldh-lrenox reached 4.1 g/L after 40 min, which was 20.5-fold higher than that of E. coligosldh. We also constructed G. oxydansgosldh and G. oxydansgosldh-lrenox strains, and they exhibited 1.2- and 2.9-fold higher conversion rates than the wild-type G. oxydans KCTC 1091. The results indicate that overcoming NADPH product inhibition using LreNOX improves chemical production in NADP+-dependent enzymatic reactions.

Identification of the enzymes responsible for 3-hydroxypropionic acid formation and their use in improving 3-hydroxypropionic acid production in Gluconobacter oxydans DSM 2003.

Gluconobacter oxydans can be efficiently used to produce 3-hydroxypropionic acid (3-HP) from 1,3-propanediol (1,3-PDO). However, the enzymes involved remain unclear. In this study, transcription analysis of two mutants of strain DSM 2003, obtained by UV-mutagenesis, revealed that membrane-bound alcohol dehydrogenase (mADH) and membrane-bound aldehyde dehydrogenase (mALDH) might be the main enzymes involved. Through deletion and complementation of the genes adhA and aldh, mADH and mALDH were verified as the main enzymes responsible for 3-HP production. Then mALDH was verified as the rate-limiting enzyme in 3-HP production. Since that overexpression of mADH had no effect on 3-HP production, whereas overexpression of mALDH increased 23.6% 3-HP production. Finally, the 3-HP titer of 45.8 g/L and the highest productivity 1.86 g/L/h were achieved when the two mutants DSM 2003/adhAB and DSM 2003/aldh were mixed at a ratio of 1:2 (cell density) and used as whole cell catalysts for 3-HP production.

Improved heterologous expression of the membrane-bound quinoprotein quinate dehydrogenase from Gluconobacter oxydans.

Gluconobacter oxydans produces 3-dehydroquinate by oxidation of quinate through a reaction catalyzed by the quinate dehydrogenase (QDH), membrane-bound, pyrroloquinoline quinone (PQQ)-dependent dehydrogenase. We previously reported the nucleotide and deduced amino acid sequence of QDH and constructed a heterologous expression system of QDH in Pseudomonas sp. (A.S. Vangnai, W. Promden, W. De-Eknamkul, K. Matsushita, H. Toyama, Biochemistry (Moscow) 75:452-459, 2010). Through this study, we aim to update the sequences of QDH and improve the heterologous expression of QDH in Gluconobacter strains using a broad-host-range plasmid. Expression of QDH using a plasmid containing a long 5'-UTR was higher than that using a plasmid with a short 5'-UTR. In addition, the usage of the putative promoter region of the membrane-bound, alcohol dehydrogenase (ADH) of Gluconobacter resulted in higher expression levels compared to the usage of the lacZ promoter. Base substitution experiments allowed to identify the correct TTG initiation codon between two possibilities, and the result of these experiments were consistent with the N-terminal amino acid sequence of the expressed QDH. However, change of the TTG codon to ATG did not increase QDH expression. Therefore, the optimal plasmid for QDH expression included the structural gene with a long 5'-UTR and the ADH promoter. Cell membrane of the recombinant Gluconobacter strain presented approximately 10-times higher specific QDH activity than that observed in the wild-type strain.

Enantioselective Hydrogen Atom Transfer: Discovery of Catalytic Promiscuity in Flavin-Dependent 'Ene'-Reductases.

Flavin has long been known to function as a single electron reductant in biological settings, but this reactivity has rarely been observed with flavoproteins used in organic synthesis. Here we describe the discovery of an enantioselective radical dehalogenation pathway for α-bromoesters using flavin-dependent 'ene'-reductases. Mechanistic experiments support the role of flavin hydroquinone as a single electron reductant, flavin semiquinone as the hydrogen atom source, and the enzyme as the source of chirality.

Biosynthesis of L-Erythrose by Assembly of Two Key Enzymes in Gluconobacter oxydans.

L-erythrose, a rare aldotetrose, possesses various pharmacological activities. However, efficient L-erythrose production is challenging. Currently, L-erythrose is produced by a two-step fermentation process from erythritol. Here, we describe a novel strategy for the production of L-erythrose in Gluconobacter oxydans (G. oxydans) by localizing the assembly of L-ribose isomerase (L-RI) to membrane-bound sorbitol dehydrogenase (SDH) via the protein-peptide interactions of the PDZ domain and PDZ ligand. To demonstrate this self-assembly, green fluorescent protein (GFP) replaced L-RI and its movement to membrane-bound SDH was observed by fluorescence microscopy. The final L-erythrose production was improved to 23.5 g/L with the stepwise metabolic engineering of G. oxydans, which was 1.4-fold higher than that obtained using coexpression of SDH and L-RI in G. oxydans. This self-assembly strategy shows remarkable potential for further improvement of L-erythrose production.

