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In the biosynthesis of the iron‐guanylylpyridinol (FeGP) cofactor, 6‐carboxymethyl‐5‐methyl‐4‐hydroxy‐2‐pyridinol (1) is 3‐methylated to form 2, then 4‐guanylylated to form 3, and converted into the full cofactor. HcgA‐G proteins catalyze the biosynthetic reactions. Herein, we report the function of two radical S‐adenosyl methionine enzymes, HcgA and HcgG, as uncovered by in vitro complementation experiments and the use of purified enzymes. In vitro biosynthesis using the cell extract from the Methanococcus maripaludis ΔhcgA strain was complemented with HcgA or precursors 1, 2 or 3. The results suggested that HcgA catalyzes the biosynthetic reaction that forms 1. We demonstrated the formation of 1 by HcgA using the 3 kDa cell extract filtrate as the substrate. Biosynthesis in the ΔhcgG system was recovered by HcgG but not by 3, which indicated that HcgG catalyzes the reactions after the biosynthesis of 3. The data indicated that HcgG contributes to the formation of CO and completes biosynthesis of the FeGP cofactor. [ABSTRACT FROM AUTHOR]

Microbial synthesis of plant‐based (−)‐menthol is of great interest because of its high demand (≈30 kiloton per year) as well as unique odor and cooling characteristics. However, this remains a great challenge due to the yet unfilled gap between (−)‐limonene and (+)‐cis‐isopulegone. Herein, the first artificial and effective system was developed for (+)‐cis‐isopulegone biosynthesis from (−)‐limonene by recruiting two bacterial enzymes to replace their inefficient counterparts from Mentha piperita, limonene‐3‐hydroxylase, and isopiperitenol dehydrogenase. A cofactor self‐regenerative recombinant Escherichia coli strain was constructed by introducing a formate dehydrogenase for nicotinamide adenine dinucleotide phosphate (NADPH) regeneration and an engineered microbial isopiperitenol dehydrogenase. The production of (+)‐cis‐isopulegone (up to 281.2 mg L−1) was improved by 36 times compared with that of the initial strain. This work lays a reliable foundation for the microbial synthesis of (−)‐menthol. [ABSTRACT FROM AUTHOR]

Microbial synthesis of (−)‐menthol, a compound of plant origin, is of great importance because of the high demand for this product and related sustainability issues. However, the total biosynthesis of (−)‐menthol from easily available feedstocks like (−)‐limonene by engineered microbial hosts is stalled by the poor protein expression or activity of several enzymes from the native (−)‐menthol biosynthesis pathway of mint (Mentha piperita). Among these unsatisfied steps, (−)‐isopiperitenol dehydrogenase (IPDH) catalyzed oxidation reaction of (−)‐trans‐isopiperitenol was one of the bottlenecks that need to be optimized. In this work, two novel bacterial enzymes with IPDH activity were discovered to replace their inefficient counterpart from plant cells in microbial (−)‐menthol synthesis. Two key residues in PaIPDH from Pseudomonas aeruginosa were mutated to PaIPDHE95F/Y199V with 4.4‐fold improved specific activity than PaIPDH. The mechanism for the beneficial mutations was elucidated by molecular dynamics simulations. PaIPDHE95F/Y199V was used to synthesize (−)‐isopiperitenone from (−)‐limonene in vivo via a self‐sufficient cofactor cascade enzyme reaction, affording a 3.7‐fold enhanced titer of (−)‐isopiperitenone compared with that obtained using the original mint IPDH (MpIPDH). The bacterial enzyme PaIPDHE95F/Y199V can be applied in the future for constructing a more efficient artificial pathway to biosynthesize (−)‐menthol in a microbial whole‐cell system. [ABSTRACT FROM AUTHOR]

