A key O-demethylase in the degradation of guaiacol by Rhodococcus opacus PD630.

Rhodococcus opacus PD630 is a high oil-producing strain with the ability to convert lignin-derived aromatics to high values, but limited research has been done to elucidate its conversion pathway, especially the upper pathways. In this study, we focused on the upper pathways and demethylation mechanism of lignin-derived aromatics metabolism by R. opacus PD630. The results of the aromatic carbon resource utilization screening showed that R. opacus PD630 had a strong degradation capacity to the lignin-derived methoxy-containing aromatics, such as guaiacol, 3,4-veratric acid, anisic acid, isovanillic acid, and vanillic acid. The gene of gcoAR, which encodes cytochrome P450, showed significant up-regulation when R. opacus PD630 grew on diverse aromatics. Deletion mutants of gcoAR and its partner protein gcoBR resulted in the strain losing the ability to grow on guaiacol, but no significant difference to the other aromatics. Only co-complementation alone of gcoAR and gcoBR restored the strain's ability to utilize guaiacol, demonstrating that both genes were equally important in the utilization of guaiacol. In vitro assays further revealed that GcoAR could convert guaiacol and anisole to catechol and phenol, respectively, with the production of formaldehyde as a by-product. The study provided robust evidence to reveal the molecular mechanism of R. opacus PD630 on guaiacol metabolism and offered a promising study model for dissecting the demethylation process of lignin-derived aromatics in microbes.IMPORTANCEAryl-O-demethylation is believed to be the key rate-limiting step in the catabolism of heterogeneous lignin-derived aromatics in both native and engineered microbes. However, the mechanisms of O-demethylation in lignin-derived aromatic catabolism remain unclear. Notably, guaiacol, the primary component unit of lignin, lacks in situ demonstration and illustration of the molecular mechanism of guaiacol O-demethylation in lignin-degrading bacteria. This is the first study to illustrate the mechanism of guaiacol metabolism by R. opacus PD630 in situ as well as characterize the purified key O-demethylase in vitro. This study provided further insight into the lignin metabolic pathway of R. opacus PD630 and could guide the design of an efficient biocatalytic system for lignin valorization.

Deciphering the transcriptional regulation of the catabolism of lignin-derived aromatics in Rhodococcus opacus PD630.

Rhodococcus opacus PD630 has considerable potential as a platform for valorizing lignin due to its innate "biological funneling" pathways. However, the transcriptional regulation of the aromatic catabolic pathways and the mechanisms controlling aromatic catabolic operons in response to different aromatic mixtures are still underexplored. Here, we identified and studied the transcription factors for aromatic degradation using GFP-based sensors and comprehensive deletion analyses. Our results demonstrate that the funneling pathways for phenol, guaiacol, 4-hydroxybenzoate, and vanillate are controlled by transcriptional activators. The two different branches of the ß-ketoadipate pathway, however, are controlled by transcriptional repressors. Additionally, promoter activity assays revealed that the substrate hierarchy in R. opacus may be ascribed to the transcriptional cross-regulation of the individual aromatic funneling pathways. These results provide clues to clarify the molecule-level mechanisms underlying the complex regulation of aromatic catabolism, which facilitates the development of R. opacus as a promising chassis for valorizing lignin.

The impact of differential lignin S/G ratios on mutagenicity and chicken embryonic toxicity.

Lignin and lignin-based materials have received considerable attention in various fields due to their promise as sustainable feedstocks. Guaiacol (G) and syringol (S) are two primary monolignols that occur in different ratios for different plant species. As methoxyphenols, G and S have been targeted as atmospheric pollutants and their acute toxicity examined. However, there is a rare understanding of the toxicological properties on other endpoints and mixture effects of these monolignols. To fill this knowledge gap, our study investigated the impact of different S/G ratios (0.5, 1, and 2) and three lignin depolymerization samples from poplar, pine, and miscanthus species on mutagenicity and developmental toxicity. A multitiered method consisted of in silico simulation, in vitro Ames test, and in vivo chicken embryonic assay was employed. In the Ames test, syringol showed a sign of mutagenicity, whereas guaiacol did not, which agreed with the T.E.S.T. simulation. For three S and G mixture and lignin monomers, mutagenic activity was related to the proportion of syringol. In addition, both S and G showed developmental toxicity in the chicken embryonic assay and T.E.S.T. simulation, and guaiacol had a severe effect on lipid peroxidation. A similar trend and comparable developmental toxicity levels were detected for S and G mixtures and the three lignin depolymerized monomers. This study provides data and insights on the differential toxicity of varying S/G ratios for some important building blocks for bio-based materials.