Characterization of membrane-bound dehydrogenases of Gluconobacter oxydans 621H using a new system for their functional expression.

Acetic acid bacteria are used in biotechnology due to their ability to incompletely oxidize a great variety of carbohydrates, alcohols, and related compounds in a regio- and stereo-selective manner. These reactions are catalyzed by membrane-bound dehydrogenases (mDHs), often with a broad substrate spectrum. In this study, the promoters of six mDHs of Gluconobacter oxydans 621H were characterized. The constitutive promoter of the alcohol dehydrogenase and the glucose-repressed promoter of the inositol dehydrogenase were used to construct a shuttle vector system for the fully functional expression of mDHs in the multi-deletion strain G. oxydans BP.9 that lacks its mDHs. This system was used to express each mDH of G. oxydans 621H, in order to individually characterize the substrates, they oxidize. From 55 tested compounds, the alcohol dehydrogenase oxidized 30 substrates and the polyol dehydrogenase 25. The substrate spectrum of alcohol dehydrogenase overlapped largely with the aldehyde dehydrogenase and partially with polyol dehydrogenase. Thus, we were able to resolve the overlapping substrate spectra of the main mDHs of G. oxydans 621H. The described approach could also be used for the expression and detailed characterization of substrates used by mDHs from other acetic acid bacteria or a metagenome.

Highly efficient bioreduction of 2-hydroxyacetophenone to (S)- and (R)-1-phenyl-1,2-ethanediol by two substrate tolerance carbonyl reductases with cofactor regeneration.

Optically pure 1-phenyl-1,2-ethanediol is a very important chiral building block and intermediate in fine chemical and pharmaceutical industries. Reduction of 2-hydroxyacetophenone provides a straightforward approach to access these important compounds. In this study, two enantiocomplementary carbonyl reductases, BDHA (2,3-butanediol dehydrogenase from Bacillus subtilis) and GoSCR (polyol dehydrogenase from Gluconobacter oxydans) were discovered for the first time to convert 2-hydroxyacetophenone (2-HAP) to (R)-1-phenyl-1,2-ethanediol ((R)-PED) and (S)-1-phenyl-1,2-ethanediol ((S)-PED) with excellent stereochemical selectivity, respectively. The two enzymes were purified and characterized. In vitro bioreduction of 2-HAP catalyzed by BDHA and GoSCR coupled with glucose dehydrogenase (GDH) from Bacillus subtilis for cofactor regeneration were demonstrated, affording both (R)-PED and (S)-PED in>99% ee and 99% conversion. Recombinant Escherichia coli whole cells co-expressing both GDH and BDHA or GoSCR genes were used to asymmetric reduction of 2-HAP to (R)-PED or (S)-PED. Under the optimized conditions, the bioreduction of 400mM (54g/L) substrate was proceeded smoothly without the external addition of cofactor, and the product (R)-PED and (S)-PED were obtained with 99% yield, >99% ee and 18.0g/L/h volumetric productivity. These results offer a practical biocatalytic method for the preparation of both (R)-PED and (S)-PED with high volumetric productivity.

Enhancement of 1,3-Dihydroxyacetone Production from Gluconobacter oxydans by Combined Mutagenesis.

Wild strain L-6 was subjected to combined mutagenesis, including UV irradiation, atmospheric and room temperature plasma, and ion beam implantation, to increase the yield of 1,3-dihydroxyacetone (DHA). With application of a high-throughput screening method, mutant Gluconobacter oxydans I-2-239 with a DHA productivity of 103.5 g/l in flask-shake fermentation was finally obtained with the starting glycerol concentration of 120 g/l, which was 115.7% higher than the wild strain. The cultivation time also decreased from 54 h to 36 h. Compared with the wild strain, a dramatic increase in enzyme activity was observed for the mutant strain, although the increase in biomass was limited. DNA and amino acid sequence alignment revealed 11 nucleotide substitutions and 10 amino acid substitutions between the sldAB of strains L-6 and I-2-239. Simulation of the 3-D structure and prediction of active site residues and PQQ binding site residues suggested that these mutations were mainly related to PQQ binding, which was speculated to be favorable for the catalyzing capacity of glycerol dehydrogenase. RT-qPCR assay indicated that the transcription levels of sldA and sldB in the mutant strain were respectively 4.8-fold and 5.4-fold higher than that in the wild strain, suggesting another possible reason for the increased DHA productivity of the mutant strain.

Enhancement of cell growth and glycolic acid production by overexpression of membrane-bound alcohol dehydrogenase in Gluconobacter oxydans DSM 2003.

