Studies on the biosynthesis of beta-phenethyl alhol in candida guilliermondii

Abstract

The occurrence of aromatic alcohols, viz., p-phenethyl alcohol (PEA), p-(4-hydroxyphenyl)ethyl alcohol (HOPEA), and p-indole ethyl alcohol (IEA) has been reported in plants and microorganisms. Among these, PEA is widely used in perfumery, as a preservative in ophthalmic solutions, and in the manufacture of semisynthetic penicillins. Recent studies have clearly established the biosynthetic capabilities of Candida species to produce PEA, HOPEA, IEA, and their corresponding hydroxy acids. In view of the reported auto-antibiotic activity of PEA, the enzymology of its biosynthesis and its regulatory aspects are of great interest. The literature pertaining to various aspects of PEA is summarized in Chapter I.

Methodology

The details of the methodology employed in studies described in Chapters III to V are presented in Chapter II.

Characterization of the Biosynthetic Pathway

C. guilliermondii produced PEA and PLA when grown in a synthetic medium containing L-phenylalanine but not ammonium sulfate as the sole nitrogen source. The following enzyme activities were detected in the cell-free preparations of C. guilliermondii grown on L-phenylalanine:

Phenylalanine aminotransferase (PAT)

Phenylpyruvate decarboxylase (PPD)

Phenylacetaldehyde reductase (PAcdR)

Phenylpyruvate reductase (PPR)

The products of these enzyme-catalyzed reactions were extracted, purified, and analyzed by UV, IR spectral studies and chromatography. Based on these results, the following pathway for the biosynthesis of PEA and PLA has been proposed (Chapter III):

L-Phenylalanine Phenylpyruvate Phenylacetaldehyde PEA

PLA

Phenylalanine aminotransferase

Phenylpyruvate decarboxylase

Phenylacetaldehyde reductase

Phenylpyruvate reductase

Regulation of Phenylalanine Aminotransferase (PAT)

The inducible nature of PAT was demonstrated. Only L-isomers of aromatic amino acids induced the enzyme synthesis. The requirement of energy supply for induction was confirmed by the use of electron-transport inhibitors. The induction was completely blocked by cycloheximide and partially by actinomycin D. Using ^14C-uracil incorporation, increased RNA synthesis in the presence of inducer was correlated with the de novo synthesis of PAT (Chapter IV).

C. guilliermondii produces more of PEA (70% yield) and less of PLA (10% yield). The induced synthesis of PAT is more sensitive to PLA (90% at 5 × 10 M) than to PEA (25% at 5 × 10 M), thus offering a plausible explanation why PLA is not accumulated in this organism.

Purification and Properties of Phenylacetaldehyde Reductase (PAcdR)

PAcdR, the terminal enzyme in the biosynthesis of PEA, has not been purified so far from prokaryotes or eukaryotes. In the present studies, it has been purified to homogeneity using several steps, including affinity chromatography on Cibacron Blue 3G-A. Various properties of this enzyme have been investigated (Chapter V).