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The addition of water to non‐activated carbon–carbon double bonds catalyzed by fatty acid hydratases (FAHYs) allows for highly regio‐ and stereoselective oxyfunctionalization of renewable oil feedstock. So far, the applicability of FAHYs has been limited to free fatty acids, mainly owing to the requirement of a carboxylate function for substrate recognition and binding. Herein, we describe for the first time the hydration of oleic acid (OA) derivatives lacking this free carboxylate by the oleate hydratase from Elizabethkingia meningoseptica (OhyA). Molecular docking of OA to the OhyA 3D‐structure and a sequence alignment uncovered conserved amino acid residues at the entrance of the substrate channel as target positions for enzyme engineering. Exchange of selected amino acids gave rise to OhyA variants which showed up to an 18‐fold improved conversion of OA derivatives, while retaining the excellent regio‐ and stereoselectivity in the olefin hydration reaction.

The utilisation of multiple host-derived carbon substrates is required by Mycobacterium tuberculosis (Mtb) to successfully sustain a tuberculosis infection thereby identifying the Mtb specific metabolic pathways and enzymes required for carbon co-metabolism as potential drug targets. Metabolic flux represents the final integrative outcome of many different levels of cellular regulation that contribute to the flow of metabolites through the metabolic network. It is therefore critical that we have an in-depth understanding of the rewiring of metabolic fluxes in different conditions. Here, we employed 13C-metabolic flux analysis using stable isotope tracers (13C and 2H) and lipid fingerprinting to investigate the metabolic network of Mtb growing slowly on physiologically relevant carbon sources in a steady state chemostat. We demonstrate that Mtb is able to efficiently co-metabolise combinations of either cholesterol or glycerol along with C2 generating carbon substrates. The uniform assimilation of the carbon sources by Mtb throughout the network indicated no compartmentalization of metabolism in these conditions however there were substrate specific differences in metabolic fluxes. This work identified that partitioning of flux between the TCA cycle and the glyoxylate shunt combined with a reversible methyl citrate cycle as the critical metabolic nodes which underlie the nutritional flexibility of Mtb. These findings provide new insights into the metabolic architecture that affords adaptability of Mtb to divergent carbon substrates.ImportanceEach year more than 1 million people die of tuberculosis (TB). Many more are infected but successfully diagnosed and treated with antibiotics, however antibiotic-resistant TB isolates are becoming ever more prevalent and so novel therapies are urgently needed that can effectively kill the causative agent. Mtb specific metabolic pathways have been identified as an important drug target in TB. However the apparent metabolic plasticity of this pathogen presents a major obstacle to efficient targeting of Mtb specific vulnerabilities and therefore it is critical to define the metabolic fluxes that Mtb utilises in different conditions. Here, we used 13C-metabolic flux analysis to measure the metabolic fluxes that Mtb uses whilst growing on potential in vivo nutrients. Our analysis identified selective use of the metabolic network that included the TCA cycle, glyoxylate shunt and methyl citrate cycle. The metabolic flux phenotypes determined in this study improves our understanding about the co-metabolism of multiple carbon substrates by Mtb identifying a reversible methyl citrate cycle and the glyoxylate shunt as the critical metabolic nodes which underlie the nutritional flexibility of Mtb.

