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ChemBioChem 22(4), 652 - 656 (2020). doi:10.1002/cbic.202000600, Broad substrate tolerance and excellent regioselectivity, as well as independence from sensitive cofactors have established benzoic acid decarboxylases from microbial sources as efficient biocatalysts. Robustness under process conditions makes them particularly attractive for preparative‐scale applications. The divalent metal‐dependent enzymes are capable of catalyzing the reversible non‐oxidative (de)carboxylation of a variety of electron‐rich (hetero)aromatic substrates analogously to the chemical Kolbe‐Schmitt reaction. Elemental mass spectrometry supported by crystal structure elucidation and quantum chemical calculations verified the presence of a catalytically relevant Mg$^{2+}$ complexed in the active site of 2,3‐dihydroxybenoic acid decarboxylase from Aspergillus oryzae (2,3‐DHBD_Ao). This unique example with respect to the nature of the metal is in contrast to mechanistically related decarboxylases, which generally have Zn$^{2+}$ or Mn$^{2+}$ as the catalytically active metal., Published by Wiley-VCH, Weinheim

Glycerol is a byproduct of biodiesel production and is generated in large amounts, which has resulted in an increased interest in its valorization. In addition to its use as an energy source directly, the chemical modification of glycerol may result in value‐added derivatives. Herein, acid phosphatases employed in the synthetic mode were evaluated for the enzymatic phosphorylation of glycerol. Nonspecific acid phosphatases could tolerate glycerol concentrations up to 80 wt % and pyrophosphate concentrations up to 20 wt % and led to product titers up to 167 g L−1 in a kinetic approach. In the complementary thermodynamic approach, phytases were able to condense glycerol and inorganic monophosphate directly. This unexpected behavior enabled the simple and cost‐effective production of rac‐glycerol‐1‐phosphate from crude glycerol obtained from a biodiesel plant. A preparative‐scale synthesis on a 100 mL‐scale resulted in the production of 16.6 g of rac‐glycerol‐1‐phosphate with a reasonable purity (≈75 %)., Good things from glycerol: The valorization of crude glycerol is achieved through regioselective enzymatic (trans)phosphorylation reactions. rac‐Glycerol‐1‐phosphate is obtained from the reaction catalyzed by nonspecific acid phosphatases with pyrophosphate or by phytases with monophosphate.

© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim In contrast to many chemical dihydroxylation methods, enzymatic epoxide hydrolysis provides an environmentally benign route to vicinal diols, which are important intermediates in the synthesis of fine chemicals and pharmaceuticals. Using epoxide hydrolases, enantiopure diols are accessible under mild conditions. In order to assess the selectivity of epoxide hydrolases on geraniol-derived oxiranes, a range of derivatives were screened against a large variety of enzyme preparations. For nearly all substrates, a matching hydrolase with excellent enantioselectivity (≥95% ee) could be found. In addition, a chemoenzymatic approach for the stereoselective synthesis of furanoid linalool oxide was developed. Combination of enzymatic enantioselective hydrolysis with stereoselective Tsuji-Trost reaction granted diastereoselective access to trans-(2R,5R)-configured linalool oxide with high diastereomeric and enantiomeric excess (97% de and 97% ee). (Figure presented.).

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.

An immobilized bi-functional redox biocatalyst was designed for the asymmetric reduction of alkenes by nicotinamide-dependent ene-reductases. The biocatalyst, which consists of co-immobilized ene-reductase and glucose dehydrogenase, was implemented in biotransformations in the presence of glucose as source of reducing equivalents and catalytic amounts of the cofactor. Enzyme co-immobilization employing glutaraldehyde activated Relizyme HA403/M as support material was performed directly from the crude cell-free extract obtained after protein overexpression in E. coli and cell lysis, avoiding enzyme purification steps. The resulting optimum catalyst showed excellent level of activity and stereoselectivity in asymmetric reduction reactions using either OYE3 from Saccharomyces cerevisiae or NCR from Zymomonas mobilis in the presence of organic cosolvents in up to 20 vol%. The bi-functional redox biocatalyst, which demonstrated remarkable reusability over several cycles, was applied in preparative-scale synthesis at 50 mM substrate concentration and provided access to three industrially relevant chiral compounds in high enantiopurity (ee up to 97 %) and in up to 42 % isolated yield. The present method highlights the potential of (co-)immobilization of ene-reductases, notorious for their poor scalability, and complements the few existing methods available for increasing productivity in asymmetric bioreduction reactions.

