Simple Plug-In Synthetic Step for the Synthesis of (-)-Camphor from Renewable Starting Materials.

Racemic camphor and isoborneol are readily available as industrial side products, whereas (1R)-camphor is available from natural sources. Optically pure (1S)-camphor, however, is much more difficult to obtain. The synthesis of racemic camphor from α-pinene proceeds via an intermediary racemic isobornyl ester, which is then hydrolyzed and oxidized to give camphor. We reasoned that enantioselective hydrolysis of isobornyl esters would give facile access to optically pure isoborneol and camphor isomers, respectively. While screening of a set of commercial lipases and esterases in the kinetic resolution of racemic monoterpenols did not lead to the identification of any enantioselective enzymes, the cephalosporin Esterase B from Burkholderia gladioli (EstB) and Esterase C (EstC) from Rhodococcus rhodochrous showed outstanding enantioselectivity (E>100) towards the butyryl esters of isoborneol, borneol and fenchol. The enantioselectivity was higher with increasing chain length of the acyl moiety of the substrate. The kinetic resolution of isobornyl butyrate can be easily integrated into the production of camphor from α-pinene and thus allows the facile synthesis of optically pure monoterpenols from a renewable side-product.

Expanding the Toolbox of R-Selective Amine Transaminases by Identification and Characterization of New Members.

Amine transaminases (ATAs) are used to synthesize enantiomerically pure amines, which are building blocks for pharmaceuticals and agrochemicals. R-selective ATAs belong to the fold type IV PLP-dependent enzymes, and different sequence-, structure- and substrate scope-based features have been identified in the past decade. However, our knowledge is still restricted due to the limited number of characterized (R)-ATAs, with additional bias towards fungal origin. We aimed to expand the toolbox of (R)-ATAs and contribute to the understanding of this enzyme subfamily. We identified and characterized four new (R)-ATAs. The ATA from Exophiala sideris contains a motif characteristic for d-ATAs, which was previously believed to be a disqualifying factor for (R)-ATA activity. The crystal structure of the ATA from Shinella is the first from a Gram-negative bacterium. The ATAs from Pseudonocardia acaciae and Tetrasphaera japonica are the first characterized (R)-ATAs with a shortened/missing N-terminal helix. The active-site charges vary significantly between the new and known ATAs, correlating with their diverging substrate scope.

Functional Expression and Characterization of a Panel of Cobalt and Iron-Dependent Nitrile Hydratases.

Nitrile hydratases (NHase) catalyze the hydration of nitriles to the corresponding amides. We report on the heterologous expression of various nitrile hydratases. Some of these enzymes have been investigated by others and us before, but sixteen target proteins represent novel sequences. Of 21 target sequences, 4 iron and 16 cobalt containing proteins were functionally expressed from Escherichia coli BL21 (DE3) Gold. Cell free extracts were used for activity profiling and basic characterization of the NHases using the typical NHase substrate methacrylonitrile. Co-type NHases are more tolerant to high pH than Fe-type NHases. A screening for activity on three structurally diverse nitriles was carried out. Two novel Co-dependent NHases from Afipia broomeae and Roseobacter sp. and a new Fe-type NHase from Gordonia hydrophobica were very well expressed and hydrated methacrylonitrile, pyrazine-carbonitrile, and 3-amino-3-(p-toluoyl)propanenitrile. The Co-dependent NHases from Caballeronia jiangsuensis and Microvirga lotononidis, as well as two Fe-dependent NHases from Pseudomonades, were-in addition-able to produce the amide from cinnamonitrile. Summarizing, seven so far uncharacterized NHases are described to be promising biocatalysts.

Pichia pastoris protease-deficient and auxotrophic strains generated by a novel, user-friendly vector toolbox for gene deletion.

