Deciphering the origin, evolution, and physiological function of the subtelomeric arylalcohol dehydrogenase gene family in the yeast Saccharomyces cerevisiae

Homology searches indicate that Saccharomyces cerevisiae strain BY4741 contains seven redundant genes that encode putative aryl-alcohol dehydrogenases (AAD). Yeast AAD genes are located in subtelomeric regions of different chromosomes, and their functional role(s) remain enigmatic. Here, we show that two of these genes, AAD4 and AAD14, encode functional enzymes that reduce aliphatic and aryl-aldehydes concomitant with the oxidation of cofactor NADPH, and that Aad4p and Aad14p exhibit different substrate preference patterns. Other yeast AAD genes are undergoing pseudogenization. The 5\u27 sequence of AAD15 has been deleted from the genome. Repair of an AAD3 missense mutation at the catalytically essential Tyr73 residue did not result in a functional enzyme. However, ancestral-state reconstruction by fusing Aad6 with Aad16 and by N-terminal repair of Aad10 restores NADPHdependent aryl-alcohol dehydrogenase activities. Phylogenetic analysis indicates that AAD genes are narrowly distributed in wood-saprophyte fungi and in yeast that occupy lignocellulosic niches. Because yeast AAD genes exhibit activity on veratraldehyde, cinnamaldehyde, and vanillin, they could serve to detoxify aryl-aldehydes released during lignin degradation. However, none of these compounds induce yeast AAD gene expression, and Aad activities do not relieve aryl-aldehyde growth inhibition. Our data suggest an ancestral role for AAD genes in lignin degradation that is degenerating as a result of yeast\u27s domestication and use in brewing, baking, and other industrial applications

Cloning, expression and characterization of an aryl-alcohol dehydrogenase from the white-rot fungus <it>Phanerochaete chrysosporium</it> strain BKM-F-1767

<p>Abstract</p> <p>Background</p> <p>The white-rot fungus <it>Phanerochaete chrysosporium</it> is among the small group of fungi that can degrade lignin to carbon dioxide while leaving the crystalline cellulose untouched. The efficient lignin oxidation system of this fungus requires cyclic redox reactions involving the reduction of aryl-aldehydes to the corresponding alcohols by aryl-alcohol dehydrogenase. However, the biochemical properties of this enzyme have not been extensively studied. These are of most interest for the design of metabolic engineering/synthetic biology strategies in the field of biotechnological applications of this enzyme.</p> <p>Results</p> <p>We report here the cloning of an aryl-alcohol dehydrogenase cDNA from the white-rot fungus <it>Phanerochaete chrysosporium</it>, its expression in <it>Escherichia coli</it> and the biochemical characterization of the encoded GST and His₆ tagged protein. The purified recombinant enzyme showed optimal activity at 37°C and at pH 6.4 for the reduction of aryl- and linear aldehydes with NADPH as coenzyme. NADH could also be the electron donor, while having a higher <it>Km</it> (220 μM) compared to that of NADPH (39 μM). The purified recombinant enzyme was found to be active in the reduction of more than 20 different aryl- and linear aldehydes showing highest specificity for mono- and dimethoxylated Benzaldehyde at positions 3, 4, 3,4 and 3,5. The enzyme was also capable of oxidizing aryl-alcohols with NADP ⁺ at 30°C and an optimum pH of 10.3 but with 15 to 100-fold lower catalytic efficiency than for the reduction reaction.</p> <p>Conclusions</p> <p>In this work, we have characterized the biochemical properties of an aryl-alcohol dehydrogenase from the white-rot fungus <it>Phanerochaete chrysosporium</it>. We show that this enzyme functions in the reductive sense under physiological conditions and that it displays relatively large substrate specificity with highest activity towards the natural compound Veratraldehyde.</p

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 (c) 2014 John Wiley & Sons, Ltd

Raspberry wine fermentation with suspended and immobilized yeast cells of two strains of Saccharomyces cerevisiae

The objectives of this study were to assess the differences in fermentative behaviour of two different strains of Saccharomyces cerevisiae (EC1118 and RC212) and to determine the differences in composition and sensory properties of raspberry wines fermented with immobilized and suspended yeast cells of both strains at 15 degrees C. Analyses of aroma compounds, glycerol, acetic acid and ethanol, as well as the kinetics of fermentation and a sensory evaluation of the wines, were performed. All fermentations with immobilized yeast cells had a shorter lag phase and faster utilization of sugars and ethanol production than those fermented with suspended cells. Slower fermentation kinetics were observed in all the samples that were fermented with strain RC212 (suspended and immobilized) than in samples fermented with strain EC1118. Significantly higher amounts of acetic acid were detected in all samples fermented with strain RC212 than in those fermented with strain EC1118 (0.282 and 0.602g/l, respectively). Slightly higher amounts of glycerol were observed in samples fermented with strain EC1118 than in those fermented with strain RC212

Process engineering for bioflavour production with metabolically active yeast - a minireview.

Flavours are biologically active molecules of large commercial interest in the food, cosmetics, detergent and pharmaceutical industry. The production of flavours can take place by either extraction from plant materials, chemical synthesis, through biological conversion of precursor molecules or through de novo biosynthesis. The latter alternatives are gaining importance through the rapidly growing fields of systems biology and metabolic engineering giving efficient production hosts for the so-called "bioflavours", which are natural flavour and/or fragrance compounds obtained with cell factories or enzymatic systems. One potential production host for bioflavour is yeast. In this mini-review, we give an overview of bioflavour production in yeast from the process engineering perspective. Two specific examples - production of 2-phenylethanol and vanillin - are used to illustrate process challenges and strategies used