Different microbial strains are able to transform oleic acid (OA) into 10-hydroxystearic acid (10-HSA) by means of the catalytic activity of the enzymes oleate hydratase (EC 4.2.1.53). Lactobacillus rhamnosus ATCC 53103 performs this biotransformation with very high stereoselectivity, affording enantiopure (R)-10-HSA. In this work, we cloned, in Escherichia coli, the oleate hydratase present in the above-mentioned probiotic strain. Our study demonstrated that the obtained recombinant hydratase retains the catalytic properties of the Lactobacillus strain but that its activity was greatly affected by the expression procedure. According to our findings, we devised a reliable procedure for the hydration of oleic acid using a recombinant E. coli whole-cell catalyst. We established that the optimal reaction conditions were pH 6.6 at 28 °C in phosphate buffer, using glycerol and ethanol as co-solvents. According to our experimental protocol, the biocatalyst does not show significant substrate inhibition as the hydration reaction can be performed at high oleic acid concentration (up to 50 g/L).

In this work, we studied the biotechnological potential of thirteen probiotic microorganisms currently used to improve human health. We discovered that the majority of the investigated bacteria are able to catalyze the hydration reaction of the unsaturated fatty acids (UFAs). We evaluated their biocatalytic activity toward the three most common vegetable UFAs, namely oleic, linoleic, and linolenic acids. The whole-cell biotransformation experiments were performed using a fatty acid concentration of 3 g/L in anaerobic conditions. Through these means, we assessed that the main part of the investigated strains catalyzed the hydration reaction of UFAs with very high regio- and stereoselectivity. Our biotransformation reactions afforded almost exclusively 10-hydroxy fatty acid derivatives with the single exception of Lactobacillus acidophilus ATCC SD5212, which converted linoleic acid in a mixture of 13-hydroxy and 10-hydroxy derivatives. Oleic, linoleic, and linolenic acids were transformed into (R)-10-hydroxystearic acid, (S)-(12Z)-10-hydroxy-octadecenoic, and (S)-(12Z,15Z)-10-hydroxy-octadecadienoic acids, respectively, usually with very high enantiomeric purity (ee > 95%). It is worth noting that the biocatalytic capabilities of the thirteen investigated strains may change considerably from each other, both in terms of activity, stereoselectivity, and transformation yields. Lactobacillus rhamnosus ATCC 53103 and Lactobacillus plantarum 299 V proved to be the most versatile, being able to efficiently and selectively hydrate all three investigated fatty acids.

Dihydrocoumarin is a natural product of great relevance for the flavour industry. In this work, we describe a study on the biotransformation of the toxic compound coumarin into natural dihydrocoumarin, recognized as safe for food aromatization. To this end, we screened a variety of yeasts and filamentous fungi, isolated from different sources, in order to evaluate their ability to reduce selectively the conjugated double bond of coumarin. Moreover, since coumarin induces cytotoxicity and therefore inhibits cell growth as well as the cell metabolic activity, we tested out different substrate concentrations. All strains were able to convert the substrate, although showing very different conversion rates and different sensitivity to the coumarin concentration. In particular, the yeasts Torulaspora delbrueckii, Kluyveromyces marxianus and the fungus Penicillium camemberti displayed the higher activity and selectivity in the substrate transformation. Among the latter strains, Kluyveromyces marxianus presented the best resistance to substrate toxicity, allowing the biotransformation process even with coumarin concentration up to 1.8 g/L.Abstract: Dihydrocoumarin is a natural product of great relevance for the flavour industry. In this work, we describe a study on the biotransformation of the toxic compound coumarin into natural dihydrocoumarin, recognized as safe for food aromatization. To this end, we screened a variety of yeasts and filamentous fungi, isolated from different sources, in order to evaluate their ability to reduce selectively the conjugated double bond of coumarin. Moreover, since coumarin induces cytotoxicity and therefore inhibits cell growth as well as the cell metabolic activity, we tested out different substrate concentrations. All strains were able to convert the substrate, although showing very different conversion rates and different sensitivity to the coumarin concentration. In particular, the yeasts Torulaspora delbrueckii, Kluyveromyces marxianus and the fungus Penicillium camemberti displayed the higher activity and selectivity in the substrate transformation. Among the latter strains, Kluyveromyces marxianus presented the best resistance to substrate toxicity, allowing the biotransformation process even with coumarin concentration up to 1.8 g/L.

The biotransformation of the aromatic amino acids phenylalanine, tyrosine and tryptophan originates a number of bioactive compounds. Yeasts are the most used microorganisms for the transformation of (L)-phenylalanine into the flavour phenylethanol. Here, we reported a study on the biotransformation of the proteogenic aminoacids phenylalanine, tyrosine and tryptophan by yeast strains belonging to Yarrowia genus. We found that the latter microorganisms, in high aerobic conditions, metabolise the aromatic amino acids (L)-phenylalanine and (L)-tyrosine with the almost exclusive formation of phenylacetic acid and 4-hydroxyphenylacetic acid, respectively. Differently, the biotransformation of (L)-tryptophan with Y. lipolytica, gave anthranilic acid as the main product. As stated by the European and USA legislations concerning natural flavour production, phenylacetic acid obtained by microbial conversion of phenylalanine of natural origin can be commercialised as a natural flavour. Accordingly, our findings were exploited in a new process, based on the Yarrowia strains-mediated biotransformation of natural (L)-phenylalanine, that allows the large-scale preparation of the high-value, natural flavour, phenylacetic acid.