Genomic, transcriptomic, and metabolic characterization of 2-Phenylethanol-resistant Saccharomyces cerevisiae obtained by evolutionary engineering.

2-Phenylethanol is an aromatic compound commonly used in the food, cosmetic, and pharmaceutical industries. Due to increasing demand for natural products by consumers, the production of this flavor by microbial fermentation is gaining interest, as a sustainable alternative to chemical synthesis or expensive plant extraction, both processes relying on the use of fossil resources. However, the drawback of the fermentation process is the high toxicity of 2-phenylethanol to the producing microorganism. The aim of this study was to obtain a 2-phenylethanol-resistant Saccharomyces cerevisiae strain by in vivo evolutionary engineering and characterize the adapted yeast at the genomic, transcriptomic and metabolic levels. For this purpose, the tolerance to 2-phenylethanol was developed by gradually increasing the concentration of this flavor compound through successive batch cultivations, leading to an adapted strain that could tolerate 3.4 g/L of 2-phenylethanol, which was about 3-times better than the reference strain. Genome sequencing of the adapted strain identified point mutations in several genes, notably in HOG1 that encodes the Mitogen-Activated Kinase of the high-osmolarity signaling pathway. As this mutation is localized in the phosphorylation lip of this protein, it likely resulted in a hyperactive protein kinase. Transcriptomic analysis of the adapted strain supported this suggestion by revealing a large set of upregulated stress-responsive genes that could be explained in great part by HOG1-dependent activation of the Msn2/Msn4 transcription factor. Another relevant mutation was found in PDE2 encoding the low affinity cAMP phosphodiesterase, the missense mutation of which may lead to hyperactivation of this enzyme and thereby enhance the stressful state of the 2-phenylethanol adapted strain. In addition, the mutation in CRH1 that encodes a chitin transglycosylase implicated in cell wall remodeling could account for the increased resistance of the adapted strain to the cell wall-degrading enzyme lyticase. Finally, the potent upregulation of ALD3 and ALD4 encoding NAD+ -dependent aldehyde dehydrogenase together with the observed phenylacetate resistance of the evolved strain suggest a resistance mechanism involving conversion of 2-phenylethanol into phenylacetaldehyde and phenylacetate implicating these dehydrogenases.

Deciphering the Origin, Evolution, and Physiological Function of the Subtelomeric Aryl-Alcohol 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' 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 NADPH-dependent 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's domestication and use in brewing, baking, and other industrial applications.IMPORTANCE Functional characterization of hypothetical genes remains one of the chief tasks of the postgenomic era. Although the first Saccharomyces cerevisiae genome sequence was published over 20 years ago, 22% of its estimated 6,603 open reading frames (ORFs) remain unverified. One outstanding example of this category of genes is the enigmatic seven-member AAD family. Here, we demonstrate that proteins encoded by two members of this family exhibit aliphatic and aryl-aldehyde reductase activity, and further that such activity can be recovered from pseudogenized AAD genes via ancestral-state reconstruction. The phylogeny of yeast AAD genes suggests that these proteins may have played an important ancestral role in detoxifying aromatic aldehydes in ligninolytic fungi. However, in yeast adapted to niches rich in sugars, AAD genes become subject to mutational erosion. Our findings shed new light on the selective pressures and molecular mechanisms by which genes undergo pseudogenization.

Biosynthesis of higher alcohol flavour compounds by the yeast Saccharomyces cerevisiae: impact of oxygen availability and responses to glucose pulse in minimal growth medium with leucine as sole nitrogen source.

Higher alcohol formation by yeast is of great interest in the field of fermented beverages. Among them, medium-chain alcohols impact greatly the final flavour profile of alcoholic beverages, even at low concentrations. It is widely accepted that amino acid metabolism in yeasts directly influences higher alcohol formation, especially the catabolism of aromatic and branched-chain amino acids. However, it is not clear how the availability of oxygen and glucose metabolism influence the final higher alcohol levels in fermented beverages. Here, using an industrial Brazilian cachaça strain of Saccharomyces cerevisiae, we investigated the effect of oxygen limitation and glucose pulse on the accumulation of higher alcohol compounds in batch cultures, with glucose (20 g/l) and leucine (9.8 g/l) as the carbon and nitrogen sources, respectively. Fermentative metabolites and CO2 /O2 balance were analysed in order to correlate the results with physiological data. Our results show that the accumulation of isoamyl alcohol by yeast is independent of oxygen availability in the medium, depending mainly on leucine, α-keto-acids and/or NADH pools. High-availability leucine experiments showed a novel and unexpected accumulation of isobutanol, active amyl alcohol and 2-phenylethanol, which could be attributed to de novo biosynthesis of valine, isoleucine and phenylalanine and subsequent outflow of these pathways. In carbon-exhausted conditions, our results also describe, for the first time, the metabolization of isoamyl alcohol, isobutanol, active amyl alcohol but not of 2-phenylethanol, by yeast strains in stationary phase, suggesting a role for these higher alcohols as carbon source for cell maintenance and/or redox homeostasis during this physiological phase.

Process engineering for bioflavour production with metabolically active yeasts - a mini-review.