bis-Tetrahydrofuran acetogenins are a class of natural products displaying highly potent and selective anti-tumor activity. Herein we report a new route to precursors of these natural products, utilizing the pseudo C2-symmetry in the central bis-tetrahydrofuran fragment. Key steps of our stereoselective chemoenzymatic strategy include the epoxide hydrolase-mediated desymmetrization of meso-epoxides and a cascade cyclization in either “inside-out” or “outside-in” direction, providing stereoselective access to the cores of both bullatacin- and rolliniastatin 1-type acetogenins with 6 stereocenters each from a common mono-epoxide precursor.

An easy to use method combining the selectivity of metal chelate affinity binding with strong covalent linking was developed for immobilization of non-specific acid phosphatases bearing a His-tag from crude cell lysate. Silica nanoparticles were grafted with aminopropyl functions which were partially transformed further with EDTA dianhydride to chelators. The heterofunctionalized nanoparticles charged with Ni2+ as the most appropriate metal ion were applied as support. First, the His-tagged phosphatases were selectively bound to the metal-chelate functions of the support. Then, the enzyme-charged silica nanoparticles were further stabilized by forming a covalent linkage between nucleophilic moieties at the enzyme surface and free amino groups of the support using neopentylglycol diglycidylether as the most effective bifunctional linking agent. The phosphatase biocatalysts obtained by this method exhibited better phosphate transfer activity with a range of alcohols and PPi as phosphate donor in aqueous medium applying batch and continuous-flow modes than the ones immobilized on conventional supports. Furthermore, this novel strategy opens up novel possibility for efficient immobilization of other His-tagged recombinant enzymes.

The biocatalytic asymmetric disproportionation of aldehydes catalyzed by horse liver alcohol dehydrogenase (HLADH) was assessed in detail on a series of racemic 2-arylpropanals. Statistical optimization by means of design of experiments (DoE) allowed the identification of critical interdependencies between several reaction parameters and revealed a specific experimental window for reaching an 'optimal compromise' in the reaction outcome. The biocatalytic system could be applied to a variety of 2-arylpropanals and granted access in a redox-neutral manner to enantioenriched (S)-profens and profenols following a parallel interconnected dynamic asymmetric transformation (PIDAT). The reaction can be performed in aqueous buffer at ambient conditions, does not rely on a sacrificial co-substrate, and requires only catalytic amounts of cofactor and a single enzyme. The high atom-efficiency was exemplified by the conversion of 75 mM of rac-2-phenylpropanal with 0.03 mol% of HLADH in the presence of ∼0.013 eq. of oxidized nicotinamide adenine dinucleotide (NAD+), yielding 28.1 mM of (S)-2-phenylpropanol in 96% ee and 26.5 mM of (S)-2-phenylpropionic acid in 89% ee, in 73% overall conversion. Isolated yield of 62% was obtained on 100 mg-scale, with intact enantiopurities.

Undesired product hydrolysis along with large amounts of waste in form of inorganic monophosphate by‐product are the main obstacles associated with the use of pyrophosphate in the phosphatase‐catalyzed synthesis of phosphate monoesters on large scale. In order to overcome both limitations, we screened a broad range of natural and synthetic organic phosphate donors with several enzymes on a broad variety of hydroxyl‐compounds. Among them, acetyl phosphate delivered stable product levels and high phospho‐transfer efficiency at the lower functional pH‐limit, which translated into excellent productivity. The protocol is generally applicable to acid phosphatases and compatible with a range of diverse substrates. Preparative‐scale transformations using acetyl phosphate synthesized from cheap starting materials yielded multiple grams of various sugar phosphates with up to 433 g L−1 h−1 space‐time yield and 75% reduction of barium phosphate waste.