Glycosidically-Bound Volatile Phenols Linked to Smoke Taint: Stability during Fermentation with Different Yeasts and in Finished Wine.

When wine grapes are exposed to smoke, there is a risk that the resulting wines may possess smoky, ashy, or burnt aromas, a wine flaw known as smoke taint. Smoke taint occurs when the volatile phenols (VPs) largely responsible for the aroma of smoke are transformed in grape into a range of glycosides that are imperceptible by smell. The majority of VP-glycosides described to date are disaccharides possessing a reducing ß-d-glucopyranosyl moiety. Here, a two-part experiment was performed to (1) assess the stability of 11 synthesized VP-glycosides towards general acid-catalyzed hydrolysis during aging, and (2) to examine whether yeast strains differed in their capacity to produce free VPs both from these model glycosides as well as from grapes that had been deliberately exposed to smoke. When fortified into both model and real wine matrices at 200 ng/g, all VP-disaccharides were stable over 12 weeks, while (42-50 ng/g) increases in free 4-ethylphenol and p-cresol were detected when these were added to wine as their monoglucosides. Guaiacol and phenol were the most abundantly produced VPs during fermentation, whether originating from natural VP-precursors in smoked-exposed Pinot Noir must, or due to fortification with synthetic VP-glycosides. Significant yeast strain-specific differences in glycolytic activities were observed for phenyl-ß-d-glycopyranoside, with two strains (RC212 and BM45) being unable to hydrolyze this model VP, albeit both were active on the guaiacyl analogue. Thus, differences in Saccharomyces cerevisiae ß-glucosidase activity appear to be influenced by the VP moiety.

Thiols Act as Methyl Traps in the Biocatalytic Demethylation of Guaiacol Derivatives.

Demethylating methyl phenyl ethers is challenging, especially when the products are catechol derivatives prone to follow-up reactions. For biocatalytic demethylation, monooxygenases have previously been described requiring molecular oxygen which may cause oxidative side reactions. Here we show that such compounds can be demethylated anaerobically by using cobalamin-dependent methyltransferases exploiting thiols like ethyl 3-mercaptopropionate as a methyl trap. Using just two equivalents of this reagent, a broad spectrum of substituted guaiacol derivatives were demethylated, with conversions mostly above 90 %. This strategy was used to prepare the highly valuable antioxidant hydroxytyrosol on a one-gram scale in 97 % isolated yield.

Isolation and identification of guaiacol producing Alicyclobacillus fastidiosus strains from orchards in Poland.

The genus Alicyclobacillus comprises a group of Gram-positive, thermo-acidophilic bacteria that are capable of producing highly resistant endospores during unfavorable environmental conditions. The members of this genus inhabit natural environments, including hot springs and soils. The main reason behind the spoilage of final commercial fruit products by Alicyclobacillus is the contamination of fruits with soil at the time of harvesting. Some of the Alicyclobacillus species, including Alicyclobacillus acidoterrestris, are categorized as spoilage bacteria due to their ability to produce off-flavor compounds (e.g., guaiacol and halophenols) that adversely affect the taste and aroma of beverages. In our study, Alicyclobacillus species were isolated from Polish orchard soils and fruits and were subjected to 16S rDNA sequencing. The results of the analysis showed that the isolated strains belonged to A. acidoterrestris and Alicyclobacillus fastidiosus species. All the three isolated strains of A. fastidiosus (f1, f2, f3) exhibited similar morphological and biochemical properties as the strain described in the literature. However, these isolated strains were able to produce guaiacol at temperatures of 20°C, 25°C, and 45°C. Thus, the strains of A. fastidiosus discovered in the present study can be included in the group of spoilage species as they possessed the gene responsible for the production of guaiacol.

Degradation of salicylic acid to catechol in Solanaceae by SA 1-hydroxylase.