Targeted gene knockouts play an important role in the study of gene function. For the generation of knockouts in the industrially important yeast Pichia pastoris, several protocols have been published to date. Nevertheless, creating a targeted knockout in P. pastoris still is a time-consuming process, as the existing protocols are labour intensive and/or prone to accumulate nucleotide mutations. In this study, we introduce a novel, user-friendly vector-based system for the generation of targeted knockouts in P. pastoris. Upon confirming the successful knockout, respective selection markers can easily be recycled. Excision of the marker is mediated by Flippase (Flp) recombinase and occurs at high frequency (≥95%). We validated our knockout system by deleting 20 (confirmed and putative) protease genes and five genes involved in biosynthetic pathways. For the first time, we describe gene deletions of PRO3 and PHA2 in P. pastoris, genes involved in proline, and phenylalanine biosynthesis, respectively. Unexpectedly, knockout strains of PHA2 did not display the anticipated auxotrophy for phenylalanine but rather showed a bradytroph phenotype on minimal medium hinting at an alternative but less efficient pathway for production of phenylalanine exists in P. pastoris. Overall, all knockout vectors can easily be adapted to the gene of interest and strain background by efficient exchange of target homology regions and selection markers in single cloning steps. Average knockout efficiencies for all 25 genes were shown to be 40%, which is comparably high.

Two N-terminally truncated variants of human ß-galactoside α2,6 sialyltransferase I with distinct properties for in vitro protein glycosylation.

Sialic acid groups of protein N-glycans are important determinants of biological activity. Exposed at the end of the glycan chain, they are potential targets for glycan remodeling. Sialyltransferases (STs; EC 2.4.99) are the enzymes that catalyze the sialic acid transfer from a CMP-activated donor on to a carbohydrate acceptor in vivo. Recombinant expression of the full-length human ß-galactoside α2,6 sialyltransferase I (ST6Gal-I) was hampered and therefore variants with truncated N-termini were investigated. We report on the distinct properties of two N-terminally truncated versions of ST6Gal-I, namely Δ89ST6Gal-I and Δ108ST6Gal-I, which were successfully expressed in human embryonic kidney cells. The different properties of these enzymes result most probably from the loss of interactions from helix α1 in the Δ108ST6Gal-I variant, which plays a role in acceptor substrate binding. The Km for N-acetyl-d-lactosamine was 10-fold increased for Δ108ST6Gal-I (84 mM) as compared to Δ89ST6Gal-I (8.3 mM). The two enzyme variants constitute a suitable tool box for the terminal modification of N-glycans. While the enzyme Δ89ST6Gal-I exhibited both ST (di-sialylation) and sialidase activity on a monoclonal antibody, the enzyme Δ108ST6Gal-I showed only ST activity with specificity for mono-sialylation.

Enzymes as Biodevelopers for Nano- And Micropatterned Bicomponent Biopolymer Thin Films.

The creation of nano- and micropatterned polymer films is a crucial step for innumerous applications in science and technology. However, there are several problems associated with environmental aspects concerning the polymer synthesis itself, cross-linkers to induce the patterns as well as toxic solvents used for the preparation and even more important development of the films (e.g., chlorobenzene). In this paper, we present a facile method to produce micro- and nanopatterned biopolymer thin films using enzymes as so-called biodevelopers. Instead of synthetic polymers, naturally derived ones are employed, namely, poly-3-hydroxybutyrate and a cellulose derivative, which are dissolved in a common solvent in different ratios and subjected to spin coating. Consequently, the two biopolymers undergo microphase separation and different domain sizes are formed depending on the ratio of the biopolymers. The development step proceeds via addition of the appropriate enzyme (either PHB-depolymerase or cellulase), whereas one of the two biopolymers is selectively degraded, while the other one remains on the surface. In order to highlight the enzymatic development of the films, video AFM studies have been performed in real time to image the development process in situ as well as surface plasmon resonance spectroscopy to determine the kinetics. These studies may pave the way for the use of enzymes in patterning processes, particularly for materials intended to be used in a physiological environment.

Restriction site free cloning (RSFC) plasmid family for seamless, sequence independent cloning in Pichia pastoris.

BACKGROUND: Tagging proteins is a standard method facilitating protein detection, purification or targeting. When tagging a certain protein of interest, it is challenging to predict which tag will give optimal results and will not interfere with protein folding, activity or yields. Ideally, multiple tags and positions are tested which however complicates molecular cloning and expression vector generation. In conventional cloning, tags are either added on PCR primers (requiring a distinct primer and PCR product per tag) or provided on the vector (typically leaving a restriction site scar). RESULTS: Here we report a vector family of 40 plasmids allowing simple, seamless fusions of a single PCR product with various N- and C-terminal tags, signal sequences and promoters. The restriction site free cloning (RSFC) strategy presented in this paper relies on seamless cloning using type IIS restriction endonucleases. After cutting out a stuffer (placeholder) fragment from the vectors, a single PCR product can be directly inserted in frame into all 40 plasmids using blunt end or TA ligations, requiring only verification of the orientation. We have established a RSFC vector family for the commonly used protein expression host Pichia pastoris and demonstrated the system with the secretory expression of horseradish peroxidase (HRP). HRP fusions to four tags (Myc, FLAG, His, Strep) and two fusion proteins (GFP and MBP) showed a 31-fold difference in volumetric activities. C-terminal tagging caused in some cases almost a complete loss of function, whereas N-terminal tags showed moderate differences. CONCLUSIONS: The RSFC vectors provide an unprecedented toolbox for expression optimization in P. pastoris. The results obtained with HRP underline the importance of comparing different tags to maximize activities of fusion proteins. In a similar fashion the RSFC strategy can be applied in other expression hosts to screen for optimal promoters, signal sequences or to facilitate the evaluation of (iso-) enzyme families.