Given the fact that the theoretically possible number of racemates is larger than that of symmetric prochiral or meso compounds,1 the development of deracemization methods, which yield a single stereoisomer from a racemate is an important topic.1–3 Enantioconvergent processes are based on the transformation of a pair of enantiomers through opposite stereochemical pathways affecting retention and inversion of configuration. Depending on the stereochemical course of enzymatic and chemical reactions, three types of deracemization protocols were recently classified by Feringa et al.4 Two chemoenzymatic methods start with a biocatalytic kinetic resolution step, which yields a hetero- or homochiral 1:1 mixture of the formed product and nonconverted substrate enantiomer. The latter is subjected to a second (non-enzymatic) transformation with retention or inversion of configuration to yield a single stereoisomeric product. Although several one-pot, two-step protocols have been successfully demonstrated,5, 10c,d they typically rely on activated species, such as sulfonates,5a–d nitrate esters,5b or Mitsunobu intermediates,5e and negatively affect the overall atom economy of the process. The most elegant method relies on one (or two) enzyme(s), which mediate the transformation of both enantiomers through stereocomplementary pathways by retention and inversion. Since the requirements of such double selectivities are very difficult to meet, successful examples are rare: This approach has been applied to the hydrolysis of epoxides using two epoxide hydrolases showing opposite enantiopreference6 or a single enzyme that catalyzes the enantioconvergent hydrolysis of enantiomers with opposite regioselectivity.7 For enzymes, the ability to act by retention or inversion is a rare feature, which has been found among epoxide hydrolases,8 dehalogenases,4, 9 and sulfatases.10 The latter catalyze the hydrolytic cleavage of (alkyl) sulfate esters by breakage of the S–O or the C–O bond leading to retention or inversion at the chiral carbon atom,10b and thus makes them prime candidates for enantioconvergent processes. So far, only a single inverting sec-alkylsulfatase (PISA1) was generated recombinantly and characterized biochemically,11 thus allowing preparative-scale applications.10c In combination with acid-catalyzed hydrolysis of the nonreacted substrate enantiomer under retention of configuration12 a chemoenzymatic two-step deracemization protocol for sec-alcohols was recently developed.10c,d However, the method suffers from serious limitations because it requires undesirably large volumes organic solvents and several molar equivalents of a strong acid (typically 2–7 equiv of p-TosOH), which pose the risk of racemization or decomposition to the functionalized substrates, especially when elevated temperatures are required for acidic hydrolysis. Moreover, it is not applicable to retaining sulfatases, because no chemical method for sulfate ester hydrolysis with inversion exists.10c So far, retaining-sulfatase activity was reported in whole cells of Rhodopirellula baltica DSM 10527,13 but the corresponding enzymes could not be identified, thus impeding the use of recombinant technology to make the enzyme available for biocatalysis. Furthermore, the retaining sulfatase of Rh. baltica would not be suitable for an enantioconvergent process with PISA1, because both proteins exhibit the same enantiopreference. During our search for a retaining sec-alkylsulfatase with an enantiopreference opposite to that of PISA1, we discovered that the arylsulfatase from Pseudomonas aeruginosa (PAS) exhibited activity on sec-alkylsulfates. PAS, which has been characterized on a molecular level,14 showed promiscuous activity on various arylic phosphates and phosphonates.15 On its standard model substrate (4-nitrophenyl sulfate), PAS exhibited a rate acceleration of kcat/kuncat 2.3×1010,16 and for a less reactive substrate the highest rate enhancement (kcat/kuncat=2×1026) of any catalytic reaction known so far has been measured.17 The stereochemical features of PAS were investigated using a series of sec-alkylsulfate esters (rac-1 a–7 a; Table ​Table1).1). The substrates 1 a–3 a bearing an acetylenic moiety on the long chain adjacent