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At least 24 aldehyde reductases from Saccharomyces cerevisiae have been characterized and most function in in situ detoxification of lignocellulosic aldehyde inhibitors, but none is classified into the polyol dehydrogenase (PDH) subfamily of the medium-chain dehydrogenase/reductase (MDR) superfamily. This study confirmed that two (2R,3R)-2,3-butanediol dehydrogenases (BDHs) from industrial (denoted Y)/laboratory (denoted B) strains of S. cerevisiae, Bdh1p(Y)/Bdh1p(B) and Bdh2p(Y)/Bdh2p(B), were members of the PDH subfamily with an NAD(P)H binding domain and a catalytic zinc binding domain, and exhibited reductive activities towards lignocellulosic aldehyde inhibitors, such as acetaldehyde, glycolaldehyde, and furfural. Especially, the highest enzyme activity towards acetaldehyde by Bdh2p(Y) was 117.95 U/mg with cofactor nicotinamide adenine dinucleotide reduced (NADH). Based on the comparative kinetic property analysis, Bdh2p(Y)/Bdh2p(B) possessed higher specific activity, substrate affinity, and catalytic efficiency towards glycolaldehyde than Bdh1p(Y)/Bdh1p(B). This was speculated to be related to their 49% sequence differences and five nonsynonymous substitutions (Ser41Thr, Glu173Gln, Ile270Leu, Ile316Met, and Gly317Cys) occurred in their conserved NAD(P)H binding domains. Compared with BDHs from a laboratory strain, Bdh1p(Y) and Bdh2p(Y) from an industrial strain displayed five nonsynonymous mutations (Thr12, Asn61, Glu168, Val222, and Ala235) and three nonsynonymous mutations (Ala34, Ile96, and Ala369), respectively. From a first analysis with selected aldehydes, their reductase activities were different from BDHs of laboratory strain, and their catalytic efficiency was higher towards glycolaldehyde and lower towards acetaldehyde. Comparative investigation of kinetic properties of BDHs from S. cerevisiae as aldehyde reductases provides a guideline for their practical applications in in situ detoxification of aldehyde inhibitors during lignocellulose bioconversion.Key Points• Two yeast BDHs have enzyme activities for reduction of aldehydes.• Overexpression of BDHs slightly improves yeast tolerance to acetaldehyde and glycolaldehyde.• Bdh1p and Bdh2p differ in enzyme kinetic properties.• BDHs from strains with different genetic backgrounds differ in enzyme kinetic properties.

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.

Acetic acid bacteria are well-known for their membrane-bound dehydrogenases rapidly oxidizing a variety of substrates in the periplasm. Since many acetic acid bacteria have not been successfully cultured in the laboratory yet, studying membrane-bound dehydrogenases directly from a metagenome of vinegar microbiota seems to be a promising way to identify novel variants of these enzymes. To this end, DNA from a mother of vinegar was isolated, sequenced, and screened for membrane-bound dehydrogenases using an in silico approach. Six metagenomic dehydrogenases were successfully expressed using an expression vector with native promoters in the acetic acid bacterium strain Gluconobacter oxydans BP.9, which is devoid of its major native membrane-bound dehydrogenases. Determining the substrates converted by these enzymes, using a whole-cell DCPIP assay, revealed one glucose dehydrogenase with an enlarged substrate spectrum additionally oxidizing aldoheptoses, D-ribose and aldotetroses, one polyol dehydrogenase with an extreme diminished spectrum but distinguishing cis and trans -1,2-cyclohexandiol and a completely new secondary alcohol dehydrogenase, which oxidizes secondary alcohols with a hydroxyl group at position 2, as long as no primary hydroxyl group is present. Three further dehydrogenases were found with substrate spectra similar to known dehydrogenases of G. oxydans 621H.

