Self-cloning yeast strains containing novel FAS2 mutations produce a higher amount of ethyl caproate in Japanese sake

Point mutation of Gly1250Ser (1250S) of the yeast fatty acid synthase gene FAS2 confers cerulenin resistance. This mutation also results in a higher production of the apple-like flavor component ethyl caproate in Japanese sake. We mutated the 1250th codon by in vitro site-directed mutagenesis to encode Ala (1250A) or Cys (1250C) and examined cerulenin resistance and ethyl caproate production. The mutated FAS2 genes were inserted into a binary plasmid vector containing a drug-resistance marker and a counter-selectable marker, GALp-GIN11M86. The plasmids were integrated into the wild-type FAS2 locus of a sake yeast strain, and the loss of the plasmid sequences from the integrants was done by growth on galactose plates, which is permissive for loss of GALp-GIN11M86. These counter-selected strains contained either the wild type or the mutated FAS2 allele but not the plasmid sequences, from which FAS2 mutant strains were selected by allele-specific PCR. The FAS2-1250C mutant produced a higher amount of ethyl caproate in sake than FAS2-1250S, while FAS2-1250A produced an ethyl caproate level intermediate between FAS2-1250S and the parental Kyokai no. 7 strain. Interestingly, these mutants only showed detectable cerulenin resistance. These 'self-cloning' yeast strains should be acceptable to the public because they can improve sake quality without the presence of extraneous DNA sequences.     

Polyploid engineering by increasing mutant gene dosage in yeasts

The yeast Saccharomyces cerevisiae, widely used for ethanol production, is one of the best-understood biological systems. Diploid strains of S. cerevisiae are preferred for industrial use due to the better fermentation efficiency, in terms of vitality and endurance as compared to those of haploid strains. Whole-genome duplications is known to promote adaptive mutations in microorganisms, and allelic variations considerably contribute to the product composition in ethanol fermentation. Although fermentation can be regulated using various strains of yeast, it is quite difficult to make fine adjustment of each component in final products. In this study, we demonstrate the use of polyploids with varying gene dosage (the number of copies of a particular gene present in a genome) in the regulation of ethanol fermentation. Ethyl caproate is one of the major flavouring agents in a Japanese alcoholic beverage called sake. A point mutation in FAS2 encoding the α subunit of fatty acid synthetase induces an increase in the amount of caproic acid, a precursor of ethyl caproate. Using the FAS2 as a model, we generated and evaluated yeast strains with varying mutant gene dosage. We demonstrated the possibility to increase mutant gene dosage via loss of heterozygosity in diploid and tetraploid strains. Productivity of ethyl caproate gradually increased with mutant gene dosage among tetraploid strains. This approach can potentially be applied to a variety of yeast strain development via growth-based screening.     

Enhanced ethyl caproate production of Chinese liquor yeast by overexpressing EHT1 with deleted FAA1

The fruity odor of Chinese liquor is largely derived from ester formation. Ethyl caproate, an ethyl ester eliciting apple-like flavor, is one of the most important esters in the strong aromatic Chinese liquor (or Luzhou-flavor liquor), which is the most popular and best-selling liquor in China. In the traditional fermentation process, ethyl caproate in strong aromatic liquor is mainly produced by aroma-producing yeast, bacteria, and mold with high esterification abilities in a mud pit at later fermentation stages at the expense of both fermentation time and grains rather than by the ethanol-fermenting yeast Saccharomyces cerevisiae. To increase the production of ethyl caproate by Chinese liquor yeast (S. cerevisiae AY15) and shorten the fermentation period, we constructed a recombinant strain EY15 by overexpressing EHT1 (encoding ethanol hexanoyl transferase), in which FAA1 (encoding acyl-CoA synthetases) was deleted. In liquid fermentation of corn hydrolysate and solid fermentation of sorghum, ethyl caproate production by EY15 was remarkably increased to 2.23 and 2.83 mg/L, respectively, which were 2.97- and 2.80-fold higher than those of the parental strain AY15. Furthermore, an increase in ethyl octanoate (52 and 43 %) and ethyl decanoate (61 and 40 %) production was observed. The differences in fermentation performance between EY15 and AY15 were negligible. This study resulted in the creation of a promising recombinant yeast strain and introduced a method that can be used for the clean production of strong aromatic Chinese liquor by ester-producing S. cerevisiae without the need for a mud pit.     