to the stereocenter were resolved with good to excellent enantioselectivities (E 59 to >200). Undesired non-enzymatic background hydrolysis of 1 a could be suppressed by addition of 20 % (v/v) of DMSO as a cosolvent.18 In contrast, the selectivities were largely lost when the acetylenic moiety was moved to the short chain (substrates 4 a–6 a). The alkyl aryl derivative 7 a gave again an excellent E value of greater than 200. All substrates converted with high enantioselectivities (1 a–3 a, 7 a) were hydrolyzed with complete retention of configuration, thus yielding S-configured alcohols and unreacted R-configured sulfate esters. To prove the stereochemical course of sulfate ester hydrolysis by PAS, enzymatic cleavage of rac-1-octyn-3-yl sulfate (6 a) was performed in an 18O-labeled buffer (label >98 %). GC/MS analysis of the alcohol 6 b formed revealed that (in contrast to inverting sec-alkylsulfatases10c) no incorporation of 18O occurred, and is consistent with the attack of the enzyme’s formylglycine nucleophile on sulfur. Hydrolysis of enantiopure (S)-6 a by PAS yielded alcohol (S)-6 b in greater than 99 % ee, thus proving that hydrolysis proceeded under strict retention of configuration. Table 1 Kinetic resolution of sulfate esters rac-1 a–7 a with retention and inversion using PAS and PISA1 Since the stereochemical features of PAS—R enantiopreference with retention—would ideally complement the S preference with inversion10c,d of PISA1,10e we optimized the enzymatic hydrolysis of the substrates 1 a–7 a with the latter enzyme (Table ​(Table1).1). Most of the substrates (1 a, 4 a–7 a) showed perfect E values of greater than 200, with only 2 a and 3 a giving insufficient selectivities. The data thus obtained enabled us to develop three types of enantioconvergent processes (Scheme 1, Table ​Table22): Table 2 Deracemization of the sulfate esters rac-1 a–7 a using retaining PAS and inverting PISA1. Scheme 1 One-pot, two-enzyme deracemization process using retaining PAS and inverting PISA1. Type A. The substrates rac-2 a and rac-3 a, where PAS was highly enantioselective, could be deracemized by a one-pot, two-step sequence using retaining PAS first, followed by non-enantioselective inverting hydrolysis with PISA1 to yield (S)-2 b and (S)-3 b in 91 and 97 % ee, respectively. Type B. For rac-4 a–6 a, where PISA1 was highly enantioselective, but PAS was not, the opposite order of events—PISA1 first, PAS second—was successful and yielded the corresponding R-configured alcohols 4 b–6 b in 94 to greater than 99 % ee Type C. The ideal single-step process using both enzymes simultaneously was designed for the substrates rac-1 a and rac-7 a. To maximize the ee value of 1 b, DMSO was used as cosolvent to suppress background hydrolysis which increased the ee value of (S)-1 b from 93 to 98 % ee. To demonstrate the applicability of this method on a preparative scale, the deracemization of rac-6 a was scaled-up (1 g, 4.4 mmol), and gave (R)-6 b as the sole product in 82 % yield upon isolation with 98 % ee. The choice of which process (Type A–C) is most suitable for the deracemization of a given substrate depends on the availability of an enantioselective sec-alkylsulfatase acting with retention or inversion of configuration. Processes of Types A and B are feasible with a single enantioselective enzyme, whereas Type C requires two enantioselective sulfatases with matching opposite enantiopreference. It should be kept in mind that Types A and B constitute kinetic resolutions,19 whereas Type C represents a parallel kinetic resolution.20 Overall, the purely enzymatic protocol excels by its significantly broader applicability compared to the chemoenzymatic procedure10c,d for the following reasons: 1) it eliminates the harsh reaction conditions required for acid-catalyzed hydrolysis, which are incompatible with sensitive functional groups and 2) it is also applicable to retaining sulfatases (such as PAS). In summary, the one-pot deracemization of sec-alcohols bearing various functional groups was achieved by enantioconvergent hydrolysis of the corresponding sulfate esters using the retaining aryl sulfatase PAS and the inverting alkyl sulfatase PISA1, which possess the required opposite enantiopreference.