Various flavoprotein oxidases were recently shown to oxidize primary thiols. Herein, this reactivity is extended to sec-thiols by using structure-guided engineering of 5-(hydroxymethyl) furfural oxidase (HMFO). The variants obtained were employed for the oxidative kinetic resolution of racemic sec-thiols, thus yielding the corresponding thioketones and nonreacted R-configured thiols with excellent enantioselectivities (E >= 200). The engineering strategy applied went beyond the classic approach of replacing bulky amino acid residues with smaller ones, as the active site was additionally enlarged by a newly introduced Thr residue. This residue established a hydrogen-bonding interaction with the substrates, as verified in the crystal structure of the variant. These strategies unlocked HMFO variants for the enantioselective oxidation of a range of sec-thiols.

A three-step one-pot biocatalytic cascade was designed for the enantioselective formal α-amination of hexanoic acid to l-norleucine. Regioselective hydroxylation by P450CLA peroxygenase to 2-hydroxyhexanoic acid was followed by oxidation to the ketoacid by two stereocomplementary dehydrogenases. Combination with final stereoselective reductive amination by amino acid dehydrogenase furnished l-norleucine in >97% ee.

The utilization of gaseous carbon dioxide instead of bicarbonate would greatly facilitate process development for enzyme catalyzed carboxylations on large scale. As proof-of-concept, 1,3-dihydroxybenzene (resorcinol) was carboxylated in the ortho-position using pressurized CO2 (~30-40 bar) catalyzed by ortho-benzoic acid decarboxylases with up to 68% conversion. Optimization studies revealed tight pH-control and enzyme stability as the most important determinants.

A series of functionalized α-chiral phosphonates was obtained by enzymatic reduction of the corresponding β-activated vinylphosphonate derivatives employing several ene-reductases of the Old Yellow Enzyme family as biocatalysts. The insufficient activation of the phosphonate group alone was compensated by introduction of an electron-withdrawing group at the β-position, which resulted in high levels of conversion (up to >99%). All active enzymes consistently furnished (R)-configurated products with exquisite stereoselectivity, thereby providing a stereo-complementary approach to current asymmetric hydrogenation protocols on similar compounds. Preparative-scale syntheses performed in aqueous buffer using formate/formate dehydrogenase for recycling of the nicotinamide cofactor granted access to β-keto-, cyano- and ester phosphonates from the (E)-isomers of α,β-unsaturated phosphonates in up to 72% isolated yield and >99% ee.

Die Verwendung von CO2 als Kohlenstoffquelle in der Synthese organischer Verbindungen entspricht den aktuellen Bemuhungen fur eine nachhaltigere Produktion von Chemikalien. Wir beschreiben hier die enzymkatalysierte para-Carboxylierung von Catecholen durch eine 3,4-Dihydroxybenzoesaure-Decarboxylase (AroY) aus der UbiD-Enzymfamilie. Die Kristallstrukturen und experimentelle Daten zeigen, dass dieses Enzym ein erst kurzlich entdecktes prenyliertes Flavin als Cofaktor benotigt (prFMN) und oxidative Prozesse zur Bildung der katalytisch aktiven prFMNiminium-Spezies fuhren. Diese Studie beschreibt die In-vitro-Rekonstitution und Aktivierung dieser prFMN-abhangigen Enzyme, welche die direkte Carboxylierung von aromatischen Catecholen unter milden Bedingungen katalysieren. Ein Reaktionsmechanismus fur den reversiblen Carboxylierungsprozess uber ein Intermediat mit einfach kovalent gebundener Chinoidform des Substrats wird vorgeschlagen, der sich von dem 1,3-Cycloadditionsmechanismus in verwandten Enzymen unterscheidet.

The major drawback of using phosphatases for transphosphorylation reactions lies in product depletion caused by the natural hydrolytic activity of the enzymes. Variants of PhoC-Mm from Morganella morganii and NSAP-Eb from Escherichia blattae were studied for their ability to maintain a high product level in the transphosphorylation of various primary alcohols. A single amino acid exchange delivered phosphatase variant PhoC-Mm G92D, which was able to catalyze the phosphorylation of primary alcohols without any major hydrolysis of the formed phosphate esters. The mutation mostly improved the affinity of the enzyme for alcohols, while rate constants of transphosphorylation and hydrolysis were decreased, overall resulting in a superior catalytic efficiency in transphosphorylation compared to hydrolysis. The presence of residual substrate alcohol at a given concentration was crucial to suppress phosphate ester hydrolysis. The present work extends the synthetic applicability of phosphatase variants beyond the previously reported nucleosides and allows preparative-scale production of various primary phosphate esters (yields up to 42%) with high enzyme productivity (TONs up to ∼66,000). Biotechnol. Bioeng. 2017;114: 2187-2195. © 2017 Wiley Periodicals, Inc.