Formation of biocompatible reversed micellar systems using phospholipids

Formation of reversed micellar systems using biocompatible components was revealed by a significant increase of water content in the organic phase. Soybean lecithin (SL), which is a mixture of different phospholipids, and phosphatidylcholine (PC) purified from soybean were used as the amphiphilic molecule. Fatty acid and fatty acid ethyl esters were used as the organic solvent. Reversed micelles were formed in the following combinations of (amphiphilic molecule)/(organic solvent): SL/ethyl caproate, SL/ethyl oleate, SL/ethyl linoleate, PC/ethyl caproate, and PC/oleic acid. Characterization of the micelles using small angle X-ray scattering analysis was presented. Reversed micelles formed in SL/ethyl caproate, SL/ethyl oleate, and PC/ethyl caproate systems were spherical. Their radius of gyration was about 40? when the water concentration in the organic phase was maximal. Maximal water concentrations in SL/ethyl caproate and PC/ethyl caproate reversed micellar systems decreased with increasing salt concentration in the aqueous phase. Micelle sizes also decreased with increased salt concentration. The extraction of protein cytochrome c using the reversed micellar system was demonstrated. Application of these reversed micellar systems will expand to pharmaceutical and food industries.     

Improved ethyl caproate production of Chinese liquor yeast by overexpressing fatty acid synthesis genes with <Emphasis Type="Italic">OPI1</Emphasis> deletion

During yeast fermentation, ethyl esters play a key role in the development of the flavor profiles of Chinese liquor. Ethyl caproate, an ethyl ester eliciting apple-like flavor, is the characteristic flavor of strong aromatic liquor, which is the best selling liquor in China. In the traditional fermentation process, ethyl caproate is mainly produced at the later fermentation stage by aroma-producing yeast, bacteria, and mold in a mud pit instead of Saccharomyces cerevisiae at the expense of grains and fermentation time. To improve the production of ethyl caproate by Chinese liquor yeast (S. cerevisiae) with less food consumption and shorter fermentation time, we constructed three recombinant strains, namely, α5-ACC1ΔOPI1, α5-FAS1ΔOPI1, and α5-FAS2ΔOPI1 by overexpressing acetyl-CoA carboxylase (ACC1), fatty acid synthase 1 (FAS1), and fatty acid synthase 2 (FAS2) with OPI1 (an inositol/choline-mediated negative regulatory gene) deletion, respectively. In the liquid fermentation of corn hydrolysate, the contents of ethyl caproate produced by α5-ACC1ΔOPI1, α5-FAS1ΔOPI1, and α5-FAS2ΔOPI1 increased by 0.40-, 1.75-, and 0.31-fold, correspondingly, compared with the initial strain α5. The contents of other fatty acid ethyl esters (FAEEs) (C8:0, C10:0, C12:0) also increased. In comparison, the content of FAEEs produced by α5-FAS1ΔOPI1 significantly improved. Meanwhile, the contents of acetyl-CoA and ethyl acetate were enhanced by α5-FAS1ΔOPI1. Overall, this study offers a promising platform for the development of pure yeast culture fermentation of Chinese strong aromatic liquor without the use of a mud pit.     

Fermentation of soybean oil deodorizer distillate with Candida tropicalis to concentrate phytosterols and to produce sterols-rich yeast cells

Phytosterols have been recovered from the deodorizer distillate produced in the final deodorization step of vegetable oil refining by various processes. The deodorizer distillate contains mainly free fatty acids (FFAs), phytosterols, and tocopherols. The presence of FFAs hinders recovery of phytosterols. In this study, fermentation of soybean oil deodorizer distillate (SODD) with Candida tropicalis 1253 was carried out. FFAs were utilized as carbon source and converted into cellular components as the yeast cells grew. Phytosterols concentration in SODD increased from 15.2 to 28.43 % after fermentation. No significant loss of phytosterols was observed during the process. Microbial fermentation of SODD is a potential approach to concentrate phytosterols before the recovery of phytosterols from SODD. During SODD fermentation, sterols-rich yeast cells were produced and the content of total sterols was as high as 6.96 %, but its major sterol was not ergosterol, which is the major sterol encountered in Saccharomyces cerevisiae. Except ergosterol, other sterols synthesized in the cells need to be identified.     