The hormone salicylic acid (SA) plays crucial roles in plant defense, stress responses, and in the regulation of plant growth and development. Whereas the biosynthetic pathways and biological functions of SA have been extensively studied, SA catabolism is less well understood. In this study, we report the identification and functional characterization of an FAD/NADH-dependent SA 1-hydroxylase from tomato (Solanum lycopersicum; SlSA1H), which catalyzes the oxidative decarboxylation of SA to catechol. Transcript levels of SlSA1H were highest in stems and its expression was correlated with the formation of the methylated catechol derivatives guaiacol and veratrole. Consistent with a role in SA catabolism, SlSA1H RNAi plants accumulated lower amounts of guaiacol and failed to produce any veratrole. Two O-methyltransferases involved in the conversion of catechol to guaiacol and guaiacol to veratrole were also functionally characterized. Subcellular localization analyses revealed the cytosolic localization of this degradation pathway. Phylogenetic analysis and functional characterization of SA1H homologs from other species indicated that this type of FAD/NADH-dependent SA 1-hydroxylases evolved recently within the Solanaceae family.

Streptomyces tunisiensis DSM 42037 mediated bioconversion of ferulic acid released from barley bran.

Streptomyces tunisiensis DSM 42037 exhibited growth capacity on a minimum medium containing 1% barley bran. This peculiar strain released 83.5% of total ferulic acid present in barley bran after 5 days of incubation and the highest amount of released ferulic acid (19 mg/L) was observed on the 3rd day of incubation. The concentrated supernatant of S. tunisiensis also released ferulic acid from the parietal arabinoxylan complex of barley bran. This strain was able to convert the free ferulic acid into 4-vinyl guaiacol (14 mg/L) and acetovanillone (12 mg/L) at molar yield of 97% and 83% respectively. The biotransformation products were successively purified by preparative thin layer and silica gel column chromatography followed by HPLC and identified by 1H nuclear magnetic resonance. Streptomyces tunisiensis DSM 42037 could have potential applications in the food, pharmaceutical and cosmetic industries thanks to its ability in biotransforming ferulic acid into 4-vinyl guaiacol and acetovanillone.

Bioproduction of 4-Vinylphenol and 4-Vinylguaiacol ß-Primeverosides Using Transformed Bamboo Cells Expressing Bacterial Phenolic Acid Decarboxylase.

Phenolic acid decarboxylase (PAD) catalyzes the decarboxylation of hydroxycinnamic acids to produce hydroxystyrenes, which serve as starting materials for the production of polymers. Bamboo (Phyllostachys nigra; Pn) cells, a suitable host for producing phenylpropanoid-derived compounds, were transformed to express PAD of Bacillus amyloliquefaciens (BaPAD). BaPAD-transformed cells accumulated several metabolites that were not detected in wild-type Pn cells or BaPAD-negative transformant. Two major metabolites were isolated from BaPAD-transformed cells, and elucidation of their chemical structures confirmed these as 4-vinylphenol ß-primeveroside (4-VPP) and 4-vinylguaiacol ß-primeveroside (4-VGP). The production titers of 4-VPP and 4-VGP reached 48 and 33 mg/L at the maximum, respectively. Feeding experiments with 4-vinylphenol (4-VP), 4-vinylguaiacol (4-VG), and their glucosides indicated that 4-VPP and 4-VGP are formed by sequential glycosylation of 4-VP and 4-VG via their corresponding glucosides. Our results demonstrate the versatility of Pn cells for producing styrene derivatives, and indicate the presence of a unique glycosylation pathway to produce 4-VPP and 4-VGP in Pn cells.

Bioconversion of ferulic acid into aroma compounds by newly isolated yeast strains of the Latin American biodiversity.

Nine yeast strains isolated from Latin American biodiversity were screened for ferulic acid (FA) consumption and conversion into aroma compounds such as vanillin, vanillic acid (VA), and 4-vinylguaiacol (VG). Selected strains (Rhodotorula mucilaginosa UFMG-CM-Y3647, UFMG-CM-Y2190, UFMG-CM-Y665) were evaluated in flask experiments to investigate the influence of the pH media on bioconversion and a two-step process was conducted to maximize the metabolites production. The effect of pH was found to be significantly important for FA bioconversion, as acidic conditions (pH < 6.0) improved VA accumulation, with highest production of 1.14 ± 0.02 and 1.25 ± 0.03 g/L shown by UFMG-CM-Y3647 and UFMG-CM-Y2190, respectively. The two-step process favored 4-VG production for most strains, being UFMG-CM-Y2190 the best producer, its cultures reaching 1.63 ± 0.09 g/L after 55 hr, showing a productivity of 29.59 ± 1.55 mg/(L·hr), as glucose affected the metabolites pool and redirected yeast metabolism. R mucilaginosa UFMG-CM-Y3647 was selected for scaled-up cultivations in a 2-L bioreactor, where pH-controlled pH 5.5 and aeration of 2.5 vvm was found to be the best condition to improve VA productivity, attaining final concentrations of 1.20 ± 0.02 g/L-1 (78% molar yield) and a productivity of 40.82 ± 0.57 mg/(L·hr).