We report the use of bifunctional starting materials (ketoacids) in a diastereoselective Passerini three-center-two-component reaction. Study of the reaction scope revealed the required structural features for stereoselectivity in the isocyanide addition. In this system, an interesting isomerization of the primary Passerini product - the α-carboxamido lactone - into an atypical product, an α-hydroxy imide, was found to occur under acidic conditions. Furthermore, enantioenriched Passerini products can be generated from an enantioenriched ketoacid obtained by chemoenzymatic synthesis.

The biocatalytic activity of a so far underexploited alkaline phosphatase, PhoK from Sphingomonas sp. BSAR-1, was extensively studied in transphosphorylation and hydrolysis reactions. The use of high-energy phosphate donors and oligophosphates as suitable phosphate donors was evaluated, as well as the hydrolytic activity on a variety of phosphate monoesters. While substrates bearing free hydroxy group displayed only moderate reactivity as acceptors for transphosphorylation by PhoK, strong hydrolytic activity on a broad variety of phosphate monoesters under alkaline conditions was observed. Site-directed mutagenesis of selected amino acid residues in the active site provided valuable insights on their involvement in enzyme catalysis. The key residue Thr89 so far postulated to engage in enzyme phosphorylation was confirmed to be crucial for catalysis and could be replaced by serine, albeit with much lower catalytic efficiency.

The utilization of carbon dioxide as a C1‐building block for the production of valuable chemicals has recently attracted much interest. Whereas chemical CO2 fixation is dominated by C−O and C−N bond forming reactions, the development of novel concepts for the carboxylation of C‐nucleophiles, which leads to the formation of carboxylic acids, is highly desired. Beside transition metal catalysis, biocatalysis has emerged as an attractive method for the highly regioselective (de)carboxylation of electron‐rich (hetero)aromatics, which has been recently further expanded to include conjugated α,β‐unsaturated (acrylic) acid derivatives. Depending on the type of substrate, different classes of enzymes have been explored for (i) the ortho‐carboxylation of phenols catalyzed by metal‐dependent ortho‐benzoic acid decarboxylases and (ii) the side‐chain carboxylation of para‐hydroxystyrenes mediated by metal‐independent phenolic acid decarboxylases. Just recently, the portfolio of bio‐carboxylation reactions was complemented by (iii) the para‐carboxylation of phenols and the decarboxylation of electron‐rich heterocyclic and acrylic acid derivatives mediated by prenylated FMN‐dependent decarboxylases, which is the main focus of this review. Bio(de)carboxylation processes proceed under physiological reaction conditions employing bicarbonate or (pressurized) CO2 when running in the energetically uphill carboxylation direction. Aiming to facilitate the application of these enzymes in preparative‐scale biotransformations, their catalytic mechanism and substrate scope are analyzed in this review.

The asymmetric bioreduction of α,β-unsaturated γ-keto esters using ene-reductases from the Old Yellow Enzyme family proceeds with excellent stereoselectivity and high conversion levels, covering a broad range of acyclic and cyclic derivatives. Various strategies were employed to provide access to both enantiomers, which are versatile precursors of bioactive molecules. The regioselectivity of hydride addition on di-activated alkenes was elucidated by isotopic labeling experiments and showed strong preference for the keto moiety as activating/binding group as opposed to the ester. Finally, chemoenzymatic synthesis of (R)-2-(2-oxocyclohexyl)acetic acid was achieved in high ee on a preparative scale combining enzymatic reduction followed by ester hydrogenolysis.