Production of the sesquiterpenoid (+)-nootkatone by metabolic engineering of Pichia pastoris.

The sesquiterpenoid (+)-nootkatone is a highly demanded and highly valued aroma compound naturally found in grapefruit, pummelo or Nootka cypress tree. Extraction of (+)-nootkatone from plant material or its production by chemical synthesis suffers from low yields and the use of environmentally harmful methods, respectively. Lately, major attention has been paid to biotechnological approaches, using cell extracts or whole-cell systems for the production of (+)-nootkatone. In our study, the yeast Pichia pastoris initially was applied as whole-cell biocatalyst for the production of (+)-nootkatone from (+)-valencene, the abundant aroma compound of oranges. Therefore, we generated a strain co-expressing the premnaspirodiene oxygenase of Hyoscyamus muticus (HPO) and the Arabidopsis thaliana cytochrome P450 reductase (CPR) that hydroxylated extracellularly added (+)-valencene. Intracellular production of (+)-valencene by co-expression of valencene synthase from Callitropsis nootkatensis resolved the phase-transfer issues of (+)-valencene. Bi-phasic cultivations of P. pastoris resulted in the production of trans-nootkatol, which was oxidized to (+)-nootkatone by an intrinsic P. pastoris activity. Additional overexpression of a P. pastoris alcohol dehydrogenase and truncated hydroxy-methylglutaryl-CoA reductase (tHmg1p) significantly enhanced the (+)-nootkatone yield to 208mg L(-1) cell culture in bioreactor cultivations. Thus, metabolically engineered yeast P. pastoris represents a valuable, whole-cell system for high-level production of (+)-nootkatone from simple carbon sources.

High-quality production of human α-2,6-sialyltransferase in Pichia pastoris requires control over N-terminal truncations by host-inherent protease activities.

BACKGROUND: α-2,6-sialyltransferase catalyzes the terminal step of complex N-glycan biosynthesis on human glycoproteins, attaching sialic acid to outermost galactosyl residues on otherwise fully assembled branched glycans. This "capping" of N-glycans is critical for therapeutic efficacy of pharmaceutical glycoproteins, making the degree of sialylation an important parameter of glycoprotein quality control. Expression of recombinant glycoproteins in mammalian cells usually delivers heterogeneous N-glycans, with a minor degree of sialylation. In-vitro chemo-enzymatic glycoengineering of the N-glycans provides an elegant solution to increase the degree of sialylation for analytical purposes but also possibly for modification of therapeutic proteins. RESULTS: Human α-2,6-sialyltransferase (ST6Gal-I) was secretory expressed in P.pastoris KM71H. ST6Gal-I featuring complete deletion of both the N-terminal cytoplasmic tail and the transmembrane domain, and also partial truncation of the stem region up to residue 108 were expressed N-terminally fused to a His or FLAG-Tag. FLAG-tagged proteins proved much more resistant to proteolysis during production than the corresponding His-tagged proteins. Because volumetric transferase activity measured on small-molecule and native glycoprotein acceptor substrates did not correlate to ST6Gal-I in the supernatant, enzymes were purified and characterized in their action on non-sialylated protein-linked and released N-glycans, and the respective N-terminal sequences were determined by automated Edman degradation. Irrespective of deletion construct used (Δ27, Δ48, Δ62, Δ89), isolated proteins showed N-terminal processing to a highly similar degree, with prominent truncations at residue 108 - 114, whereby only Δ108ST6Gal-I retained activity. FLAG-tagged Δ108ST6Gal-I was therefore produced and obtained with a yield of 4.5 mg protein/L medium. The protein was isolated and shown by MS to be intact. Purified enzyme exhibited useful activity (0.18 U/mg) for sialylation of different substrates. CONCLUSIONS: Functional expression of human ST6Gal-I as secretory protein in P.pastoris necessitates that N-terminal truncations promoted by host-inherent proteases be tightly controlled. N-terminal FLAG-Tag contributes extra stability to the N-terminal region as compared to N-terminal His-Tag. Proteolytic degradation proceeds up to residues 108 - 114 and of the resulting short-form variants, only Δ108ST6Gal-I seems to be active. FLAG-Δ108ST6Gal-I transfers sialic acids to monoclonal antibody substrate with sufficient yields, and because it is stably produced in P.pastoris, it is identified here as an interesting glycoengineering catalyst.