Flavours are biologically active molecules of large commercial interest in the food, cosmetics, detergent and pharmaceutical industries. The production of flavours can take place by either extraction from plant materials, chemical synthesis, biological conversion of precursor molecules or 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. Yeasts are potential production hosts for bioflavours. In this mini-review, we give an overview of bioflavour production in yeasts from the process-engineering perspective. Two specific examples, production of 2-phenylethanol and vanillin, are used to illustrate the process challenges and strategies used.

Physiological and biochemical characteristics of the ethyl tiglate production pathway in the yeast Saprochaete suaveolens.

A yeast identified as Saprochaete suaveolens was investigated for its capacity to produce a large panel of flavouring molecules. With a production of 32 compounds including 28 esters, S. suaveolens seems to be a good producer of fruity flavours and fragrances and especially of unsaturated esters, such as ethyl tiglate. Physiological and biochemical analyses were performed in this study in an attempt to comprehend the metabolic route to the formation of this compound. We show that the accumulation of ethyl tiglate by S. suaveolens is specifically induced by isoleucine. However, and contrary to S. cerevisiae, which harbours a classical Ehrlich pathway leading to the production of 2-methylbutanol from isoleucine, our results provide phenotypic and enzymological evidence of ethyl tiglate biosynthesis in S. suaveolens through the catabolism of this amino acid by the ß-oxidation pathway, which generates tiglyl-CoA as a probable intermediate. A kinetic analysis of this flavour molecule during growth of S. suaveolens on glucose and isoleucine showed a phase of production of ethyl tiglate that culminated concurrently with isoleucine exhaustion, followed by a disappearance of this compound, likely due to reassimilation by the yeast.

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 °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 0.602 g/l, respectively). Slightly higher amounts of glycerol were observed in samples fermented with strain EC1118 than in those fermented with strain RC212.

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.

Xylosylation as an effective means for reducing yeast growth inhibition by 2-phenylethanol.

Microbial bioproduction processes of 2-phenylethanol, an important rose-like flavor and fragrance compound that occurs naturally in the essential oils of many flowers and plants, are hindered by the growth inhibition it exerts towards the producing microorganism, mainly yeast. We show here for the first time that glycosylation of 2-phenylethanol with xylose increased the inhibitory concentration inducing 50% decrease of the yeast Saccharomyces cerevisiae strain BY4741 growth rate (IC50 ) from 14 mM (1.71 g/L) 2-phenylethanol (2PE) to 100 mM (25.5 g/L) 2-phenylethyl ß-D-xylopyranoside (X-2PE). More interestingly, the IC10 was only 3 mM (0.37 g/L) for 2PE and 86 mM (21.9 g/L) for X-2PE. Xylosylation of 2-phenylethanol can offer therefore an effective means for reducing yeast growth inhibition in microbial bioproduction processes of this important flavor and fragrance compound.

Influence of nitrogen supply on the production of higher alcohols/esters and expression of flavour-related genes in cachaça fermentation.

This study provides the first attempt to analyse the influence of ammonium supplements on sugar-cane juice fermentation and the flavour profile in a cachaça industrial process. The objective was to find a relationship between higher alcohol/ester content and the transcription levels of the main genes involved in production of these compounds under cachaça fermentation. Sugar-cane juice with a low amount of assimilable nitrogen (81 mg N/L), was further supplemented with mid-range or high concentrations of ammonium sulfate. Overall, higher alcohol production was reduced by ammonium supplementation, and this can be correlated with a general downregulation of genes encoding decarboxylases and dehydrogenases of the Ehrlich pathway. The production of acetate esters was enhanced by mid-range ammonium supplementation and the production of acyl esters by high ammonium supplementation. The acyl esters could be correlated with expression of alcohol acyl-transferase EEB1 and the acyl esterase IAH1.

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

BACKGROUND: The white-rot fungus Phanerochaete chrysosporium 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. RESULTS: We report here the cloning of an aryl-alcohol dehydrogenase cDNA from the white-rot fungus Phanerochaete chrysosporium, its expression in Escherichia coli 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 Km (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. CONCLUSIONS: In this work, we have characterized the biochemical properties of an aryl-alcohol dehydrogenase from the white-rot fungus Phanerochaete chrysosporium. 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.

Role of ethylene in the biosynthetic pathway of aliphatic ester aroma volatiles in Charentais Cantaloupe melons.

Compared to other melon types, Cantaloupe Charentais melons are highly aromatic with a major contribution to the aroma being made by aliphatic and branched esters. Using a transgenic line in which the synthesis of the plant hormone ethylene has been considerably lowered by antisense ACC oxidase mRNA (AS), the aliphatic ester pathway steps at which ethylene exerts its regulatory role were found. The data show that the production of aliphatic esters such as hexyl and butyl acetate was blocked in AS fruit and could be reversed by ethylene. Using fruit discs incubated in the presence of various precursors, the steps at which ester formation was inhibited in AS fruit was shown to be the reduction of fatty acids and aldehydes, the last step of acetyl transfer to alcohols being unaffected. However, treating AS fruit with the ethylene antagonist 1-methylcyclopropene resulted in about 50% inhibition of acetyl transfer activity, indicating that this portion of activity was ethylene-dependent and this was supported by the low residual ethylene concentration of AS fruit discs (around 2 microl l(-1)). In conclusion, the reduction of fatty acids and aldehydes appears essentially to be ethylene-dependent, whilst the last step of alcohol acetylation has ethylene-dependent and ethylene-independent components, probably corresponding to differentially regulated alcohol acetyltransferases.