Heat resistance and genomics of spoilage Alicyclobacillus spp. Isolated from fruit juice and fruit-based beverages.

Alicyclobacillus acidoterrestris is a spore-forming bacterium of importance to the fruit juice industry due to its remarkable heat resistance and production of guaiacol taint. Whole genome sequencing analysis reveals species demarcation corresponds to the two major genotypic groups to which A. acidoterrestris isolates belong. Heat resistance was significantly different between genotypic groups 1 and 2 with D90 values of 15.5 and 9.3 min, respectively (p < 0.01). Comparison of squalene-hopene cyclase (shc) encoding sequences reveals non-synonymous changes and the alteration of glutamine residues. Glutamine absence may link to the stability reinforcement of the enzyme structure against thermal denaturation. Genomic islands harbouring heavy metal resistance genes are found in the majority of genotypic group 1 genomes (63%) but occurs in only one genome (5%) of genotypic group 2. Distribution of the genomic islands in the genotypic groups 1 and 2 is also consistent with phylogenetic trees and ANI and dDDH values. Subsequently, we propose genotypic group 1 as a new species closely related to A. acidoterrestris that possesses enhanced heat resistance.

Characterization of alkylguaiacol-degrading cytochromes P450 for the biocatalytic valorization of lignin.

Cytochrome P450 enzymes have tremendous potential as industrial biocatalysts, including in biological lignin valorization. Here, we describe P450s that catalyze the O-demethylation of lignin-derived guaiacols with different ring substitution patterns. Bacterial strains Rhodococcus rhodochrous EP4 and Rhodococcus jostii RHA1 both utilized alkylguaiacols as sole growth substrates. Transcriptomics of EP4 grown on 4-propylguaiacol (4PG) revealed the up-regulation of agcA, encoding a CYP255A1 family P450, and the aph genes, previously shown to encode a meta-cleavage pathway responsible for 4-alkylphenol catabolism. The function of the homologous pathway in RHA1 was confirmed: Deletion mutants of agcA and aphC, encoding the meta-cleavage alkylcatechol dioxygenase, grew on guaiacol but not 4PG. By contrast, deletion mutants of gcoA and pcaL, encoding a CYP255A2 family P450 and an ortho-cleavage pathway enzyme, respectively, grew on 4-propylguaiacol but not guaiacol. CYP255A1 from EP4 catalyzed the O-demethylation of 4-alkylguaiacols to 4-alkylcatechols with the following apparent specificities (kcat/KM): propyl > ethyl > methyl > guaiacol. This order largely reflected AgcA's binding affinities for the different guaiacols and was the inverse of GcoAEP4's specificities. The biocatalytic potential of AgcA was demonstrated by the ability of EP4 to grow on lignin-derived products obtained from the reductive catalytic fractionation of corn stover, depleting alkylguaiacols and alkylphenols. By identifying related P450s with complementary specificities for lignin-relevant guaiacols, this study facilitates the design of these enzymes for biocatalytic applications. We further demonstrated that the metabolic fate of the guaiacol depends on its substitution pattern, a finding that has significant implications for engineering biocatalysts to valorize lignin.

Insight into multicopper oxidase laccase from Myrothecium verrucaria ITCC-8447: a case study using in silico and experimental analysis.