Protein expression in Pichia pastoris: recent achievements and perspectives for heterologous protein production.

Pichia pastoris is an established protein expression host mainly applied for the production of biopharmaceuticals and industrial enzymes. This methylotrophic yeast is a distinguished production system for its growth to very high cell densities, for the available strong and tightly regulated promoters, and for the options to produce gram amounts of recombinant protein per litre of culture both intracellularly and in secretory fashion. However, not every protein of interest is produced in or secreted by P. pastoris to such high titres. Frequently, protein yields are clearly lower, particularly if complex proteins are expressed that are hetero-oligomers, membrane-attached or prone to proteolytic degradation. The last few years have been particularly fruitful because of numerous activities in improving the expression of such complex proteins with a focus on either protein engineering or on engineering the protein expression host P. pastoris. This review refers to established tools in protein expression in P. pastoris and highlights novel developments in the areas of expression vector design, host strain engineering and screening for high-level expression strains. Breakthroughs in membrane protein expression are discussed alongside numerous commercial applications of P. pastoris derived proteins.

Characterization of a multifunctional α2,3-sialyltransferase from Pasteurella dagmatis.

A new multifunctional α2,3-sialyltransferase has been discovered in Pasteurella dagmatis. The enzyme, in short PdST, was identified from the P. dagmatis genome by sequence similarity with sialyltransferases of glycosyltransferase family GT-80. In addition to its regioselective sialyltransferase activity (5.9 U/mg; pH 8.0), purified PdST is alternatively active at low pH as α2,3-sialidase (0.5 U/mg; pH 4.5) and α2,3-trans-sialidase (1.0 U/mg; pH 4.5). It also shows cytidine-5'-monophosphate N-acetyl-neuraminic (CMP-Neu5Ac) hydrolase activity (3.7 U/mg; pH 8.0) when no sialyl acceptor substrate is present in the reaction. After sialyltransferase PmST1 from P. multocida, PdST is the second member of family GT-80 to display this remarkable catalytic promiscuity. A unique feature of PdST, however, is a naturally occurring Ser-to-Thr substitution within a highly conserved Y(112)DDGS(116) sequence motif. In PmST1, the equivalent Ser(143) is involved in binding of the CMP-Neu5Ac donor substrate. Reversion of the natural mutation in a T116S-PdST variant resulted in a marked increase in α2,3-trans-sialidase side activity (4.0 U/mg; pH 4.5), whereas the major sialyltransferase activity was lowered (3.8 U/mg; pH 8.0). The Michaelis-Menten constant for CMP-Neu5Ac was decreased 4-fold in T116S mutant when compared with wild-type PdST (KM=1.1 mM), indicating that residue 116 of PdST contributes to a delicate balance between substrate binding and catalytic activity. D-Galactose and various ß-D-galactosides function as sialyl acceptors from CMP-Neu5Ac, whereas other hexoses (e.g. D-glucose) are inactive. Structure comparison was used to rationalize the particular acceptor substrate specificity of PdST in relation to other GT-80 sialyltransferases that show strict α2,3-regioselectivity, but are flexible in using α/ß-galactosides for sialylation.

Surface engineering of a cutinase from Thermobifida cellulosilytica for improved polyester hydrolysis.

