Enantiocomplementary Bioreduction of 1-(Arylsulfanyl)propan-2-ones.

This study explored the enantiocomplementary bioreduction of substituted 1-(arylsulfanyl)propan-2-ones in batch mode using four wild-type yeast strains and two different recombinant alcohol dehydrogenases from Lactobacillus kefir and Rhodococcus aetherivorans. The selected yeast strains and recombinant alcohol dehydrogenases as whole-cell biocatalysts resulted in the corresponding 1-(arylsulfanyl)propan-2-ols with moderate to excellent conversions (60-99%) and high selectivities (ee > 95%). The best bioreductions-in terms of conversion (>90%) and enantiomeric excess (>99% ee)-at preparative scale resulted in the expected chiral alcohols with similar conversion and selectivity to the screening reactions.

Structure-based reshaping of a new ketoreductase from Sphingobacterium siyangense SY1 toward α-haloacetophenones.

Ketoreductases play an indispensable role in the asymmetric synthesis of chiral drug intermediates, and an in-depth understanding of their substrate selectivity can improve the efficiency of enzyme engineering. In this endeavor, a new short-chain dehydrogenase/reductase (SDR) SsSDR1 identified from Sphingobacterium siyangense SY1 by gene mining method was successfully cloned and functionally expressed in Escherichia coli. Its activity against halogenated acetophenones has been tested and the results illustrated that SsSDR1-WT exhibits high activity for 3,5-bis(trifluoromethyl)acetophenone (1f), an important precursor in the synthesis of aprepitant. In addition, SsSDR1-WT showed obvious substrate preference for acetophenones without α-halogen substitution compared to their α-halogen analogs. To explore the structural basis of substrate selectivity, the X-ray crystal structures of SsSDR1-WT in its apo form and the complex structure with NAD were resolved. Taking 2-chloro-1-(3, 4-difluorophenyl) ethanone (1i) as the representative α-haloacetophenone, the key sites affecting substrate selectivity of SsSDR1-WT were identified and through the rational remodeling of the cavities C1 and C2 of SsSDR1, an excellent mutant I144A/S153L with significantly improved activity against α-halogenated acetophenones was obtained. The asymmetric catalysis of 1f and 1i was performed at the scale of 50 mL, and the space-time yields (STY) of the two were 1200 and 6000 g/L∙d, respectively. This study not only provides valuable biocatalysts for halogenated acetophenones, but also yields insights into the relationship between the substrate-binding pocket and substrate selectivity.

Bioinformatic prediction of the stereoselectivity of modular polyketide synthase: an update of the sequence motifs in ketoreductase domain.

Polyketides are a major class of natural products, including bioactive medicines such as erythromycin and rapamycin. They are often rich in stereocenters biosynthesized by the ketoreductase (KR) domain within the polyketide synthase (PKS) assembly line. Previous studies have identified conserved motifs in KR sequences that enable the bioinformatic prediction of product stereochemistry. However, the reliability and applicability of these prediction methods have not been thoroughly assessed. In this study, we conducted a comprehensive bioinformatic analysis of 1,762 KR sequences from cis-AT PKSs to reevaluate the residues involved in conferring stereoselectivity. Our findings indicate that the previously identified fingerprint motifs remain valid for KRs in ß-modules from actinobacteria, but their reliability diminishes for KRs from other module types or taxonomic origins. Additionally, we have identified several new motifs that exhibit a strong correlation with the stereochemical outcomes of KRs. These updated fingerprint motifs for stereochemical prediction not only enhance our understanding of the enzymatic mechanisms governing stereocontrol but also facilitate accurate stereochemical prediction and genome mining of polyketides derived from modular cis-AT PKSs.

Sustainable asymmetric synthesis of diltiazem precursor enabled by recombinant Escherichia coli whole cells co-expressing an engineered ketoreductase and glucose dehydrogenase.