Effect of rosuvastatin on the concentration of each fatty acid in the fraction of free fatty acids and total lipids in human plasma: The role of cholesterol homeostasis

Each fatty acid (FA) or class of FAs has a different behavior in the pathologies of atherosclerosis. The aim of this study was to investigate changes in the concentration of each fatty acid in the fraction of free fatty acids (FFAs) and total lipids in human plasma after short-term therapy with rosuvastatin as a cholesterol-lowering statin drug. Six hypercholesterolemic men on a habitual diet were studied in a randomized, double-blind, and crossover process. They received 20 mg rosuvastatin or placebo in random order, each for 4 weeks and after 2 weeks of washout period, they received another medication (placebo or rosuvastatin) for another period of 4 weeks. Rosuvastatin treatment significantly decreased the absolute concentrations of saturated and monounsaturated FAs in the total FAs as well as in FFAs. Long chain polyunsaturated fatty acids with 20 and 22 carbon atoms in the molecule had no significant change in the fraction of FFAs. Rosuvastatin is directly involved in cholesterol biosynthesis and indirectly through cholesterol homeostasis in the biosynthesis of other plasma lipids.In conclusion, our findings show that rosuvastatin treatment leads to significant changes in the concentration of each fatty acid, except for long-chain polyunsaturated fatty acids in FFAs. Our observations indicate that cholesterol homeostasis through its regulatory mechanisms appears to be the main cause of changes in the concentration of each plasma fatty acid during rosuvastatin treatment. These changes can be a source of beneficial consequences, in addition to lowering low-density lipoprotein cholesterol in cardiovascular diseases.     

Changes in ester compounds and higher alcohols of awamori during aging

The total contents of ethyl acetate, ethyl caproate, ethyl caprylate, isoamyl caproate, ethyl caprate, isoamyl caprylate, ethyl laurate, isoamyl caprate, ethyl myristate, ethyl palmitate, ethyl stearate, ethyl oleate, ethyl linoleate and β-phenethyl acetate in awamori ranged from 70.67 to 569.72 mg per l a t 100 per cent alcohol. The ratio of ethyl acetate content to the total content of 14 ester compounds (ethyl acetate/total esters) ranged from 0.59 to 0.86, and that of ethyl caprylate to the total esters was from 0.98 ×10⁻² to 8.26 × 10⁻². Ethyl acetate was the main component of ester compounds followed in descending order by ethyl caprate, ethyl palmitate and ethyl caprylate. Of the higher alcohols, awamori contained only β-phenethyl alcohol in significant quantity.On aging in kame 13 of 16 ester compounds tended to decrease distinctly and the remaining 3 components to show no distinct change, while 3 of 5 higher alcohols tended to increase distinctly. On aging in non-porous containers, however, 3 of 16 ester compounds decreased distinctly, and 3 of 5 higher alcohols increased distinctly. In the process of aging, esters underwent hydrolysis in kame but not in non-porous containers.     

Yeast-immobilized SPV device for koji quality control in sake brewing process

The malted rice, koji, is an indispensable material for the brewing of sake. It saccharifies rice starch and supplies vitamins for the yeast in sake brewing. Since the quality of sake depends strongly on the quality of koji, quality control of koji is very important in the brewing. There are some methods to measure the activity of enzymes and the quantity of vitamins with the quality of koji. None of these methods, however, directly relate to the yeast metabolism. We constructed a sensor system to monitor the yeast metabolism in sake brewing by use of immobilized Saccharomyces cerevisiae and a Surface PhotoVoltage device (SPV). In this system, S. cerevisiae K701 and K9, designed for use in sake brewing by the Brewing Society of Japan, were employed as immobilized microbe. The pH change due to the production of organic acids in sake brewing is measured using the SPV. A linear relationship was observed between decrease in the photocurrent (the metabolism response) and the concentration to less than 60 mM of glucose (r=0.990). Then we measured the koji extract and observed the difference of response between K701 and K9 which corresponded to the productivity of acidic substances by batch test.     

