Search

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]

A carbonyl reductase variant, SmCRM5, from Serratia marcescens was obtained through structure-guided directed evolution. The variant showed improved specific activity (U mg−1) towards most of the 16 tested substrates and gave high stereoselectivities of up to 99% in the asymmetric synthesis of 13 γ-/δ-lactones. In particular, SmCRM5 showed a 13.8-fold higher specific activity towards the model substrate, i.e., 5-oxodecanoic acid, and gave (R)-δ-decalactone in 99% ee with a space–time yield (STY) of 301 g L−1 d−1. The preparative synthesis of six δ-lactones in high yields and with high enantiopurities showed the feasibility of the biocatalytic synthesis of these high-value-added chemicals, providing a cost-effective and green alternative to noble-metal catalysis. [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]

Oleate hydratase catalyzes the hydration of unsaturated fatty acids, giving access to C 10 -functionalization of oleic acid. The resultant 10-hydroxystearic acid is a key material for the synthesis of many biomass-derived value-added products. Herein, we report the engineering of an oleate hydratase from Paracoccus aminophilus (Pa OH) with significantly improved catalytic efficiency (from 33 s−1 mM−1 to 119 s−1 mM−1), as well as 3.4 times increased half-life at 30 °C. The structural mechanism regarding the impact of mutations on the improved catalytic activity and thermostability was elucidated with the aid of molecular dynamics simulation. The practical feasibility of the engineered Pa OH variant F233L/F122L/T15 N was demonstrated through the pilot synthesis of 10-hydroxystearic acid and 10-oxostearic acid via an optimized multi-enzymatic cascade reaction, with space-time yields of 540 g L−1 day−1 and 160 g L−1 day−1, respectively. Image 1 Make fatty acid valuable by 'adding water' : In this paper, comprehensive protein engineering studies were carried out to reshape an oleate hydratase from Paracoccus aminophilus , resulting in significantly improved activites and thermostabilities. The best variant provides an access to C10-functionalization of oleic acid by simple hydration, which forms value-added building blocks useful for production of flavours and biodegradable materials. • An efficient high-throughput screening method for evaluating the activity of oleate hydratase was developed. • Directed evolution of an oleate hydratase led to significantly improved thermostability as well as catalytic efficiency. • Molecular dynamic simulations elucidated the molecular basis of beneficial mutagenesis. [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]