As a key synthetic intermediate of the cardiovascular drug diltiazem, methyl (2R,3S)-3-(4-methoxyphenyl) glycidate ((2R,3S)-MPGM) (1) is accessible via the ring closure of chlorohydrin (3S)-methyl 2-chloro-3-hydroxy-3-(4-methoxyphenyl)propanoate ((3S)-2). We report the efficient reduction of methyl 2-chloro-3-(4-methoxyphenyl)-3-oxo-propanoate (3) to (3S)-2 using an engineered enzyme SSCRM2 possessing 4.5-fold improved specific activity, which was obtained through the structure-guided site-saturation mutagenesis of the ketoreductase SSCR by reliving steric hindrance and undesired interactions. With the combined use of the co-expression fine-tuning strategy, a recombinant E. coli (pET28a-RBS-SSCRM2 /pACYCDuet-GDH), co-expressing SSCRM2 and glucose dehydrogenase, was constructed and optimized for protein expression. After optimizing the reaction conditions, whole-cell-catalyzed complete reduction of industrially relevant 300 g L-1 of 3 was realized, affording (3S)-2 with 99% ee and a space-time yield of 519.1 g∙L-1 ∙d-1 , representing the highest record for the biocatalytic synthesis of (3S)-2 reported to date. The E-factor of this biocatalytic synthesis was 24.5 (including water). Chiral alcohol (3S)-2 generated in this atom-economic synthesis was transformed to (2R,3S)-MPGM in 95% yield with 99% ee.

Electron withdrawing group-dependent substrate inhibition of an α-ketoamide reductase from Saccharomyces cerevisiae.

Aldo-keto reductases remain enzymes of interest in biocatalysis due to their ability to reduce carbonyls to alcohols stereospecifically. Based on genomic sequence, we identified aldo-keto reductases of a S. cerevisiae strain extracted from an ancient amber sample. One of the putative enzymes, AKR 163, displays 99% identity with α-amide ketoreductases from the S288C and YJM248 S. cerevisiae strains, which have been investigated for biocatalytic applications. To further investigate AKR 163, we successfully cloned, expressed in E.coli as a glutathione-S-transferase fusion protein, and affinity purified AKR 163. Kinetic studies revealed that AKR 163 experiences strong substrate inhibition by substrates containing halogen atoms or other electron withdrawing groups adjacent to the reactive carbonyl, with Ki values ranging from 0.29 to 0.6 mM and KM values ranging from 0.38 to 0.9 mM at pH 8.0. Substrates without electron withdrawing groups do not display substrate inhibition kinetics and possess much larger KM values between 83 and 260 mM under the same conditions. The kcat values ranged from 0.5 to 2.5s-1 for substrates exhibiting substrate inhibition and 0.22 to 0.52s-1 for substrates that do not engage in substrate inhibition. Overall, the results are consistent with rate-limiting dissociation of the NADP+ cofactor after hydride transfer when electron withdrawing groups are present and activating the reduction step. This process leads to a buildup of enzyme-NADP+ complex that is susceptible to binding and inhibition by a second substrate molecule.

Computationally Supported Inversion of Ketoreductase Stereoselectivity.

Whereas directed evolution and rational design by structural inspection are established tools for enzyme redesign, computational methods are less mature but have the potential to predict small sets of mutants with desired properties without laboratory screening of large libraries. We have explored the use of computational enzyme redesign to change the enantioselectivity of a highly thermostable alcohol dehydrogenase from Thermus thermophilus in the asymmetric reduction of ketones. The enzyme reduces acetophenone to (S)-1-phenylethanol. To invert the enantioselectivity, we used an adapted CASCO workflow which included Rosetta for enzyme design and molecular dynamics simulations for ranking. To correct for unrealistic binding modes, we used Boltzmann weighing of binding energies computed by a linear interaction energy approach. This computationally cheap method predicted four variants with inverted enantioselectivity, each with 6-8 mutations around the substrate-binding site, causing only modest reduction (2- to 7-fold) of kcat /KM values. Laboratory testing showed that three variants indeed had inverted enantioselectivity, producing (R)-alcohols with up to 99 % enantiomeric excess. The broad substrate range allowed reduction of acetophenone derivatives with full conversion to highly enantioenriched alcohols. The results demonstrate the use of computational methods to control ketoreductase stereoselectivity in asymmetric transformations with minimal experimental screening.