High-performance liquid chromatography analysis of naturally occurring D-amino acids in sake

We measured all of the D- and L-amino acids in 141 bottles of sakes using HPLC. We used two precolumn derivatization methods of amino acid enantiomer detection with o-phthalaldehyde and N-acetyl-L-cysteine, as well as (+)-1-(9-fluorenyl)ethyl chloroformate/1-aminoadamantane and one postcolumn derivatization method with o-phthalaldehyde and N-acetyl-L-cysteine. We found that the sakes contained the D-amino acids forms of Ala, Asn, Asp, Arg, Glu, Gln, His, Ile, Leu, Lys, Ser, Tyr, Val, Phe, and Pro. We were not able to detect D-Met, D-Thr D-Trp in any of the sakes analyzed. The most abundant D-Ala, D-Asp, and D-Glu ranged from 66.9 to 524.3 μM corresponding to relative 34.4, 12.0, and 14.6% D-enantiomer. The basic parameters that generally determine the taste of sake such as the sake meter value (SMV; "Nihonshudo"), acidity ("Sando"), amino acid value ("Aminosando"), alcohol content by volume, and rice species of raw material show no significant relationship to the D-amino acid content of sake. The brewing water ("Shikomimizu") and brewing process had effects on the D-amino acid content of the sakes: the D-amino acid contents of the sakes brewed with deep-sea water "Kaiyoushinosousui", "Kimoto yeast starter", "Yamahaimoto", and the long aging process "Choukijukusei" are high compared with those of other sakes analyzed. Additionally, the D-amino acid content of sakes that were brewed with the adenine auxotroph of sake yeast ("Sekishoku seishu kobo", Saccharomyces cerevisiae) without pasteurization ("Hiire") increased after storage at 25 °C for three months.     

Reassessment of Brain Free Fatty Acid Liberation During Global Ischemia and Its Attenuation by Barbiturate Anesthesia

Abstract: We previously reported that whole-brain free fatty acids (FFA) rose almost linearly for up to 1 h after decapitation of unanesthetized rats and was significantly attenuated by pentobarbital anesthesia. However, our values for total FFA and arachidonic, stearic, oleic, and palmitic acids were severalfold higher than those obtained by previous investigators. Based upon the suggestion that this may be due to FFAs released from di- and triglycerides in the quantitation of FFAs, we have now analyzed and improved our procedures for TLC separation of FFA and reassessed the accumulation of FFA in whole brain during decapitation ischemia in unanesthetized and pentobarbital-anesthetized rats. FFA levels in whole brain after 0.5 min of ischemia were one-half to one- fourth the levels previously reported after 1 min of ischemia. The rise in FFA between 0.5 and 60 min of ischemia was 9-fold for total FFA, and between 7 and 12-fold for each of the FFAs quantitated. Pentobarbital significantly attenuated the rise of all FFAs with, however, greater effects on oleic and palmitic acids than previously reported.     

Effect of increased yeast alcohol acetyltransferase activity on flavor profiles of wine and distillates