The transformation of (abundant) oxygenated biomass-derived building blocks via chemo-enzymatic methods is a valuable concept for accessing useful compounds, as it combines the high selectivity of enzymes and the versatility of chemical catalysts. In this work, we demonstrate a straightforward combination of a phenolic acid decarboxylase (PAD) and palladium on charcoal (Pd/C) that affords the flavor compound 4-ethylguaiacol from ferulic acid. The use of a two-phase system proved to be advantageous in terms of enzyme activity, stability, and volumetric productivity and allows us to carry out the hydrogenation step directly in the organic layer containing exclusively the intermediate, vinylguaiacol. The enzymatic decarboxylation step in the biphasic system afforded 89% conversion of 100 mM (19 g L–1) ferulic acid with an isolated yield of 75%. By extracting 4-vinylguaiacol continuously into the organic phase, conversion was enhanced to 92% using 170 mM (33 g L–1) ferulic acid, which was only possible in the...

The biocatalytic oxidative tandem decarboxylation of C7–C18 dicarboxylic acids to terminal C5–C16 dienes was catalyzed by the P450 monooxygenase OleT with conversions up to 29 % for 1,11-dodecadiene (0.49 g L–1). The sequential nature of the cascade was proven by the fact that decarboxylation of intermediate C6–C11 ω-alkenoic acids and heptanedioic acid exclusively gave nonconjugated 1,4-pentadiene; scale-up allowed the isolation of 1,15-hexadecadiene and 1,11-dodecadiene; the system represents a short and green route to terminal dienes from renewable dicarboxylic acids.

The biocatalytic asymmetric disproportionation of aldehydes catalyzed by horse liver alcohol dehydrogenase (HLADH) was assessed in detail on a series of racemic 2‐arylpropanals. Statistical optimization by means of design of experiments (DoE) allowed the identification of critical interdependencies between several reaction parameters and revealed a specific experimental window for reaching an ′optimal compromise′ in the reaction outcome. The biocatalytic system could be applied to a variety of 2‐arylpropanals and granted access in a redox‐neutral manner to enantioenriched (S)‐profens and profenols following a parallel interconnected dynamic asymmetric transformation (PIDAT). The reaction can be performed in aqueous buffer at ambient conditions, does not rely on a sacrificial co‐substrate, and requires only catalytic amounts of cofactor and a single enzyme. The high atom‐efficiency was exemplified by the conversion of 75 mM of rac‐2‐phenylpropanal with 0.03 mol% of HLADH in the presence of ∼0.013 eq. of oxidized nicotinamide adenine dinucleotide (NAD+), yielding 28.1 mM of (S)‐2‐phenylpropanol in 96% ee and 26.5 mM of (S)‐2‐phenylpropionic acid in 89% ee, in 73% overall conversion. Isolated yield of 62% was obtained on 100 mg‐scale, with intact enantiopurities.

Fungal ferulic acid decarboxylases (FDCs) belong to the UbiD-family of enzymes and catalyse the reversible (de)carboxylation of cinnamic acid derivatives through the use of a prenylated flavin cofactor. The latter is synthesised by the flavin prenyltransferase UbiX. Herein, we demonstrate the applicability of FDC/UbiX expressing cells for both isolated enzyme and whole-cell biocatalysis. FDCs exhibit high activity with total turnover numbers (TTN) of up to 55000 and turnover frequency (TOF) of up to 370 min−1. Co-solvent compatibility studies revealed FDC's tolerance to some organic solvents up 20 % v/v. Using the in-vitro (de)carboxylase activity of holo-FDC as well as whole-cell biocatalysts, we performed a substrate profiling study of three FDCs, providing insights into structural determinants of activity. FDCs display broad substrate tolerance towards a wide range of acrylic acid derivatives bearing (hetero)cyclic or olefinic substituents at C3 affording conversions of up to >99 %. The synthetic utility of FDCs was demonstrated by a preparative-scale decarboxylation.

l-Tyrosine derivatives were obtained in >97% ee via a biocatalytic one-pot two-step cascade using substituted benzenes, pyruvate, and NH3 as starting materials. In the first step, monosubstituted arenes were regioselectively hydroxylated in the o-position by monooxygenase P450 BM3 (using O2 as oxidant with NADPH-recycling) to yield the corresponding phenols, which subsequently underwent C–C coupling and simultaneous asymmetric amination with pyruvate and NH3 using tyrosine phenol lyase to furnish l-DOPA surrogates in up to 5.2 g L–1. Instead of analytically pure arenes, crude aromatic gasoline blends containing toluene were used to yield 3-methyl-l-tyrosine in excellent yield (2 g L–1) and >97% ee.