Carbohydrates are much more than just a source of energy as they also mediate a variety of recognition processes that are central to human health. As such, saccharides can be applied in the food and pharmaceutical industries to stimulate our immune system (e.g., prebiotics), to control diabetes (e.g., low-calorie sweeteners), or as building blocks for anticancer and antiviral drugs (e.g., L-nucleosides). Unfortunately, only a small number of all possible monosaccharides are found in nature in sufficient amounts to allow their commercial exploitation. Consequently, so-called rare sugars have to be produced by (bio)chemical processes starting from cheap and widely available substrates. Three enzyme classes that can be used for rare sugar production are keto–aldol isomerases, epimerases, and oxidoreductases. In this review, the recent developments in rare sugar production with these biocatalysts are discussed.

Membrane-bound, pyrroloquinoline quinone (PQQ)-dependent glycerol dehydrogenase (GLDH, or polyol dehydrogenase) of Gluconobacter sp. oxidizes various secondary alcohols to produce the corresponding ketones, such as oxidation of D-sorbitol to L-sorbose in vitamin C production. Substrate specificity of GLDH is considered limited to secondary alcohols in the D-erythro configuration at the next to the last carbon. Here, we suggest that L-ribose, D- and L-lyxoses, and L-tagatose are also substrates of GLDH, but these sugars do not meet the substrate specificity rule of GLDH. The oxygen consumption activity of wild-type Gluconobacter frateurii cell membranes depends on several kinds of sugars as compared with that of the membranes of a GLDH-negative variant. Biotransformation of those sugars with the membranes was examined to determine the reaction products. A time course measuring the pH in the reaction mixture and the increase or decrease in substrates and products on TLC suggested that oxidation products of L-lyxose and L-tagatose were ketones with unknown structures, but those of L-ribose and D-lyxose were acids. The oxidation product of L-ribose was purified and revealed to be L-ribonate by HRMS and NMR analysis. Biotransformation of L-ribose with the membranes and also with the whole cells produced L-ribonate in nearly stoichiometric amounts, indicating that the specific oxidation site in L-ribose is recognized by GLDH. Since purified GLDH produced L-ribonate without any intermediate-like compounds, we propose here a reaction model where the first carbon in the pyranose form of L-ribose is oxidized by GLDH to L-ribonolactone, which is further hydrolyzed spontaneously to produce L-ribonate.

Background Whole cells of Gluconobacter oxydans are widely used in various biocatalytic processes. Sorbitol at high concentrations is commonly used in complex media to prepare biocatalysts. Exploiting an alternative process for preparation of biocatalysts with low cost substrates is of importance for industrial applications. Results G. oxydans 621H was confirmed to have the ability to grow in mineral salts medium with glycerol, an inevitable waste generated from industry of biofuels, as the sole carbon source. Based on the glycerol utilization mechanism elucidated in this study, the major polyol dehydrogenase (GOX0854) and the membrane-bound alcohol dehydrogenase (GOX1068) can competitively utilize glycerol but play no obvious roles in the biocatalyst preparation. Thus, the genes related to these two enzymes were deleted. Whole cells of G. oxydans ∆GOX1068∆GOX0854 can be prepared from glycerol with a 2.4-fold higher biomass yield than that of G. oxydans 621H. Using whole cells of G. oxydans ∆GOX1068∆GOX0854 as the biocatalyst, 61.6 g L −1 xylonate was produced from 58.4 g L −1 xylose at a yield of 1.05 g g −1 . Conclusion This process is an example of efficient preparation of whole cells of G. oxydans with reduced cost. Besides xylonate production from xylose, other biocatalytic processes might also be developed using whole cells of metabolic engineered G. oxydans prepared from glycerol.