The oxidation activity of multicopper-oxidases overlaps with different substrates of laccases and bilirubin oxidases, thus in the present study an integrated approach of bioinformatics using homology modeling, docking, and experimental validation was used to confirm the type of multicopper-oxidase in Myrothecium verrucaria ITCC-8447. The result of peptide sequence of M. verrucaria ITCC-8447 enabled to predict the 3 D-structure of multicopper-oxidase. It was overlapped with the structure of laccase and root mean square deviation (RMSD) was 1.53 Å for 533 and, 171 residues. The low binding energy with azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) (-5.64) as compared to bilirubin (-4.39) suggested that M. verrucaria ITCC-8447 have laccase-like activity. The experimental analysis confirmed high activity with laccase specific substrates, phenol (18.3 U/L), ampyrone (172.4 U/L) and, ampyrone phenol coupling (50 U/L) as compared to bilirubin oxidase substrate bilirubin (16.6 U/L). In addition, lowest binding energy with ABTS (-5.64), syringaldazine SYZ (-4.83), guaiacol GCL (-4.42), and 2,6-dimethoxyphenol DMP (-4.41) confirmed the presence of laccase. Further, complete remediation of two hazardous model pollutants i.e., phenol and resorcinol (1.5 mM) after 12 h of incubation and low binding energy of -4.32 and, -4.85 respectively confirmed its removal by laccase. The results confirmed the presence of laccase in M. verrucaria ITCC-8447 and its effective bioremediation potential.

One-Pot Synthesis of Adipic Acid from Guaiacol in Escherichia coli.

Adipic acid is one of the most important small molecules in the modern chemical industry. However, the damaging environmental impact of the current industrial synthesis of adipic acid has necessitated the development of greener, biobased approaches to its manufacture. Herein we report the first one-pot synthesis of adipic acid from guaiacol, a lignin-derived feedstock, using genetically engineered whole-cells of Escherichia coli. The reaction is mild, efficient, requires no additional additives or reagents, and produces no byproducts. This study demonstrates how modern synthetic biology can be used to valorize abundant feedstocks into industrially relevant small molecules in living cells.

Production of Substituted Styrene Bioproducts from Lignin and Lignocellulose Using Engineered Pseudomonas putida KT2440.

Ferulic acid is a renewable chemical found in lignocellulose from grasses such as wheat straw and sugarcane. Pseudomonas putida is able to liberate and metabolize ferulic acid from plant biomass. Deletion of the hydroxycinnamoyl-CoA hydratase-lyase gene (ech) produced a strain of P. putida unable to utilize ferulic and p-coumaric acid, which is able to accumulate ferulic acid and p-coumaric acid from wheat straw or sugar cane bagasse. Further engineering of this strain saw the replacement of ech with the phenolic acid decarboxylase padC, which converts p-coumaric and ferulic acid into 4-vinylphenol and the flavor agent 4-vinylguaiacol, respectively. The engineered strain containing padC is able to generate 4-vinylguaiacol and 4-vinylphenol from media containing lignocellulose or Green Value Protobind lignin as feedstock, and does not require the addition of an exogenous inducer molecule. Biopolymerization of 4-vinylguaiacol and 4-vinylcatechol styrene products is also carried out, using Trametes versicolor laccase, to generate "biopolystyrene" materials on small scale.

Phenolic acid decarboxylase of Aspergillus luchuensis plays a crucial role in 4-vinylguaiacol production during awamori brewing.

Aspergillus luchuensis has been used to produce awamori, a distilled liquor, in Okinawa, Japan. Vanillin, derived from ferulic acid (FA) in rice grains, is one of the characteristic flavors in aged and matured awamori, known as kusu. Decarboxylation of FA leads to the production of 4-vinylguaiacol (4-VG), which is converted to vanillin by natural oxidization. However, the mechanism underlying FA conversion to 4-VG has remained unknown in awamori brewing. In our previous studies, we showed that phenolic acid decarboxylase from A. luchuensis (AlPAD) could catalyze the conversion of FA to 4-VG, and that AlPAD is functionally expressed during koji making (Maeda et al., J. Biosci. Bioeng., 126, 162-168, 2018). In this study, to understand the contribution of AlPAD to 4-VG production in awamori brewing, we created an alpad disruptant (Δalpad) and compared its 4-VG productivity to that of the wild-type strain. The amount of 4-VG in the distillate of moromi prepared with the wild-type strain showed a significant increase, proportional to the time required for koji making. In the Δalpad strain, the amount of 4-VG was very small and remained unchanged during the koji making. In an awamori brewing test using koji harvested 42-66 h after inoculation, the contribution of AlPAD to 4-VG production was in the range of 88-94 %. These results indicate that AlPAD plays a key role in 4-VG production during awamori brewing.