Anthranoyl-CoA monooxygenase/reductase (ACMR) participates in an unusual pathway for the degradation of aromatic compounds in Azoarcus evansii. It catalyzes the monooxygenation of anthranoyl-CoA to 5-hydroxyl-2-aminobenzoyl-CoA and the subsequent reduction to the dearomatized product 2-amino-5-oxo-cyclohex-1-ene-1-carbonyl-CoA. The two reactions occur in separate domains, termed the monooxygenase and reductase domain. Both domains were reported to utilize FAD as a cofactor for hydroxylation and reduction, respectively. We have heterologously expressed ACMR in Escherichia coli BL21 and found that the monooxygenase domain contains FAD. However, the reductase domain utilizes FMN and not FAD for the reduction of the intermediate 5-hydroxyl-2-aminobenzoyl-CoA. A homology model for the reductase domain predicted a topology similar to the Old Yellow Enzyme family, which exclusively bind FMN, in accordance with our results. Binding studies with 2-aminobenzoyl-CoA (AbCoA) and p-hydroxybenzaldehyde (pHB) as probes for the monooxygenase and reductase domain, respectively, indicated that two functionally distinct and independent active sites exist. Given the homodimeric quartenary structure of ACMR and the compact shape of the dimer as determined by small-angle X-ray scattering experiments we propose that the monooxygenase and reductase domain of opposite peptide chains are involved in the transformation of anthranoyl-CoA to 2-amino-5-oxo-cyclohex-1-ene-1-carbonyl-CoA.

Die enzymatische oxidative Decarboxylierung von linearen kurzkettigen Fettsauren (C4:0–C9:0) unter Verwendung der P450-Monooxygenase OleT, O2 und NAD(P)H als Elektronendonor ermoglicht die Synthese terminaler C3- bis C8-Alkene mit Produkttitern von bis zu 0.93 g L−1 und TTNs >2000. Essentiell fur diesen Prozess war die Entwicklung einer effizienten Elektronentransferkette unter Verwendung von Putidaredoxin (CamAB) und NAD(P)H-Regeneration basierend auf Glukose, Ameisensaure oder Phosphonat. Mithilfe dieses Reaktionssystems lassen sich auf biokatalytischem Weg industriell wichtige Alkene, wie z. B. Propen und 1-Octen zum ersten Mal vollstandig aus nachwachsenden Rohstoffen herstellen.

Utilization of gaseous carbon dioxide as a C1-building block in the biocatalytic ortho-carboxylation of a phenol., The utilization of gaseous carbon dioxide instead of bicarbonate would greatly facilitate process development for enzyme catalyzed carboxylations on a large scale. As a proof-of-concept, 1,3-dihydroxybenzene (resorcinol) was carboxylated in the ortho-position using pressurized CO2 (∼30–40 bar) catalyzed by ortho-benzoic acid decarboxylases with up to 68% conversion. Optimization studies revealed tight pH-control and enzyme stability as the most important determinants.

The promiscuous regio- and stereoselective hydration of 4-hydroxystyrenes catalyzed by ferulic acid decarboxylase from Enterobacter sp. (FDC_Es) depends on bicarbonate bound in the active site, which serves as a proton relay activating a water molecule for nucleophilic attack on a quinone methide electrophile. This “cofactor” is crucial for achieving improved conversions and high stereoselectivities for (S)-configured benzylic alcohol products. Similar effects were observed with simple aliphatic carboxylic acids as additives. A rational redesign of the active site by replacing the bicarbonate or acetate “cofactor” with a newly introduced side-chain carboxylate from an adjacent amino acid yielded mutants that efficiently acted as C=C hydratases. A single-point mutation of valine 46 to glutamate or aspartate improved the hydration activity by 40% and boosted the stereoselectivity 39-fold in the absence of bicarbonate or acetate.