Modeling and comparison of the structures of the two closely related cutinases Thc_Cut1 and Thc_Cut2 from Thermobifida cellulosilytica DSM44535 revealed that dissimilarities in their electrostatic and hydrophobic surface properties in the vicinity to the active site could be responsible for pronounced differences in hydrolysis efficiencies of polyester (i.e., PET, polyethyleneterephthalate). To investigate this hypothesis in more detail, selected amino acids of surface regions outside the active site of Thc_Cut2, which hydrolyzes PET much less efficiently than Thc_Cut1 were exchanged by site-directed mutagenesis. The mutants were expressed in E. coli BL21-Gold(DE3), purified and characterized regarding their specific activities and kinetic parameters on soluble substrates and their ability to hydrolyze PET and the PET model substrate bis(benzoyloxyethyl) terephthalate (3PET). Compared to Thc_Cut2, mutants carrying Arg29Asn and/or Ala30Val exchanges showed considerable higher specific activity and higher kcat /KM values on soluble substrates. Exchange of the positively charged arginine (Arg19 and Arg29) located on the enzyme surface to the non-charged amino acids serine and asparagine strongly increased the hydrolysis activity for 3PET and PET. In contrast, exchange of the uncharged glutamine (Glu65) by the negatively charged glutamic acid lead to a complete loss of hydrolysis activity on PET films. These findings clearly demonstrate that surface properties (i.e., amino acids located outside the active site on the protein surface) play an important role in PET hydrolysis.

Fusion of binding domains to Thermobifida cellulosilytica cutinase to tune sorption characteristics and enhancing PET hydrolysis.

A cutinase from Thermomyces cellullosylitica (Thc_Cut1), hydrolyzing the synthetic polymer polyethylene terephthalate (PET), was fused with two different binding modules to improve sorption and thereby hydrolysis. The binding modules were from cellobiohydrolase I from Hypocrea jecorina (CBM) and from a polyhydroxyalkanoate depolymerase from Alcaligenes faecalis (PBM). Although both binding modules have a hydrophobic nature, it was possible to express the proteins in E. coli . Both fusion enzymes and the native one had comparable kcat values in the range of 311 to 342 s(-1) on pNP-butyrate, while the catalytic efficiencies kcat/Km decreased from 0.41 s(-1)/ µM (native enzyme) to 0.21 and 0.33 s(-1)/µM for Thc_Cut1+PBM and Thc_Cut1+CBM, respectively. The fusion enzymes were active both on the insoluble PET model substrate bis(benzoyloxyethyl) terephthalate (3PET) and on PET although the hydrolysis pattern was differed when compared to Thc_Cut1. Enhanced adsorption of the fusion enzymes was visible by chemiluminescence after incubation with a 6xHisTag specific horseradish peroxidase (HRP) labeled probe. Increased adsorption to PET by the fusion enzymes was confirmed with Quarz Crystal Microbalance (QCM-D) analysis and indeed resulted in enhanced hydrolysis activity (3.8× for Thc_Cut1+CBM) on PET, as quantified, based on released mono/oligomers.

Characterization of two bacterial hydroxynitrile lyases with high similarity to cupin superfamily proteins.

Hydroxynitrile lyases (HNLs) catalyze the cleavage of cyanohydrins. In the reverse reaction, they catalyze the formation of carbon-carbon bonds by enantioselective condensation of hydrocyanic acid with carbonyls. In this study, we describe two proteins from endophytic bacteria that display activity in the cleavage and the synthesis reaction of (R)-mandelonitrile with up to 74% conversion of benzaldehyde (enantiopreference ee 89%). Both showed high similarity to proteins of the cupin superfamily which so far were not known to exhibit HNL activity.

Crystallization of the novel S-adenosyl-L-methionine-dependent C-methyltransferase CouO from Streptomyces rishiriensis and preliminary diffraction data analysis.

Recombinant Q9F8T9 protein from Streptomyces rishiriensis (CouO), an S-adenosyl-L-methionine-dependent C-methyltransferase, has been successfully cloned, expressed and purified. CouO was crystallized from a single condition in the Morpheus crystallization screen. A vitrified crystal diffracted to 2.05 Å resolution and belonged to space group P2(1), with unit-cell parameters a = 33.02, b = 82.87, c = 76.77 Å, ß = 96.93°.

Crystallization of a novel metal-containing cupin from Acidobacterium sp. and preliminary diffraction data analysis.