Ketoreductase Catalyzed (Dynamic) Kinetic Resolution for Biomanufacturing of Chiral Chemicals.

Biocatalyzed asymmetric reduction of ketones is an environmentally friendly approach and one of the most cost-effective routes for producing chiral alcohols. In comparison with the well-studied reduction of prochiral ketones to generate chiral alcohols with one chiral center, resolution of racemates by ketoreductases (KREDs) to produce chiral compounds with at least two chiral centers is also an important strategy in asymmetric synthesis. The development of protein engineering and the combination with chemo-catalysts further enhanced the application of KREDs in the efficient production of chiral alcohols with high stereoselectivity. This review discusses the advances in the research area of KRED catalyzed asymmetric synthesis for biomanufacturing of chiral chemicals with at least two chiral centers through the kinetic resolution (KR) approach and the dynamic kinetic resolution (DKR) approach.

Thermostabilizing ketoreductase ChKRED20 by consensus mutagenesis at dimeric interfaces.

Protein stability is crucial in enzymatic catalysis. To improve the efficiency in the searching for thermostablizing mutations, we applied a sequence consensus approach focusing on dimeric interface residues of ketoreductase ChKRED20. The strategy returned a success rate of 43%, revealing 9 beneficial mutations from 21 candidates with improved kinetic or thermodynamic stability. Several combinatorial mutants were then constructed, and mutant M8K displayed the highest thermostability, with a melting temperature (Tm) of 89 °C and a half-inactivation temperature (T50) of 93.4 °C, both of over 35 °C increase compared to the wild-type. M8K could remain stable for at least 7 days at its optimal reaction temperature of 55 °C. Its inactivation half-life (t1/2) was 110 min at 90 °C, while the wild-type was 18.6 min at 60 °C. The results were interpreted in the context of structural and molecular dynamic simulation analysis, which revealed the addition of intramolecular interactions, decreased conformational flexibility and increased compactness, all in agreement with the observed effect.

Chemoenzymatic Cascades for the Enantioselective Synthesis of ß-Hydroxysulfides Bearing a Stereocentre at the C-O or C-S Bond by Ketoreductases.

Chiral ß-hydroxysulfides are an important class of organic compounds which find broad application in organic and pharmaceutical chemistry. Herein we describe the development of novel biocatalytic and chemoenzymatic methods for the enantioselective synthesis of ß-hydroxysulfides by exploiting ketoreductase (KRED) enzymes. Four KREDs were discovered from a pool of 384 enzymes identified and isolated through a metagenomic approach. KRED311 and KRED349 catalysed the synthesis of ß-hydroxysulfides bearing a stereocentre at the C-O bond with opposite absolute configurations and excellent ee values by novel chemoenzymatic and biocatalytic-chemical-biocatalytic (bio-chem-bio) cascades starting from commercially available thiophenols/thiols and α-haloketones/alcohols. KRED253 and KRED384 catalysed the enantioselective synthesis of ß-hydroxysulfides bearing a stereocentre at the C-S bond with opposite enantioselectivities by dynamic kinetic resolution (DKR) of racemic α-thioaldehydes.

A Coupled Ketoreductase-Diaphorase Assay for the Detection of Polyethylene Terephthalate-Hydrolyzing Activity.