The functionalization of bio‐based chemicals is essential to allow valorization of natural carbon sources. An atom‐efficient biocatalytic oxidative cascade was developed for the conversion of saturated fatty acids to α‐ketoacids. Employment of P450 monooxygenase in the peroxygenase mode for regioselective α‐hydroxylation of fatty acids combined with enantioselective oxidation by α‐hydroxyacid oxidase(s) resulted in internal recycling of the oxidant H2O2, thus minimizing degradation of ketoacid product and maximizing biocatalyst lifetime. The O2‐dependent cascade relies on catalytic amounts of H2O2 and releases water as sole by‐product. Octanoic acid was converted under mild conditions in aqueous buffer to 2‐oxooctanoic acid in a simultaneous one‐pot two‐step cascade in up to >99 % conversion without accumulation of hydroxyacid intermediate. Scale‐up allowed isolation of final product in 91 % yield and the cascade was applied to fatty acids of various chain lengths (C6:0 to C10:0).

The biocatalytic reduction of activated CC-bonds is dominated by ene-reductases from the Old Yellow Enzyme family, which gained broad practical use owing to exquisite stereoselectivity combined with wide substrate scope. Protein diversity is fostered by mining distinct protein classes and by implementing protein engineering techniques. Recent efforts are focusing on expanding the chemical complexity of the product portfolio, either through substrate functionalization or design of multi-step reactions. This review also highlights unusual chemistries catalyzed by ene-reductases and presents emerging methodologies developed to bypass the need of natural nicotinamide cofactors.

The major drawback of using phosphatases for transphosphorylation reactions lies in product depletion caused by the natural hydrolytic activity of the enzymes. Variants of PhoC-Mm from Morganella morganii and NSAP-Eb from Escherichia blattae were studied for their ability to maintain a high product level in the transphosphorylation of various primary alcohols. A single amino acid exchange delivered phosphatase variant PhoC-Mm G92D, which was able to catalyze the phosphorylation of primary alcohols without any major hydrolysis of the formed phosphate esters. The mutation mostly improved the affinity of the enzyme for alcohols, while rate constants of transphosphorylation and hydrolysis were decreased, overall resulting in a superior catalytic efficiency in transphosphorylation compared to hydrolysis. The presence of residual substrate alcohol at a given concentration was crucial to suppress phosphate ester hydrolysis. The present work extends the synthetic applicability of phosphatase variants beyond the previously reported nucleosides and allows preparative-scale production of various primary phosphate esters (yields up to 42%) with high enzyme productivity (TONs up to ∼66,000). Biotechnol. Bioeng. 2017;114: 2187-2195. © 2017 Wiley Periodicals, Inc.

The catalytic promiscuity of a ferulic acid decarboxylase from Enterobacter sp. (FDC_Es) and phenolic acid decarboxylases (PADs) for the asymmetric conjugate addition of water across the C=C bond of hydroxystyrenes was extended to the N‐, C‐ and S‐nucleophiles methoxyamine, cyanide and propanethiol to furnish the corresponding addition products in up to 91% ee. The products obtained from the biotransformation employing the most suitable enzyme/nucleophile pairs were isolated and characterized after optimizing the reaction conditions. Finally, a mechanistic rationale supported by quantum mechanical calculations for the highly (S)‐selective addition of cyanide is proposed.

Angewandte Chemie / International edition 56(44), 13893 - 13897 (2017). doi:10.1002/anie.201708091, The utilization of CO$-2$ as a carbon source for organic synthesis meets the urgent demand for more sustainability in the production of chemicals. Herein, we report on the enzyme-catalyzed para-carboxylation of catechols, employing 3,4-dihydroxybenzoic acid decarboxylases (AroY) that belong to the UbiD enzyme family. Crystal structures and accompanying solution data confirmed that AroY utilizes the recently discovered prenylated FMN (prFMN) cofactor, and requires oxidative maturation to form the catalytically competent prFMN4$^{iminium}$ species. This study reports on the in vitro reconstitution and activation of a prFMN-dependent enzyme that is capable of directly carboxylating aromatic catechol substrates under ambient conditions. A reaction mechanism for the reversible decarboxylation involving an intermediate with a single covalent bond between a quinoid adduct and cofactor is proposed, which is distinct from the mechanism of prFMN-associated 1,3-dipolar cycloadditions in related enzymes., Published by Wiley-VCH, Weinheim