Membrane-bound alcohol dehydrogenase (mADH) was overexpressed in Gluconobacter oxydans DSM 2003, and the effects on cell growth and glycolic acid production were investigated. The transcription levels of two terminal ubiquinol oxidases (bo3 and bd) in the respiratory chain of the engineered strain G. oxydans-adhABS were up-regulated by 13.4- and 3.8-fold, respectively, which effectively enhanced the oxygen uptake rate, resulting in higher resistance to acid. The cell biomass of G. oxydans-adhABS could increase by 26%-33% when cultivated in a 7L bioreactor. The activities of other major membrane-bound dehydrogenases were also increased to some extent, particularly membrane-bound aldehyde dehydrogenase (mALDH), which is involved in the catalytic oxidation of aldehydes to the corresponding acids and was 1.26-fold higher. Relying on the advantages of the above, G. oxydans-adhABS could produce 73.3gl-1 glycolic acid after 45h of bioconversion with resting cells, with a molar yield 93.5% and a space-time yield of 1.63gl-1h-1. Glycolic acid production could be further improved by fed-batch fermentation. After 45h of culture, 113.8gl-1 glycolic acid was accumulated, with a molar yield of 92.9% and a space-time yield of 2.53gl-1h-1, which is the highest reported glycolic acid yield to date.

A highly efficient sorbitol dehydrogenase from Gluconobacter oxydans G624 and improvement of its stability through immobilization.

A sorbitol dehydrogenase (GoSLDH) from Gluconobacter oxydans G624 (G. oxydans G624) was expressed in Escherichia coli BL21(DE3)-CodonPlus RIL. The complete 1455-bp codon-optimized gene was amplified, expressed, and thoroughly characterized for the first time. GoSLDH exhibited Km and kcat values of 38.9 mM and 3820 s(-1) toward L-sorbitol, respectively. The enzyme exhibited high preference for NADP(+) (vs. only 2.5% relative activity with NAD(+)). GoSLDH sequencing, structure analyses, and biochemical studies, suggested that it belongs to the NADP(+)-dependent polyol-specific long-chain sorbitol dehydrogenase family. GoSLDH is the first fully characterized SLDH to date, and it is distinguished from other L-sorbose-producing enzymes by its high activity and substrate specificity. Isothermal titration calorimetry showed that the protein binds more strongly to D-sorbitol than other L-sorbose-producing enzymes, and substrate docking analysis confirmed a higher turnover rate. The high oxidation potential of GoSLDH for D-sorbitol was confirmed by cyclovoltametric analysis. Further, stability of GoSLDH significantly improved (up to 13.6-fold) after cross-linking of immobilized enzyme on silica nanoparticles and retained 62.8% residual activity after 10 cycles of reuse. Therefore, immobilized GoSLDH may be useful for L-sorbose production from D-sorbitol.

Overexpression of membrane-bound gluconate-2-dehydrogenase to enhance the production of 2-keto-D-gluconic acid by Gluconobacter oxydans.

BACKGROUND: 2-keto-D-gluconic acid (2KGA) is widely used as a chemical intermediate in the cosmetic, pharmaceutical and environmental industries. Several microbial fermentation processes have been developed for production of 2KGA but these suffer from substrate/product inhibition, byproduct formation and low productivity. In previous work, we showed that 2KGA can be specifically produced from glucose (Glu) or gluconic acid (GA) by resting wild-type Gluconobacter oxydans DSM2003 cells, although substrate concentration was relatively low. In this study, we attempted to improve 2KGA productivity by G. oxydans DSM2003 by overexpressing the ga2dh gene, which encodes the membrane-bound gluconate-2-dehydrogenase enzyme (GA2DH). RESULTS: The ga2dh gene was overexpressed in G. oxydans DSM2003 under the control of three promoters, P tufB , P ga2dh or P ghp0169 , respectively. Among the recombinant strains obtained, G. oxydans_tufB_ga2dh showed a similar growth rate to that of the control strain and displayed the highest specific productivity of 2KGA from GA, which was increased nearly twofold compared with that of the control strain during batch biotransformation. When biocatalysis conditions were optimized, with provision of sufficient oxygen during biotransformation, up to 480 g/L GA was completely utilized over 45 h by resting cells of G. oxydans_tufB_ga2dh and 453.3 g/L 2KGA was produced. A productivity of 10.07 g/L/h and a yield of 95.3 % were obtained. Overexpression of the ga2dh gene also significantly improved the conversion of Glu to 2KGA. Under optimized conditions, 270 g/L Glu was converted to 321 g/L 2KGA over 18 h, with a yield of 99.1 % and a productivity of 17.83 g/L/h. The glucose concentrations during the batch biotransformation and the 2KGA productivities achieved in this study were relatively high compared with the results of previous studies. CONCLUSIONS: This study developed an efficient bacterial strain (G. oxydans_tufB_ga2dh) for the production of 2KGA by overexpressing the ga2dh gene in G. oxydans. Supply of sufficient oxygen enhanced the positive effect of gene overexpression on 2KGA production. Gluconobacter oxydans_tufB_ga2dh is thus a competitive species for use in 2KGA production.