In the last two decades, several PET-degrading enzymes from already known microorganisms or metagenomic sources have been discovered to face the growing environmental concern of polyethylene terephthalate (PET) accumulation. However, there is a limited number of high-throughput screening protocols for PET-hydrolyzing activity that avoid the use of surrogate substrates. Herein, a microplate fluorescence screening assay was described. It was based on the coupled activity of ketoreductases (KREDs) and diaphorase to release resorufin in the presence of the products of PET degradation. Six KREDs were identified in a commercial panel that were able to use the PET building block, ethylene glycol, as substrate. The most efficient KRED, KRED61, was combined with the diaphorase from Clostridium kluyveri to monitor the PET degradation reaction catalyzed by the thermostable variant of the cutinase-type polyesterase from Saccharomonospora viridis AHK190. The PET degradation products were measured both fluorimetrically and by HPLC, with excellent correlation between both methods.

Characterization of Pyridomycin B Reveals the Formation of Functional Groups in Antimycobacterial Pyridomycin.

Pyridomycin, a cyclodepsipeptide with potent antimycobacterial activity, specifically inhibits the InhA enoyl reductase of Mycobacterium tuberculosis. Structure-activity relationship studies indicated that the enolic acid moiety in the pyridomycin core system is an important pharmacophoric group, and the natural configuration of the C-10 hydroxyl contributes to the bioactivity of pyridomycin. The ring structure of pyridomycin was generated by the nonribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) hybrid system (PyrE-PyrF-PyrG). Bioinformatics analysis reveals that short-chain dehydrogenase/reductase (SDR) family protein Pyr2 functions as a 3-oxoacyl acyl carrier protein (ACP) reductase in the pyridomycin pathway. Inactivation of pyr2 resulted in accumulation of pyridomycin B, a new pyridomycin analogue featured with enol moiety in pyridyl alanine moiety and a saturated 3-methylvaleric acid group. The elucidated structure of pyridomycin B suggests that rather than functioning as a post-tailoring enzyme, Pyr2 catalyzes ketoreduction to form the C-10 hydroxyl group in pyridyl alanine moiety and the double bond formation of the enolic acid moiety derived from isoleucine when the intermediate assembled by PKS-NRPS machinery is still tethered to the last NRPS module in a special energy-saving manner. Ser-His-Lys residues constitute the active site of Pyr2, which is different from the typically conserved Tyr-based catalytic triad in the majority of SDRs. Site-directed mutation identified that His154 in the active site is a critical residue for pyridomycin B production. These findings will improve our understanding of pyridomycin biosynthetic logic, identify the missing link for the double bound formation of enol ester in pyridomycin, and enable the creation of chemical diversity of pyridomycin derivatives. IMPORTANCE Tuberculosis (TB) is one of the world's leading causes of death by infection. Recently, pyridomycin, the antituberculous natural product from Streptomyces has garnered considerable attention for being determined as a target inhibitor of InhA enoyl reductase of Mycobacterium tuberculosis. In this study, we report a new pyridomycin analogue from mutant HTT12, demonstrate the essential role of a previously ignored gene pyr2 in pyridomycin biosynthetic pathway, and imply that Pyr2 functions as a trans ketoreductase (KR) contributing to the formation of functional groups of pyridomycin utilizing a distinct catalytic mechanism. As enol moiety are important for pharmaceutical activities of pyridomycin, our work would expand our understanding of the mechanism of SDR family proteins and set the stage for future bioengineering of new pyridomycin derivatives.

An NADPH-Dependent Ketoreductase Catalyses the Tetracyclic to Pentacyclic Skeletal Rearrangement in Chartreusin Biosynthesis.

Redox tailoring enzymes play key roles in generating structural complexity and diversity in type II polyketides. In chartreusin biosynthesis, the early 13 C-labeling experiments and bioinformatic analysis suggest the unusual aglycone is originated from a tetracyclic anthracyclic polyketide. Here, we demonstrated that the carbon skeleton rearrangement from a linear anthracyclic polyketide to an angular pentacyclic biosynthetic intermediate requires two redox enzymes. The flavin-dependent monooxygenase ChaZ catalyses a Baeyer-Villiger oxidation on resomycin C to form a seven-membered lactone. Subsequently, a ketoreductase ChaE rearranges the carbon skeleton and affords the α-pyrone containing pentacyclic intermediate in an NADPH-dependent manner via tandem reactions including the reduction of the lactone carbonyl group, Aldol-type reaction, followed by a spontaneous γ-lactone ring formation, oxidation and aromatization. Our work reveals an unprecedented function of a ketoreductase that contributes to generate structural complexity of aromatic polyketide.