Addressing the challenges associated with the development of in vitro biocatalytic carboxylate reductions for potential applications, important aspects of the co-factor regeneration systems and strategies for minimizing over-reduction were investigated. The ATP recycling can be performed with similarly high efficiency exploiting the polyphosphate source by combining Meiothermus ruber polyphosphate kinase and adenylate kinase or with Sinorhizobium meliloti polyphosphate kinase instead of the latter. Carboxylate reductions with the enzyme candidates used in this work allow operating at co-factor concentrations of adenosine 5'-triphosphate and β-nicotinamide adenine dinucleotide 2'-phosphate of 100 μM and, thereby, reducing the amounts of alcohols formed by side activities in the enzyme preparations. This study confirmed the expected benefits of carboxylic acid reductases in chemoselectively reducing the carboxylates to the corresponding aldehydes while leaving reductively-sensitive nitro, ester and cyano groups intact.

Lipoproteins play a central role in the development of atherosclerosis. High and low-density lipoproteins (HDL and LDL), known as ‘good’ and ‘bad’ cholesterol, respectively, remove and/or deposit lipids into the artery wall. Hence, insight into lipid exchange processes between lipoproteins and cell membranes is of particular importance in understanding the onset and development of cardiovascular disease. In order to elucidate the impact of phospholipid tail saturation and the presence of cholesterol in cell membranes on these processes, neutron reflection was employed in the present investigation to follow lipid exchange with both HDL and LDL against model membranes. Mirroring clinical risk factors for the development of atherosclerosis, lower exchange was observed in the presence of cholesterol, as well as when using an unsaturated phospholipid, compared to faster exchange when using a fully saturated phospholipid. These results highlight the importance of membrane composition on the interaction with lipoproteins, chiefly the saturation level of the lipids and presence of cholesterol, and provide novel insight into factors of importance for build-up and reversibility of atherosclerotic plaque. In addition, the correlation between the results and well-established clinical risk factors suggests that the approach taken can be employed also for understanding a broader set of risk factors including, e.g., effects of triglycerides and oxidative stress, as well as local effects of drugs on atherosclerotic plaque formation.

We report the synthesis of diverse β-hydroxy-α,α-dialkyl-α-amino acids with perfect stereoselectivity for the α-quaternary center through the action of l- and d-specific threonine aldolases. A wide variety of aliphatic and aromatic aldehydes were accepted by the enzymes and conversions up to >80 % were obtained. In the case of d-selective threonine aldolase from Pseudomonas sp., generally higher diastereoselectivities were observed. The applicability of the protocol was demonstrated by performing enzymatic reactions on preparative scale. Using the d-threonine aldolase from Pseudomonas sp., (2R,3S)-2-amino-3-(2-fluorophenyl)-3-hydroxy-2-methylpropanoic acid was generated in preparative amounts in one step with a diastereomeric ratio >100 favoring the syn-product. A Birch-type reduction enabled the reductive removal of the β-hydroxy group from (2S)-2-amino-3-hydroxy-2-methyl-3-phenylpropanoic acid to generate enantiopure l-α-methyl-phenylalanine via a two-step chemo-enzymatic transformation.

Cholesterol is an essential component of mammalian membranes and is known to induce a series of physicochemical changes in the lipid bilayer. Such changes include the formation of liquid-ordered phases with an increased thickness and a configurational order as compared to liquid-disordered phases. For saturated lipid membranes, cholesterol molecules localize close to the lipid head group-tail interface. However, the presence of polyunsaturated lipids was recently shown to promote relocation of cholesterol toward the inner interface between the two bilayer leaflets. Here, neutron reflection is used to study the location of cholesterol (both non-deuterated and per-deuterated versions are used) within supported lipid bilayers composed of a natural mixture of phosphatidylcholine (PC). The lipids were produced in a genetically modified strain of Escherichia coli and grown under specific deuterated conditions to give an overall neutron scattering length density (which depends on the level of deuteration) of the lipids matching that of D

Deuteration of biomolecules has a major impact on both quality and scope of neutron scattering experiments. Cholesterol is a major component of mammalian cells, where it plays a critical role in membrane permeability, rigidity and dynamics, and contributes to specific membrane structures such as lipid rafts. Cholesterol is the main cargo in low and high-density lipoprotein complexes (i.e. LDL, HDL) and is directly implicated in several pathogenic conditions such as coronary artery disease which leads to 17 million deaths annually. Neutron scattering studies on membranes or lipid-protein complexes exploiting contrast variation have been limited by the lack of availability of fully deuterated biomolecules and especially perdeuterated cholesterol. The availability of perdeuterated cholesterol provides a unique way of probing the structural and dynamical properties of the lipoprotein complexes that underly many of these disease conditions. Here we describe a procedure for in vivo production of perdeuterated recombinant cholesterol in lipid-engineered Pichia pastoris using flask and fed-batch fermenter cultures in deuterated minimal medium. Perdeuteration of the purified cholesterol was verified by mass spectrometry and its use in a neutron scattering study was demonstrated by neutron reflectometry measurements using the FIGAROO instrument at the ILL.

Hydratases provide access to secondary and tertiary alcohols by regio- and/or stereospecifically adding water to carbon-carbon double bonds. Thereby, hydroxy groups are introduced without the need for costly cofactor recycling, and that makes this approach highly interesting on an industrial scale. Here we present the first crystal structure of a recombinant oleate hydratase originating from Elizabethkingia meningoseptica in the presence of flavin adenine dinucleotide (FAD). A structure-based mutagenesis study targeting active site residues identified E122 and Y241 as crucial for the activation of a water molecule and for protonation of the double bond, respectively. Moreover, we also observed that two-electron reduction of FAD results in a sevenfold increase in the substrate hydration rate. We propose the first reaction mechanism for this enzyme class that explains the requirement for the flavin cofactor and the involvement of conserved amino acid residues in this regio- and stereoselective hydration.