Acetic acid bacteria such as Gluconobacter oxydans are used in several biotechnological processes due to their ability to perform rapid incomplete regio- and stereo-selective oxidations of a great variety of carbohydrates, alcohols, and related compounds by their membrane-bound dehydrogenases. In order to understand the growth physiology of industrial strains such as G. oxydans ATCC 621H that has high substrate oxidation rates but poor growth yields, we compared its genome sequence to the genome sequence of strain DSM 3504 that reaches an almost three times higher optical density. Although the genome sequences are very similar, DSM 3504 has additional copies of genes that are absent from ATCC 621H. Most importantly, strain DSM 3504 contains an additional type II NADH dehydrogenase (ndh) gene and an additional triosephosphate isomerase (tpi) gene. We deleted these additional paralogs from DSM 3504, overexpressed NADH dehydrogenase in ATCC 621H, and monitored biomass and the concentration of the representative cell components as well as O₂and CO₂transfer rates in growth experiments on mannitol. The data revealed a clear competition of membrane-bound dehydrogenases and NADH dehydrogenase for channeling electrons in the electron transport chain of Gluconobacter and an important role of the additional NADH dehydrogenase for increased growth yields. The less active the NADH dehydrogenase is, the more active is the membrane-bound polyol dehydrogenase. These results were confirmed by introducing additional ndh genes via plasmid pAJ78 in strain ATCC 621H, which leads to a marked increase of the growth rate.

ABSTRACT The genus Gluconobacter comprises some of the most frequently used microorganisms when it comes to biotechnological applications. Not only has it been involved in \"historical\" production processes, such as vinegar production, but in the last decades many bioconversion routes for special and rare sugars involving Gluconobacter have been developed. Among the most recent are the biotransformations involved in the production of L-ribose and miglitol, both very promising pharmaceutical lead molecules. Most of these processes make use of Gluconobacter's membrane-bound polyol dehydrogenases. However, recently other enzymes have also caught the eye of industrial biotechnology. Among them are dextran dextrinase, capable of transglucosylating substrate molecules, and intracellular NAD-dependent polyol dehydrogenases, of interest for co-enzyme regeneration. As such, Gluconobacter is an important industrial microbial strain, but it also finds use in other fields of biotechnology, such as biosensor-technology. This review aims to give an overview of the myriad of applications for Gluconobacter, with a special focus on some recent developments.

Polyol dehydrogenases (PDHs) play a pivotal role in the biotransformation between rare sugar and alcohol. Among these PDHs, mannitol 2-dehydrogenase (MDH, EC 1.1.1.67), galactitol 2-dehydrogenase (GDH, EC 1.1.1.16), ribitol 2-dehydrogenase (RDH, EC 1.1.1.56), xylitol 4-dehydrogenase (XDH, EC 1.1.1.14), and arabitol 2-dehydrogenase (ArDH, EC 1.1.1.12) are the most studied. MDH can catalyze the transformation between d -fructose and mannitol as well as the transformation between d -arabitol and d -xylulose. In addition to MDH, the other PDHs including RDH, GDH, ArDH, and XDH are also important tools for the production of rare sugars including d -tagatose, allitol, d -xylulose, and l -xylulose. Concerning the intermediate function of PDH in the linkage of rare sugar and sugar alcohols, this review attempts to conclude their catalytic properties and potential applications.

The first hyperthermophilic d-arabitol dehydrogenase from Thermotoga maritima was heterologously purified from Escherichia coli. The protein was purified with and without a Strep-tag. The enzyme exclusively catalyzed the NAD(H)-dependent oxidoreduction of d-arabitol, d-xylitol, d-ribulose, or d-xylulose. A twofold increase of catalytic rates was observed upon addition of Mg^sup 2+^ or K^sup +^. Interestingly, only the tag-less protein was thermostable, retaining 90% of its activity after 90 min at 85 °C. However, the tag-less form of d-arabitol dehydrogenase had similar kinetic parameters compared to the tagged enzyme, demonstrating that the Strep-tag was not deleterious to protein function but decreased protein stability. A single band at 27.6 kDa was observed on SDS-PAGE and native PAGE revealed that the protein formed a homohexamer and a homododecamer. The enzyme catalyzed oxidation of d-arabitol to d-ribulose and therefore belongs to the class of d-arabitol 2-dehydrogenases, which are typically observed in yeast and not bacteria. The product d-ribulose is a rare ketopentose sugar that has numerous industrially applications. Given its thermostability and specificity, d-arabitol 2-dehydrogenase is a desirable biocatalyst for the production of rare sugar precursors. [PUBLICATION ABSTRACT]