Application of Ketoreductase in Asymmetric Synthesis of Pharmaceuticals and Bioactive Molecules: An Update (2018-2020).

With the rapid development of genomic DNA sequencing, recombinant DNA expression, and protein engineering, biocatalysis has been increasingly and widely adopted in the synthesis of pharmaceuticals, bioactive molecules, fine chemicals, and agrochemicals. In this review, we have summarized the most recent advances achieved (2018-2020) in the research area of ketoreductase (KRED)-catalyzed asymmetric synthesis of chiral secondary alcohol intermediates to pharmaceuticals and bioactive molecules. In the first part, synthesis of chiral alcohols with one stereocenter through the bioreduction of four different ketone classes, namely acyclic aliphatic ketones, benzyl or phenylethyl ketones, cyclic aliphatic ketones, and aryl ketones, is discussed. In the second part, KRED-catalyzed dynamic reductive kinetic resolution and reductive desymmetrization are presented for the synthesis of chiral alcohols with two contiguous stereocenters.

Biosynthesis of 6-Hydroxymellein Requires a Collaborating Polyketide Synthase-like Enzyme.

The polyketide synthase (PKS)-like protein TerB, consisting of inactive dehydratase, inactive C-methyltransferase, and functional ketoreductase domains collaborates with the iterative non reducing PKS TerA to produce 6-hydroxymellein, a key pathway intermediate during the biosynthesis of various fungal natural products. The catalytically inactive dehydratase domain of TerB appears to mediate productive interactions with TerA, demonstrating a new mode of trans-interaction between iterative PKS components.

Structural insights into the desymmetrization of bulky 1,2-dicarbonyls through enzymatic monoreduction.

Benzil reductases are dehydrogenases preferentially active on aromatic 1,2-diketones, but the reasons for this peculiar substrate recognition have not yet been clarified. The benzil reductase (KRED1-Pglu) from the non-conventional yeast Pichia glucozyma showed excellent activity and stereoselectivity in the monoreduction of space-demanding aromatic 1,2-dicarbonyls, making this enzyme attractive as biocatalyst in organic chemistry. Structural insights into the stereoselective monoreduction of 1,2-diketones catalyzed by KRED1-Pglu were investigated starting from its 1.77 Å resolution crystal structure, followed by QM and classical calculations; this study allowed for the identification and characterization of the KRED1-Pglu reactive site. Once identified the recognition elements involved in the stereoselective desymmetrization of bulky 1,2-dicarbonyls mediated by KRED1-Pglu, a mechanism was proposed together with an in silico prediction of substrates reactivity.

Molecular Basis for Polyketide Ketoreductase-Substrate Interactions.

Polyketides are a large class of structurally and functionally diverse natural products with important bioactivities. Many polyketides are synthesized by reducing type II polyketide synthases (PKSs), containing transiently interacting standalone enzymes. During synthesis, ketoreductase (KR) catalyzes regiospecific carbonyl to hydroxyl reduction, determining the product outcome, yet little is known about what drives specific KR-substrate interactions. In this study, computational approaches were used to explore KR-substrate interactions based on previously solved apo and mimic cocrystal structures. We found five key factors guiding KR-substrate binding. First, two major substrate binding motifs were identified. Second, substrate length is the key determinant of substrate binding position. Third, two key residues in chain length specificity were confirmed. Fourth, phosphorylation of substrates is critical for binding. Finally, packing/hydrophobic effects primarily determine the binding stability. The molecular bases revealed here will help further engineering of type II PKSs and directed biosynthesis of new polyketides.