ω-Transaminase (TA) catalyzed asymmetric syntheses of amines were carried out in the one enzyme systems with wild-type enzymes (S)-TA from Pseudomonas aeruginosa, (S)-TA from Paracoccus denitrificans and (R)-TA from Aspergillus terreus. The scope of amine donors and aromatic carbonyl substrates was thoroughly explored. Among the range of potential amino donors, 2-propylamine, 2-butylamine and 1-phenylethylamine were found as promising candidates, which gave superior conversions in the amination reactions compared to other donors. Various prochiral aromatic ketones were accepted as substrates by the investigated enzymes. In most cases, good to excellent conversions (up to 98%) to the amine products with excellent e.e.-values (>99.9% for (S) or (R)) were obtained by the action of a single enzyme and an appropriate amino donor. (S)-TA from Paracoccus denitrificans was found to accept bulky ketones, e.g. 1-indanone, α- and β-tetralone or 2-acetonaphthone, in the asymmetric amination. In some cases the enantiomeric excesses in the amination reactions were dependent on the amino donor. Moreover, the influence of the pH, temperature and cosolvents on the outcome of reactions was additionally investigated.

The C-terminal activation of peptides as prerequisite for the formation or ligation of peptide fragments is often associated with the problem of epimerization. We report that ruthenium-catalyzed alkyne addition with (+)-2,3-O-isopropylidene-2,3-dihydroxy-1,4-bis(diphenylphosphino)butane as ligand allows the racemization-free synthesis of peptide enol esters tolerating a wide range of functional groups. The transformation can be performed in a variety of different solvents addressing the solubility issues imposed by peptides with varying amino acid side chain patterns. We show that peptide enol esters with an amide motif in the enol moiety are excellent acyl donors for the peptide condensation with other peptide fragments in organic solvents using serine endopeptidase subtilisin A as catalyst. The reported combination of transition metal catalysis with enzymatic peptide ligations adds an important tool for the racemization-free synthesis and ligation of peptides which is compatible even with unprotected amino acid side chains.

Transaminases are useful biocatalysts for the production of amino acids and chiral amines as intermediates for a broad range of drugs and fine chemicals. Here, we describe the discovery and characterisation of new transaminases from microorganisms which were enriched in selective media containing (R)-amines as sole nitrogen source. While most of the candidate proteins were clearly assigned to known subgroups of the fold IV family of PLP-dependent enzymes by sequence analysis and characterisation of their substrate specificity, some of them did not fit to any of these groups. The structure of one of these enzymes from Curtobacterium pusillum, which can convert d-amino acids and various (R)-amines with high enantioselectivity, was solved at a resolution of 2.4 Å. It shows significant differences especially in the active site compared to other transaminases of the fold IV family and thus indicates the existence of a new subgroup within this family. Although the discovered transaminases were not able to convert ketones in a reasonable time frame, overall, the enrichment-based approach was successful, as we identified two amine transaminases, which convert (R)-amines with high enantioselectivity, and can be used for a kinetic resolution of 1-phenylethylamine and analogues to obtain the (S)-amines with e.e.s >99%.

In this study we investigated the ability of amidases to hydrolyse polyurethane polyester co-polymers. In order to improve enzyme adsorption, a polyamidase from Nocardia farcinica (PA) was fused to a polymer binding module from a polyhydroxyalkanoate depolymerase from Alcaligenes faecalis (PA_PBM). The activity of these enzymes and of various commercially available amidases on a synthesized soluble model substrate was compared. The recombinant native PA showed the highest activity of 10.5 U/mg followed by PA_PBM with an activity of 1.13 U/mg. Both enzymes were able to cleave the urethane bond in polyurethane-polyesters with different degree of crystallinity as shown by FTIR. According to LC-TOF analysis the monomer 4,4′-diaminodiphenylmethane (MDA) and the oligomers 4-hydroxybutyl (3-(3-aminobenzyl)phenyl)carbamate [B], bis(4-hydroxybutyl) (methylenebis(3,1-phenylene))dicarbamate [C] and 4-(((3-(3-(((4-hydroxybutoxy)carbonyl)amino)benzyl)phenyl)carbamoyl)oxy)butyl (4-hydroxybutyl) adipate [D] were released. The polymer with a higher content of the rigid segment, MDA, was hydrolysed to a lower extent. Interestingly, despite the lower activity on the soluble model substrate, the PA_PBM fusion enzyme was up to 4 times more active on the polymer when compared with the native enzyme, confirming the relevance of enzyme adsorption for efficient hydrolysis.

