M. M. W. Etschmann

Biotechnological production of 2-phenylethanol

Abstract. 2-Phenylethanol (2-PE) is an important flavour and fragrance compound with a rose-like odour. Most of the worlds annual production of several thousand tons is synthesised by chemical means but, due to increasing demand for natural flavours, alternative production methods are being sought. Harnessing the Ehrlich pathway of yeasts by bioconversion of L-phenylalanine to 2-PE could be an option, but in situ product removal is necessary due to product inhibition. This review describes the microbial production of 2-PE, and also summarizes the chemical syntheses and the market situation.

Cell factory applications of the yeast Kluyveromyces marxianus for the biotechnological production of natural flavour and fragrance molecules

Kluyveromyces marxianus is emerging as a new platform organism for the production of flavour and fragrance (F&F) compounds. This food‐grade yeast has advantageous traits, such as thermotolerance and rapid growth, that make it attractive for cell factory applications. The major impediment to its development has been limited fundamental knowledge of its genetics and physiology, but this is rapidly changing. K. marxianus produces a wide array of volatile molecules and contributes to the flavour of a range of different fermented beverages. Advantage is now being taken of this to develop strains for the production of metabolites such as 2‐phenylethanol and ethyl acetate. Strains that were selected from initial screens were used to optimize processes for production of these F&F molecules. Most developments have focused on optimizing growth conditions and the fermentation process, including product removal, with future advancement likely to involve development of new strains through the application of evolutionary or rational engineering strategies. This is being facilitated by new genomic and molecular tools. Furthermore, synthetic biology offers a route to introduce new biosynthetic pathways into this yeast for F&F production. Consumer demand for biologically‐synthesized molecules for use in foods and other products creates an opportunity to exploit the unique potential of K. marxianus for this cell factory application. Copyright

Synthesis of green note aroma compounds by biotransformation of fatty acids using yeast cells coexpressing lipoxygenase and hydroperoxide lyase

Green notes are substances that characterize the aroma of freshly cut grass, cucumbers, green apples, and foliage. In plants, they are synthesized by conversion of linolenic or linoleic acid via the enzymes lipoxygenase (LOX) and hydroperoxide lyase (HPL) to short-chained aldehydes. Current processes for production of natural green notes rely on plant homogenates as enzyme sources but are limited by low enzyme concentration and low specificity. In an alternative approach, soybean LOX2 and watermelon HPL were overexpressed in Saccharomyces cerevisiae. After optimization of the expression constructs, a yeast strain coexpressing LOX and HPL was applied in whole cell biotransformation experiments. Whereas addition of linolenic acid to growing cultures of this strain yielded no products, we were able to identify high green note concentrations when resting cells were used. The primary biotransformation product was 3(Z)-hexenal, a small amount of which isomerized to 2(E)-hexenal. Furthermore, both aldehydes were reduced to the corresponding green note alcohols by endogenous yeast alcohol dehydrogenase to some extent. As the cosolvent ethanol was the source of reducing equivalents for green note alcohol formation, the hexenal/hexenol ratio could be influenced by the use of alternative cosolvents. Further investigations to identify the underlying mechanism of the rather low biocatalyst stability revealed a high toxicity of linolenic acid to yeast cells. The whole cell catalyst containing LOX and HPL enzyme activity described here can be a promising approach towards a highly efficient microbial green note synthesis process.