Efficient and long-term vanillin production from 4-vinylguaiacol using immobilized whole cells expressing Cso2 protein.

Vanillin is a well-known fragrant, flavoring compound. Previously, we established a method of coenzyme-independent vanillin production via an oxygenase from Caulobacter segnis ATCC 21756, called Cso2, that converts 4-vinylguaiacol to vanillin and formaldehyde using oxygen. In this study, we found that reactive oxygen species inhibited the catalytic activity of Cso2, and the addition of catalase increased vanillin production. Since Escherichia coli harbors catalases, we used E. coli cells expressing Cso2 to produce vanillin. Cell immobilization in calcium alginate enabled the long-term use of the E. coli cells for vanillin production. Thus, we demonstrate the possibility of using immobilized E. coli cells for both continuous and repeated batch vanillin production without any coenzymes.

Characterization of the metabolites of gigantol in rat, dog, monkey, and human hepatocytes using ultra-high-performance liquid chromatography coupled with high-resolution mass spectrometry.

RATIONALE: Gigantol (3',4-dihydroxy-3,5'-dimethoxybibenzyl) is a bibenzyl compound isolated from Dendrobii Caulis that has been widely used as a medicinal herb in China. To fully understand the mechanism of action of gigantol, it is necessary to determine its metabolic profile. METHODS: Gigantol at a concentration of 20 µM was incubated with hepatocytes (rat, dog, monkey, and human) at 37°C. After 120 min incubation, the samples were analyzed using liquid chromatography coupled with electrospray ionization tandem mass spectrometry. The structures of the metabolites were characterized by their molecular masses, product ions, and retention times. RESULTS: A total of 17 metabolites were detected and structurally identified. The metabolism involved the following pathways: (a) oxidation to form quinone-methide species and subsequently conjugation with glutathione (GSH); (b) demethylation to form demethylated gigantol, which was further conjugated with GSH; (c) hydroxylation to yield hydroxyl-gigantol followed by glucuronidation or GSH conjugation; and (d) glucuronidation to form glucuronide conjugates. Glucuronidation was the primary metabolic pathway in all tested species. CONCLUSIONS: Hydroxylation, demethylation, glucuronidation, and GSH conjugation were the major metabolic pathways of gigantol. This study provides new information on the metabolic profiles of gigantol and helps us understand the disposition of the compound.

Bioinspired design of an artificial peroxidase: introducing key residues of native peroxidases into F43Y myoglobin with a Tyr-heme cross-link.

Inspired by the structural features of native peroxidases, an artificial peroxidase was rationally designed using F43Y myoglobin with a Tyr-heme cross-link by further introduction of key residues, including both a distal Arg and a Trp close to the heme group, which exhibits an enhanced peroxidase activity similar to the most efficient native horseradish peroxidase. This study provides a simple approach for design of artificial heme enzymes by the combination of catalytic elements of native enzymes with the post-translational modifications of heme proteins.

Tetrad analysis of sake yeast and identification of an RFLP marker for the absence of phenolic off-flavour production.

Mating is a promising breeding method for industrial yeast. Although sake yeast has a low spore-formation ability, segregants exhibiting a mating type have been isolated from sake yeast K7. Here, we constructed zygotes from a cross between those segregants and a laboratory yeast strain. Because most sake and brewing yeast strains are prototrophs, we developed a PCR-based method to confirm that mating had taken place based on genome sequencing data and differences in nucleotide sequences between the two parental strains. The mated strain, termed S. cerevisiae MITOY123, showed restored spore-formation ability, unlike most sake and brewing yeast strains. By using the mated yeast strain MITOY123, it was possible to carry out tetrad analysis for the trait of the absence of off-flavour due to phenolic products such as 4-vinylguiacol (4-VG) in sake yeast K7. This tetrad analysis indicated that a single genetic region around the gene PAD1 is responsible for the absence of phenolic off-flavour in sake yeast K7. In order to aid the breeding of sake and brewing yeast strains by mating, we also identified a restriction fragment length polymorphism (RFLP) marker for the absence of phenolic off-flavour production in strains derived from sake yeast K7. Collectively, our data show that it is possible to breed new sake and brewing yeast strains by mating and to test for the absence of phenolic off-flavour production in resultant strains easily by RFLP analysis.