The triterpenoid (+)-ambrein is a natural precursor for (-)-ambrox, which constitutes one of the most sought-after fragrances and fixatives for the perfume industry. (+)-Ambrein is a major component of ambergris, an intestinal excretion of sperm whales that is found only serendipitously. Thus, the demand for (-)-ambrox is currently mainly met by chemical synthesis. A recent study described for the first time the applicability of an enzyme cascade consisting of two terpene cyclases, namely squalene-hopene cyclase from Alicyclobacillus acidocaldarius (AaSHC D377C) and tetraprenyl-β-curcumene cyclase from Bacillus megaterium (BmeTC) for in vitro (+)-ambrein production starting from squalene. Yeasts, such as Pichia pastoris, are natural producers of squalene and have already been shown in the past to be excellent hosts for the biosynthesis of hydrophobic compounds such as terpenoids. By targeting a central enzyme in the sterol biosynthesis pathway, squalene epoxidase Erg1, intracellular squalene levels in P. pastoris could be strongly enhanced. Heterologous expression of AaSHC D377C and BmeTC and, particularly, development of suitable methods to analyze all products of the engineered strain provided conclusive evidence of whole-cell (+)-ambrein production. Engineering of BmeTC led to a remarkable one-enzyme system that was by far superior to the cascade, thereby increasing (+)-ambrein levels approximately 7-fold in shake flask cultivation. Finally, upscaling to 5 L bioreactor yielded more than 100 mg L−1 of (+)-ambrein, demonstrating that metabolically engineered yeast P. pastoris represents a valuable, whole-cell system for high-level production of (+)-ambrein. Keywords: Pichia pastoris, Metabolic engineering, Terpene cyclase, Triterpenoid, Squalene, (+)-ambrein

Conjugate additions of carbon nucleophiles to appropriate acceptor molecules were investigated with respect to the synthetic potential and stereochemistry of the products. Reactions of short-chain α,β-unsaturated ketones and mono-substituted nitroalkenes with CH-acidic carboxylic ester derivatives were catalyzed by various immobilized lipases. Using methyl nitroacetate complete conversion with methyl vinyl ketone and trans-β-nitrostyrene was obtained. The reactions proceeded without enantioselectivity. Evidence for enzyme catalysis is provided by the observation that after denaturation of Candida antarctica lipase B or inhibition no reaction took place. Docking studies with the chiral addition product methyl 2-methyl-2-nitro-5-oxohexanoate did not reveal any specific binding mode for this reaction product, which would have been the requirement for stereoselective additions. These results support the experimental findings that the conjugate addition takes place without enantiopreference.

Many synthetically useful reactions are catalyzed by cofactor-dependent enzymes. As cofactors represent a major cost factor, methods for efficient cofactor regeneration are required especially for large-scale synthetic applications. In order to generate a novel and efficient host chassis for bioreductions, we engineered the methanol utilization pathway of Pichia pastoris for improved NADH regeneration. By deleting the genes coding for dihydroxyacetone synthase isoform 1 and 2 (DAS1 and DAS2), NADH regeneration via methanol oxidation (dissimilation) was increased significantly. The resulting Δdas1 Δdas2 strain performed better in butanediol dehydrogenase (BDH1) based whole-cell conversions. While the BDH1 catalyzed acetoin reduction stopped after 2 h reaching ~50% substrate conversion when performed in the wild type strain, full conversion after 6 h was obtained by employing the knock-out strain. These results suggest that the P. pastoris Δdas1 Δdas2 strain is capable of supplying the actual biocatalyst with the cofactor over a longer reaction period without the over-expression of an additional cofactor regeneration system. Thus, focusing the intrinsic carbon flux of this methylotrophic yeast on methanol oxidation to CO2 represents an efficient and easy-to-use strategy for NADH-dependent whole-cell conversions. At the same time methanol serves as co-solvent, inductor for catalyst and cofactor regeneration pathway expression and source of energy.

