A multistep enzyme catalysis was successfully implemented to produce long-chain α,ω-dicarboxylic and ω-hydroxycarboxylic acids from renewable fatty acids and plant oils. Sebacic acid as well as ω-hydroxynonanoic acid and ω-hydroxytridec-11-enoic acid were produced from oleic and ricinoleic acid.

Oxyfunctionalization of plant oils such as olive oil and soybean oil into C9 carboxylic acids (e.g., n-nonanoic acid and 9-hydroxynonanoic acid) was investigated. The biotransformation was composed of hydrolysis of plant oils by the Thermomyces lanuginosus lipase (TLL) and C9−C10 double-bond cleavage in unsaturated fatty acids by a serial reaction of a fatty acid double bond-hydratase of Stenotrophomonas maltophilia, an alcohol dehydrogenase of Micrococcus luteus, and a Baeyer−Villiger monooxygenase (BVMO) of Pseudomonas putida KT2440 expressed in Escherichia coli. The newly cloned oleate hydratase allowed one to produce 10-hydroxyoctadecanoic acid and 10hydroxyoctadec-12-enoic acid at a high rate from oleic acid and linoleic acid, respectively, which are major fatty acid constituents of many plant oils. Furthermore, overexpression of a long chain fatty acid transporter FadL in the recombinant E. coli led to a significant increase of whole-cell biotransformation rates of oleic acid and linoleic acid into the corresponding esters. The resulting esters (the BVMO reaction products) were hydrolyzed in situ by TLL, generating nonanoic acid, non-3-enoic acid, and 9-hydroxynonanoic acid, which can be further oxidized to 1,9-nonanedioic acid. This study demonstrated that industrially relevant C9 carboxylic acids could be produced from olive oil or soybean oil by simultaneous enzyme/whole-cell biocatalysis.

Ap ractical chemoenzymatic method for the synthesis of 9-hydroxynonanoic acid and1 ,9-nonanedioic acid (i.e., azelaic acid) from oleic acid [(9Z)-octadec-9-enoic acid] was investigated. Biotransformation of oleic acid into 9-(nonanoyloxy)nonanoic acid via 10-hydroxyoctadecanoic acid and 10keto-octadecanoic acid was driven by aC -9 double bond hydratase from Stenotrophomonas maltophilia, an alcohol dehydrogenase from Micrococcus luteus, and aB aeyer-Villiger monooxygenase (BVMO) from Pseudomonas putida KT2440, which was expressedi nr ecombinant Escherichia coli.A fter productiono ft he ester (i.e. ,t he BVMO reactionp roduct), the compound wasc hemically hydrolyzed to nnonanoic acid and9 -hydroxynonanoic acid because n-nonanoic acid is toxic to E. coli.T he ester was also converted into 9-hydroxynonanoic acid and the n-nonanoic acid methyle ster, which can be oxygenated into the 9-hydroxynonanoic acid methyl ester by the AlkBGT from P. putida GPo1. Finally,9 -hydroxynonanoic acid was chemically oxidized to azelaic acid with ah ighy ield under fairly mild reactionc onditions.F or example,w hole-cell biotransformation at ah igh cell density (i.e., 10 gd ry cells/L) allowed the final ester product concentration and volumetric productivity to reach2 5mMa nd 2.8 mM h À1 ,r espectively.T he overall molar yield of azelaic acid from oleic acid was 58%, based on the biotransformation and chemical transformation conversion yields of 84% and 68%, respectively.Scheme 2. Chemical conversion of the ester intermediate (4)toa zelaic acid (7).

Baeyer-Villiger monooxygenases (BVMOs) are able to catalyze regiospecific Baeyer-Villiger oxygenation of a variety of cyclic and linear ketones to generate the corresponding lactones and esters, respectively. However, the enzymes are usually difficult to express in a functional form in microbial cells and are rather unstable under process conditions hindering their large-scale applications. Thereby, we investigated engineering of the BVMO from Pseudomonas putida KT2440 and the gene expression system to improve its activity and stability for large-scale biotransformation of ricinoleic acid (1) into the ester (i.e., (Z)-11-(heptanoyloxy)undec-9-enoic acid) (3), which can be hydrolyzed into 11-hydroxyundec-9-enoic acid (5) (i.e., a precursor of polyamide-11) and n-heptanoic acid (4). The polyionic tag-based fusion engineering of the BVMO and the use of a synthetic promoter for constitutive enzyme expression allowed the recombinant Escherichia coli expressing the BVMO and the secondary alcohol dehydrogenase of Micrococcus luteus to produce the ester (3) to 85 mM (26.6 g/L) within 5 h. The 5 L scale biotransformation process was then successfully scaled up to a 70 L bioreactor; 3 was produced to over 70 mM (21.9 g/L) in the culture medium 6 h after biotransformation. This study demonstrated that the BVMO-based whole-cell reactions can be applied for large-scale biotransformations.

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