The distinctive flavor of wine, brandy, and other grape-derived alcoholic beverages is affected by many compounds, including esters produced during alcoholic fermentation. The characteristic fruity odors of the fermentation bouquet are primarily due to a mixture of hexyl acetate, ethyl caproate (apple-like aroma), iso-amyl acetate (banana-like aroma), ethyl caprylate (apple-like aroma), and 2-phenylethyl acetate (fruity, flowery flavor with a honey note). The objective of this study was to investigate the feasibility of improving the aroma of wine and distillates by overexpressing one of the endogenous yeast genes that controls acetate ester production during fermentation. The synthesis of acetate esters by the wine yeast Saccharomyces cerevisiae during fermentation is ascribed to at least three acetyltransferase activities, namely, alcohol acetyltransferase (AAT), ethanol acetyltransferase, and iso-amyl AAT. To investigate the effect of increased AAT activity on the sensory quality of Chenin blanc wines and distillates from Colombar base wines, we have overexpressed the alcohol acetyltransferase gene (ATF1) of S. cerevisiae. The ATF1 gene, located on chromosome XV, was cloned from a widely used commercial wine yeast strain of S. cerevisiae, VIN13, and placed under the control of the constitutive yeast phosphoglycerate kinase gene (PGK1) promoter and terminator. Chromoblot analysis confirmed the integration of the modified copy of ATF1 into the genome of three commercial wine yeast strains (VIN7, VIN13, and WE228). Northern blot analysis indicated constitutive expression of ATF1 at high levels in these yeast transformants. The levels of ethyl acetate, iso-amyl acetate, and 2-phenylethyl acetate increased 3- to 10-fold, 3.8- to 12-fold, and 2- to 10-fold, respectively, depending on the fermentation temperature, cultivar, and yeast strain used. The concentrations of ethyl caprate, ethyl caprylate, and hexyl acetate only showed minor changes, whereas the acetic acid concentration decreased by more than half. These changes in the wine and distillate composition had a pronounced effect on the solvent or chemical aroma (associated with ethyl acetate and iso-amyl acetate) and the herbaceous and heads-associated aromas of the final distillate and the solvent or chemical and fruity or flowery characters of the Chenin blanc wines. This study establishes the concept that the overexpression of acetyltransferase genes such as ATF1 could profoundly affect the flavor profiles of wines and distillates deficient in aroma, thereby paving the way for the production of products maintaining a fruitier character for longer periods after bottling.     

Genetic engineering of a sake yeast producing no urea by successive disruption of arginase gene

Urea is reported to be a main precursor of ethyl carbamate (ECA), which is suspected to be a carcinogen, in wine and sake. In order to minimize production of urea, arginase-deficient mutants (delta car1/delta car1) were constructed from a diploid sake yeast, Kyokai no. 9, by successive disruption of the two copies of the CAR1 gene. First, the yeast strain was transformed with plasmid pCAT2 (delta car1 SMR1), and strains heterozygous for CAR1 gene were isolated on sulfometuron methyl plates. Successively, the other CAR1 gene was disrupted by transformation with plasmid pCAT1 (delta car1 G418r) and the resulting car1 mutants were isolated on a G418 plate. Arginase assay of the total cell lysate of the mutants showed that 70% of transformants isolated on G418 plates had no detectable enzyme activity, possibly as a result of the disruption of the two copies of the CAR1 gene. Further genomic Southern analysis confirmed this result. We could brew sake containing no urea with the delta car1/delta car1 homozygous mutant. It is of additional interest that no ECA was detected in the resulting sake, even after storage for 5 months at 30 degrees C. This molecular biological study suggests that ECA in sake originates mainly from urea that is produced by the arginase.     

Genetic engineering of a sake yeast producing no urea by successive disruption of arginase gene.

Urea is reported to be a main precursor of ethyl carbamate (ECA), which is suspected to be a carcinogen, in wine and sake. In order to minimize production of urea, arginase-deficient mutants (delta car1/delta car1) were constructed from a diploid sake yeast, Kyokai no. 9, by successive disruption of the two copies of the CAR1 gene. First, the yeast strain was transformed with plasmid pCAT2 (delta car1 SMR1), and strains heterozygous for CAR1 gene were isolated on sulfometuron methyl plates. Successively, the other CAR1 gene was disrupted by transformation with plasmid pCAT1 (delta car1 G418r) and the resulting car1 mutants were isolated on a G418 plate. Arginase assay of the total cell lysate of the mutants showed that 70% of transformants isolated on G418 plates had no detectable enzyme activity, possibly as a result of the disruption of the two copies of the CAR1 gene. Further genomic Southern analysis confirmed this result. We could brew sake containing no urea with the delta car1/delta car1 homozygous mutant. It is of additional interest that no ECA was detected in the resulting sake, even after storage for 5 months at 30 degrees C. This molecular biological study suggests that ECA in sake originates mainly from urea that is produced by the arginase.     