An increasing demand for non-petroleum-based products is envisaged in the near future. Carboxylic acids such as citric acid, succinic acid, fatty acids, and many others are available in abundance from renewable resources and they could serve as economic precursors for bio-based products such as polymers, aldehyde building blocks, and alcohols. However, we are confronted with the problem that carboxylic acid reduction requires a high level of energy for activation due to the carboxylate's thermodynamic stability. Catalytic processes are scarce and often their chemoselectivity is insufficient. This review points at bio-alternatives: currently known enzyme classes and organisms that catalyze the reduction of carboxylic acids are summarized. Two totally distinct biocatalyst lines have evolved to catalyze the same reaction: aldehyde oxidoreductases from anaerobic bacteria and archea, and carboxylate reductases from aerobic sources such as bacteria, fungi, and plants. The majority of these enzymes remain to be identified and isolated from their natural background in order to evaluate their potential as industrial biocatalysts.

Enzymes of the non-conventional yeast Yarrowia lipolytica seem to be tailor-made for the conversion of lipophilic substrates. Herein, we cloned and overexpressed the Zn-dependent alcohol dehydrogenase ADH2 from Yarrowia lipolytica in Escherichia coli. The purified enzyme was characterized in vitro. The substrate scope for YlADH2 mediated oxidation and reduction was investigated spectrophotometrically and the enzyme showed a broader substrate range than its homolog from Saccharomyces cerevisiae. A preference for secondary compared to primary alcohols in oxidation direction was observed for YlADH2. 2-Octanone was investigated in reduction mode in detail. Remarkably, YlADH2 displays perfect (S)-selectivity and together with a highly (R)-selective short chain dehydrogenase/ reductase from Yarrowia lipolytica it is possible to access both enantiomers of 2-octanol in >99% ee with Yarrowia lipolytica oxidoreductases.

Non-aqueous metal-oxygen batteries depend critically on the reversible formation/decomposition of metal oxides on cycling. Irreversible parasitic reactions cause poor rechargeability, efficiency, and cycle life and have predominantly been ascribed to the reactivity of reduced oxygen species with cell components. These species, however, cannot fully explain the side reactions. Here we show that singlet oxygen forms at the cathode of a lithium-oxygen cell during discharge and from the onset of charge, and accounts for the majority of parasitic reaction products. The amount increases during discharge, early stages of charge, and charging at higher voltages, and is enhanced by the presence of trace water. Superoxide and peroxide appear to be involved in singlet oxygen generation. Singlet oxygen traps and quenchers can reduce parasitic reactions effectively. Awareness of the highly reactive singlet oxygen in non-aqueous metal-oxygen batteries gives a rationale for future research towards achieving highly reversible cell operation.

The co‐catabolism of multiple host‐derived carbon substrates is required by Mycobacterium tuberculosis (Mtb) to successfully sustain a tuberculosis infection. However, the metabolic plasticity of this pathogen and the complexity of the metabolic networks present a major obstacle in identifying those nodes most amenable to therapeutic interventions. It is therefore critical that we define the metabolic phenotypes of Mtb in different conditions. We applied metabolic flux analysis using stable isotopes and lipid fingerprinting to investigate the metabolic network of Mtb growing slowly in our steady‐state chemostat system. We demonstrate that Mtb efficiently co‐metabolises either cholesterol or glycerol, in combination with two‐carbon generating substrates without any compartmentalisation of metabolism. We discovered that partitioning of flux between the TCA cycle and the glyoxylate shunt combined with a reversible methyl citrate cycle is the critical metabolic nodes which underlie the nutritional flexibility of Mtb. These findings provide novel insights into the metabolic architecture that affords adaptability of bacteria to divergent carbon substrates and expand our fundamental knowledge about the methyl citrate cycle and the glyoxylate shunt.