In this review we examine the fascinating array of microbial and enzymatic transformations of ferulic acid. Ferulic acid is an extremely abundant, preformed phenolic aromatic chemical found widely in nature. Ferulic acid is viewed as a commodity scale, renewable chemical feedstock for biocatalytic conversion to other useful aromatic chemicals. Most attention is focused on bioconversions of ferulic acid itself. Topics covered include cinnamoyl side-chain cleavage; nonoxidative decarboxylation; mechanistic details of styrene formation; purification and characterization of ferulic acid decarboxylase; conversion of ferulic acid to vanillin; O-demethylation; and reduction reactions. Biotransformations of vinylguaiacol are discussed, and selected biotransformations of vanillic acid including oxidative and nonoxidative decarboxylation are surveyed. Finally, enzymatic oxidative dimerization and polymerization reactions are reviewed.

A ferulic acid decarboxylase enzyme which catalyzes the decarboxylation of ferulic acid to 4-hydroxy-3-methoxystyrene was purified from Pseudomonas fluorescens UI 670. The enzyme requires no cofactors and contains no prosthetic groups. Gel filtration estimated an apparent molecular mass of 40.4 (±-t6%) kDa, whereas sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed a molecular mass of 20.4 kDa, indicating that ferulic acid decarboxylase is a homodimer in solution. The purified enzyme displayed an optimum temperature range of 27 to 30"C, exhibited an optimum pH of 7.3 in potassium phosphate buffer, and had a Km of 7.9 mM for ferulic acid. This enzyme also decarboxylated 4-hydroxycinnamic acid but not 2-or 3-hydroxycinnamic acid, indicating that a hydroxy group para to the carboxylic acid-containing side chain is required for the enzymatic reaction. The enzyme was inactivated by Hg2', Cu2+, p-chloromercuribenzoic acid, and N-ethylmaleimide, suggesting that sulfhydryl groups are necessary for enzyme activity. Diethyl pyrocarbonate, a histidine-specific inhibitor, did not affect enzyme activity.Ferulic acid (compound 1 in Fig. 1) is an extremely abundant, lignin-related aromatic acid of interest as a renewable resource for the production of useful aromatic chemicals. We are exploiting the use of enzymes and microbial transformations as a means of generating value-added products from ferulic acid (17,18). An understanding of the biochemical and enzymatic processes involved in ferulic acid biotransformations is required as a theoretical basis for the ultimate development of biocatalytic processes for the production of large amounts of ferulic acid-derived aromatic chemicals. Whole-cell bioconversions of ferulic acid to 4-hydroxy-3-methoxystyrene (compound 3 in Fig. 1) have been reported to occur in bacteria and fungi (1,2,17,18,(23)(24)(25). Some properties of the enzymes decarboxylating 4-hydroxycinnamic acid have been studied with crude cell extracts from an Aerobacter sp. (11) and with a partially purified enzyme from Cladosporium phlei (13). Surprisingly, no work on the purification and characterization of the enzyme(s) catalyzing the decarboxylation of ferulic acid to 4-hydroxy-3-methoxystyrene has been reported to date.We have identified a strain of Pseudomonas fluorescens (UI-670) that efficiently transforms ferulic acid (compound 1) to 4-hydroxy-3-methoxystyrene (compound 3) (Fig. 1). Deuterium labeling experiments confirmed the intermediacy of compound 2 in the decarboxylation reaction (17). In this report, we describe the purification and characterization of a ferulic acid decarboxylase from P. fluorescens. MATERIALS AND METHODSMaterials and reagents. trans-Ferulic acid (4-hydroxy-3-methoxy-cinnamic acid, 99%), trans-cinnamic acid, hydrocinnamic acid, cis-2-methoxycinnamic acid, 2-carboxycinnamic acid, iodoacetamide, p-chloromercuribenzoic acid, N-ethylmaleimide, and diethyl pyrocarbonate were purchased from Aldrich Chemical Company (Milwaukee, Wis. Microorganism and culture conditions. The P. ...

1992) based on the 'H-nuclear magnetic resonance spectral analysis of diastereomeric S-(+)-O-acetylmandelate esters of hydroxystearates. This report describes the stereochemistries of microbial hydrations of oleic acid to 10-hydroxystearic acid by Nocardia aurantia (also known as Rhodococcus rhodochrous) ATCC 12674, Nocardia restrictus ATCC 14887, Mycobacterium fortuitum UI-53387, Pseudomonas species strain NRRL-2994, Pseudomonas species strain NRRL B-3266, and baker's yeast. 10(R)-hydroxystearic acid isolated from Pseudomonas species strain NRRL-2994 was the standard for use in the 'H-nuclear magnetic resonance spectral technique to permit simple assignments of the absolute configurations of 10-hydroxystearic acid produced by different microorganisms. While the R. rhodochrous ATCC 12674-mediated hydration of oleic acid gave mixtures of enantiomers 10(R)-hydroxystearic acid and 10(S)-hydroxystearic acid, Pseudomonas species strain NRRL-B-3266 produced optically pure 10(R)-hydroxystearic acid. The remaining microorganisms stereoselectively hydrated oleic acid to 10(R)-hydroxystearic acid containing between 2 and 18% of the contaminating 10(S)-hydroxystearic acid.

Saccharomyces cerevisiae (dry baker's yeast) and Pseudomonasfluorescens were used to convert trans-ferulic acid into 4-hydroxy-3-methoxystyrene in 96 and 89% yields, respectively. The metabolites were isolated by solid-phase extraction and analyzed by thin-layer chromatography and high-performance liquid chromatography. The identities of the metabolites were determined by 'H-and "3C-nuclear magnetic resonance * Corresponding author.

Resting cells of Saccharomyces cerevisiae (baker's yeast, type II; Sigma) were used to convert oleic acid into 10-hydroxyoctadecanoic acid with a 45% yield. Nocardia aurantia (ATCC 12674), Nocardia sp. (NRRL 5646), and Mycobacteriumfortuitum (UI 53378) all converted oleic acid into 10-oxo-octadecanoic acid with 65, 55, and 80% yields, respectively. Structures of all metabolites were suggested by 'H and 13C nuclear magnetic resonance and by infrared and mass spectrometry. Structures of isomeric hydroxystearate and oxostearate derivatives and the stereochemical purity of hydroxystearates are difficult to prove unambiguously unless authentic standard compounds are available for spectral comparison. We describe the use of the chemical Baeyer-Villiger oxidation technique with 10-oxo-octadecanoic acid followed by mass spectral analysis of neutral extracts as a simple method to confirm the position of oxo-functional groups in the structures of fatty acid ketones. We further introduce a simple method based on 'H nuclear magnetic resonance analysis of diastereomeric S-(+)-O-acetylmandelate esters of hydroxystearates as a means of ascertaining stereochemical purities of hydroxy fatty acids.

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A short formal synthesis of squalamine is described, utilizing the biotransformation product 2, which is available in one step from commercially available 3-keto-23,24-bisnorchol-4-en-22-ol (1). Regioselective C-22 oxidation and C-24 sulfation of the corresponding alcohols in the presence of a free C-7 alcohol make for an efficient preparation of squalamine intermediate 11.

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