New method for GC/FID and GC-C-IRMS analysis of plasma free fatty acid concentration and isotopic enrichment

A simple, direct and accurate method for the determination of concentration and enrichment of free fatty acids (FFAs) in human plasma was developed. The validation and comparison to a conventional method are reported. Three amide derivatives, dimethyl, diethyl and pyrrolidide, were investigated in order to achieve optimal resolution of the individual fatty acids. This method involves the use of dimethylamine/Deoxo-Fluor to derivatize plasma free fatty acids to their dimethylamides. This derivatization method is very mild and efficient, and is selective only towards FFAs so that no separation from a total lipid extract is required. The direct method gave lower concentrations for palmitic acid and stearic acid and increased concentrations for oleic acid and linoleic acid in plasma as compared to methyl ester derivative after thin-layer chromatography. The [(13)C]palmitate isotope enrichment measured using direct method was significantly higher than that observed with the BF(3)/MeOH-TLC method. The present method provided accurate and precise measures of concentration as well as enrichment when analyzed with gas chromatography combustion-isotope ratio-mass spectrometry.     

Genome editing to generate nonfoam-forming sake yeast strains

ABSTRACT

Mutations frequently occur during breeding of sake yeasts and result in unexpected phenotypes. Here, genome editing tools were applied to develop an ideal nonfoam-forming sake yeast strain, K7GE01, which had homozygous awa1?/awa1? deletion alleles that were responsible for nonfoam formation and few off-target mutations. High-dimensional morphological phenotyping revealed no detectable morphological differences between the genome-edited strain and its parent, while the canonical nonfoam-forming strain, K701, showed obvious morphological changes. Small-scale fermentation tests also showed differences between components of sake produced by K7GE01 and K701. The K7GE01 strain produced sake with significant differences in the concentrations of ethyl acetate, malic acid, lactic acid, and acetic acid, while K701 produced sake with more differences. Our results indicated genuine phenotypes of awa1?/awa1? in sake yeast isolates and showed the usefulness of genome editing tools for sake yeast breeding.     

浓香型白酒糟醅及窖泥产香功能菌的研究进展

浓香型白酒主要香气物为己酸乙酯、乳酸乙酯、乙酸乙酯和丁酸乙酯四大酯类,白酒微生物发酵过程中酯类合成主要是酸与醇在酯化酶的作用下产生,因此产香功能菌包括香气及其前体物产生菌和酯化酶产生菌.本文综述了浓香型白酒糟醅及窖泥主要产香功能菌的来源、组成、鉴定方法、功能特性、相互关系及其在浓香型白酒生产过程中的应用等方面的研究进展,旨为浓香型白酒重要微生物的研究提供思路,为其发酵过程控制提供理论依据.     

Taste-guided fractionation and instrumental analysis of hydrophobic compounds in sake

The taste-active hydrophobic compounds in a charcoal-untreated sake sample were subjected to a taste dilution analysis (TDA). All of the high-TDA factor fractions showed a bitter or astringent taste in common, but their taste characters were different. The taste-active compounds of the high-TDA factor fractions were purified by taste-guided fractionation, using RP-HPLC and an instrumental analysis. From each of the seven fractions, ferulic acid, ethyl ferulate, tryptophol, three previously reported bitter-tasting peptides, and two novel ethyl esters of the peptides of 10 amino acid residues were identified. All the identified compounds had a similar taste character to that of the TDA fractions analyzed. Ethyl ferulate and the ethyl ester of the peptides showed a moderately bitter taste. The concentration of the identified compounds in seven jyunmai-type sake samples was determined. This concentration was decreased dose dependently by a charcoal treatment which is commonly applied in the final step of sake manufacture, notably with the compounds of high hydrophobicity.