Enantioconvergent hydrolysis by epoxide hydrolase is a promising method for the synthesis of important vicinal diols. However, the poor regioselectivity of the naturally occurring enzymes results in low enantioconvergence in the enzymatic hydrolysis of styrene oxides. Herein, modulated residue No. 263 was redesigned based on structural information and a smart variant library was constructed by site‐directed modification using an "optimized amino acid alphabet" to improve the regioselectivity of epoxide hydrolase from Vigna radiata (VrEH2). The regioselectivity coefficient (r) of variant M263Q for the R‐isomer of meta‐substituted styrene oxides was improved 40–63‐fold, and variant M263V also exhibited higher regioselectivity towards the R‐isomer of para‐substituted styrene oxides compared with the wild type, which resulted in improved enantioconvergence in hydrolysis of styrene oxide scaffolds. Structural insight showed the crucial role of residue No. 263 in modulating the substrate binding conformation by altering the binding surroundings. Furthermore, increased differences in the attacking distance between nucleophilic residue Asp101 and the two carbon atoms of the epoxide ring provided evidence for improved regioselectivity. Several high‐value vicinal diols were readily synthesized (>88% yield, 90%–98% ee) by enantioconvergent hydrolysis using the reprogrammed variants. These findings provide a successful strategy for enhancing the enantioconvergence of native epoxide hydrolases through key single‐site mutation and more powerful enzyme tools for the enantioconvergent hydrolysis of styrene oxide scaffolds into single (R)‐enantiomers of chiral vicinal diols. [ABSTRACT FROM AUTHOR]

Chiral amines are common drug building blocks and important active pharmaceutical ingredients. Preparing these functionalized compounds from simple materials, such as alkanes, is of great interest. We recently developed an artificial bioamination cascade for the C−H amination of cyclic alkanes by combining P450 monooxygenase, alcohol dehydrogenase, and amine dehydrogenase. Herein, this system has been extended to the synthesis of chiral aromatic amines. In the first hydroxylation step, process optimization increased the conversion to 77 %. Two stereoselectively complementary alcohol dehydrogenases and an amine dehydrogenase were selected for the bioconversion of aromatic hydrocarbons to amines. The amination reaction was optimized with respect to cofactor addition and enzyme dosage. Isopropanol was added to decrease ketone intermediate accumulation in the amination step, which further enhanced the overall conversion. This cascade system converted a panel of hydrocarbon substrates into the corresponding amines with excellent optical purity (>99 % ee) and moderate conversion ratios (13–53 %). [ABSTRACT FROM AUTHOR]

Structurally diverse γ‐ and δ‐lactones were efficiently synthesized stereoselectively using an engineered carbonyl reductase from Serratia marcescens (SmCRV4). SmCRV4 exhibited improved activity (up to 500‐fold) and thermostability toward 14 γ‐/δ‐keto acids and esters, compared with the wild‐type enzyme, with 110‐fold enhancement in catalytic efficiency (kcat/Km) toward methyl 4‐oxodecanoate. The preparative synthesis of alkyl and aromatic γ‐ and δ‐lactones with 95 %–>99 % ee and 78 %–90 % yields was demonstrated. The highest space‐time yield, 1175 g L−1 d−1, was achieved for (R)‐γ‐decalactone. [ABSTRACT FROM AUTHOR]

Biocatalysts originating from Badamu ( Prunus communis) have been applied to catalyze the asymmetric synthesis of ( R)-4-methylsulfanylmandelonitrile, a key building block of thiamphenicol and florfenicol. Here, four hydroxynitrile lyase (HNL) isozymes from Badamu were cloned and heterologously expressed in Pichia pastoris. The biochemical properties and catalytic performances of these isozymes were comprehensively explored to evaluate their efficiency and selectivity in asymmetric synthesis. Among then, PcHNL5 was identified with outstanding activity and enantioselectivity in asymmetric hydrocyanation. Under the optimized mild biphasic reaction conditions, seventeen prochiral aromatic aldehydes were converted to valuable chiral cyanohydrins with good yields (up to 94%) and excellent optical purities (up to >99.9% ee), which provide a facile access to numerous chiral amino alcohols, hypoglycemic agents, angiotension converting enzyme (ACE) inhibitors and β-blockers. This work therefore underlines the importance of discovering the most potent biocatalyst among a group of isozymes for converting unnatural substrates into value-added products. [ABSTRACT FROM AUTHOR]