Semi-Continuous Flow Biocatalysis with Affinity Co-Immobilized Ketoreductase and Glucose Dehydrogenase.

The co-immobilization of ketoreductase (KRED) and glucose dehydrogenase (GDH) on highly cross-linked agarose (sepharose) was studied. Immobilization of these two enzymes was performed via affinity interaction between His-tagged enzymes (six histidine residues on the N-terminus of the protein) and agarose matrix charged with nickel (Ni2+ ions). Immobilized enzymes were applied in a semicontinuous flow reactor to convert the model substrate; α-hydroxy ketone. A series of biotransformation reactions with a substrate conversion of >95% were performed. Immobilization reduced the requirement for cofactor (NADP+) and allowed the use of higher substrate concentration in comparison with free enzymes. The immobilized system was also tested on bulky ketones and a significant enhancement in comparison with free enzymes was achieved.

Site directed mutagenesis as a precision tool to enable synthetic biology with engineered modular polyketide synthases.

Modular polyketide synthases (PKSs) are a multidomain megasynthase class of biosynthetic enzymes that have great promise for the development of new compounds, from new pharmaceuticals to high value commodity and specialty chemicals. Their colinear biosynthetic logic has been viewed as a promising platform for synthetic biology for decades. Due to this colinearity, domain swapping has long been used as a strategy to introduce molecular diversity. However, domain swapping often fails because it perturbs critical protein-protein interactions within the PKS. With our increased level of structural elucidation of PKSs, using judicious targeted mutations of individual residues is a more precise way to introduce molecular diversity with less potential for global disruption of the protein architecture. Here we review examples of targeted point mutagenesis to one or a few residues harbored within the PKS that alter domain specificity or selectivity, affect protein stability and interdomain communication, and promote more complex catalytic reactivity.

Early Steps in the Biosynthetic Pathway of Rishirilide B.

The biological active compound rishirilide B is produced by Streptomyces bottropensis. The cosmid cos4 contains the complete rishirilide B biosynthesis gene cluster. Its heterologous expression in the host Streptomyces albus J1074 led to the production of rishirilide B as a major compound and to small amounts of rishirilide A, rishirilide D and lupinacidin A. In order to gain more insights into the biosynthesis, gene inactivation experiments and gene expression experiments were carried out. This study lays the focus on the functional elucidation of the genes involved in the early biosynthetic pathway. A total of eight genes were deleted and six gene cassettes were generated. Rishirilide production was not strongly affected by mutations in rslO2, rslO6 and rslH. The deletion of rslK4 and rslO3 led to the formation of polyketides with novel structures. These results indicated that RslK4 and RslO3 are involved in the generation or selection of the starter unit for rishirilide biosynthesis. In the rslO10 mutant strain, two novel compounds were detected, which were also produced by a strain containing solely the genes rslK1, rslK2, rslK3, rslK4, and rslA. rslO1 and rslO4 mutants predominately produce galvaquinones. Therefore, the ketoreductase RslO10 is involved in an early step of rishirilide biosynthesis and the oxygenases RslO1 and RslO4 are most probably acting on an anthracene moiety. This study led to the functional elucidation of several genes of the rishirilide pathway, including rslK4, which is involved in selecting the unusual starter unit for polyketide synthesis.

A Sequential Umpolung/Enzymatic Dynamic Kinetic Resolution Strategy for the Synthesis of γ-Lactones.

Combining biological and small-molecule catalysts under a chemoenzymatic manifold presents a series of significant advantages to the synthetic community. We report herein the successful development of a two-step/single flask synthesis of γ-lactones through the merger of Umpolung catalysis with a ketoreductase-catalyzed dynamic kinetic resolution, reduction, and cyclization. This combined approach delivers highly enantio- and diastereoenriched heterocycles and demonstrates the feasibility of integrating NHC catalysis with enzymatic processes.