Recombinant AciX9_0562 from Acidobacterium sp. MP5ACTX9 (UniProt ID E8WYN5) containing sequence motifs characteristic of the RmlC-type cupins superfamily and containing Pfam motif PF07883 has been successfully cloned, expressed and purified. AciX9_0562 crystallized in a number of conditions from the Morpheus protein crystallization screen. The best crystal diffracted to 2.7 Å resolution (space group C222(1); unit-cell parameters a = 125.29, b = 254.63, c = 82.99 Å). Structure solution was facilitated by the automated molecular-replacement pipeline BALBES. The initial solution was automatically rebuilt using the PHENIX AutoBuild wizard, with final R and R(free) values of 0.23 and 0.26, respectively. The structure is currently undergoing manual refinement.

Plasmid-Based Gene Knockout Strategy with Subsequent Marker Recycling in Pichia pastoris.

Gene knockout is a key technology in the development of cell factories and basic research alike. The methylotrophic yeast Pichia pastoris is typically employed as a producer of proteins and of fine chemicals, due to its ability to accumulate high cell densities in conjunction with a set of strong inducible promoters. However, protocols for genome engineering in this host are still cumbersome and time-consuming. Moreover, extensive genome engineering raises the need for a multitude of selection markers, which are limited in P. pastoris. In this chapter, we describe a fast and efficient method for gene disruption in P. pastoris that utilizes marker recycling to enable repetitive genome engineering cycles. A set of ready-to-use knockout vectors simplifies cloning procedures and facilitates quick knockout generation.

Metagenomic analysis of the 1-aminocyclopropane-1-carboxylate deaminase gene (acdS) operon of an uncultured bacterial endophyte colonizing Solanum tuberosum L.

Deamination of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) is a key plant-beneficial trait found in many plant growth-promoting bacteria. In this study, we analysed ACC deaminase genes (acdS) of bacterial endophytes colonizing field-grown potato plants. PCR analysis revealed the presence of two types of acdS genes, the dominant one showing high homology to an acdS gene derived from Pseudomonas fluorescens. Construction, functional screening and sequence analysis of metagenomic libraries revealed clones containing the acdS gene identified in the PCR library. Sequence analysis of one metagenomic clone identified the entire acdS operon of an uncultivated endophyte and revealed that the acdS gene is coupled upstream with an acdR transcriptional regulator gene as previously found in P. putida strain UW4 (Grichko and Glick 2000). However, in-silico analysis of 195 fully sequenced, acdS-containing bacterial genomes revealed that the majority of strains, including numerous strains belonging to the genus Pseudomonas, do not contain an acdR regulatory gene in the vicinity of the acdS gene or elsewhere in the genome. The acdR (+)-acdS (+) operon was exclusively found in several Alpha- and Betaproteobacteria most prominently in the genus Burkholderia.

Engineering of choline oxidase from Arthrobacter nicotianae for potential use as biological bleach in detergents.

In order to engineer the choline oxidase from Arthrobacter nicotianae (An_CodA) for the potential application as biological bleach in detergents, the specific activity of the enzyme toward the synthetic substrate tris-(2-hydroxyethyl)-methylammonium methylsulfate (MTEA) was improved by methods of directed evolution and rational design. The best mutants (up to 520% wt-activity with MTEA) revealed mutations in the FAD- (A21V, G62D, I69V) and substrate-binding site (S348L, V349L, F351Y). In a separate screening of a library comprising of randomly mutagenised An_CodA, with the natural substrate choline, four mutations were identified, which were further combined in one clone. The constructed clone showed improved activity towards both substrates, MTEA and choline. Mapping these mutation sites onto the structural model of An_CodA revealed that Phe351 is positioned right in the active site of An_CodA and very likely interacts with the bound substrate. Ala21 is part of an alpha-helix which interacts with the diphosphate moiety of the flavin cofactor and might influence the activity and specificity of the enzyme.

Hydrogen-Driven Cofactor Regeneration for Stereoselective Whole-Cell C=C Bond Reduction in Cupriavidus necator.

The coupling of recombinantly expressed oxidoreductases to endogenous hydrogenases for cofactor recycling permits the omission of organic cosubstrates as sacrificial electron donors in whole-cell biotransformations. This increases atom efficiency and simplifies the reaction. A recombinant ene-reductase was expressed in the hydrogen-oxidizing proteobacterium Cupriavidus necator H16. In hydrogen-driven biotransformations, whole cells catalyzed asymmetric C=C bond reduction of unsaturated cyclic ketones with stereoselectivities up to >99 % enantiomeric excess. The use of hydrogen as a substrate for growth and cofactor regeneration is particularly attractive because it represents a strategy for improving atom efficiency and reducing side product formation associated with the recycling of organic cofactors.