Characterization and isolation of mutants producing increased amounts of isoamyl acetate derived from hygromycin B-resistant sake yeast

Hygromycin B is an aminoglycoside antibiotic that inhibits protein synthesis in prokaryotes and eukaryotes. Twenty-four hygromycin B-resistants mutants were isolated from sake yeast, and were divided into three different degrees of strength according to hygromycin B resistance. Three of four hygromycin B strongly resistant mutants produced increased amounts of isoamyl acetate in sake brewing test, although isoamyl alcohol levels remained unchanged. Many hygromycin B-resistants mutants showed higher E/A ratios than K-701 in culture with koji extract medium. Strain HMR-18 produced the largest amount of isoamyl acetate, and its alcohol acetyltransferase (AATFase) activity was 1.3-fold that of K-701. DNA microarray analysis showed that many genes overexpressed in HMR-18 were involved in stress responses (heat shock, low pH, and so on) but HMR-18 showed thermo- and acid-sensitivity. It was strongly resistant to hygromycin B and another aminoglycoside antibiotic, G418.     

Histone deacetylases in sake yeast affect fermentation characteristics

ABSTRACT

Yeast histone deacetylases (HDAC) affect the production of alcoholic beverages. In this study, we evaluated the sake fermentation characteristics when using HDAC gene-disrupted yeast strain Kyokai No. 701. Flavor components of the sake product were significantly changed. RPD3 or HDA1 disruption increased twofold the amount of isoamyl acetate, and isoamyl alcohol levels also increased in the rpd3Δ strain. To determine the contribution of Rpd3L and Rpd3S complexes to sake characteristics, a gene responsible for Rpd3L and/or Rpd3S formation was also disrupted. Disruption of DEP1 or SDS3 that is an essential component of Rpd3L led to increased isoamyl alcohol production similar to that of the rpd3Δ strain, but the efficiency of isoamyl alcohol esterification was not affected. In addition, Rpd3 and Hda1 may regulate the responsiveness to oxygen in isoamyl acetate production. We conclude that HDAC genes regulate the production of flavor components during sake fermentation.

Abbreviations: HDAC: Histone deacetylase; HAT: histone acetyltransferase; K701: sake yeast Kyokai No. 701; PCR: polymerase chain reaction; HPLC: high performance liquid chromatography; E/A: Ester/Alcohol; BCAA: branched chain-amino acid; Atf: alcohol acetyltransferase.     

Expression Levels of the Yeast Alcohol Acetyltransferase Genes ATF1, Lg-ATF1, and ATF2 Control the Formation of a Broad Range of Volatile Esters

Volatile aroma-active esters are responsible for the fruity character of fermented alcoholic beverages such as beer and wine. Esters are produced by fermenting yeast cells in an enzyme-catalyzed intracellular reaction. In order to investigate and compare the roles of the known Saccharomyces cerevisiae alcohol acetyltransferases, Atf1p, Atf2p and Lg-Atf1p, in volatile ester production, the respective genes were either deleted or overexpressed in a laboratory strain and a commercial brewing strain. Subsequently, the ester formation of the transformants was monitored by headspace gas chromatography and gas chromatography combined with mass spectroscopy (GC-MS). Analysis of the fermentation products confirmed that the expression levels of ATF1 and ATF2 greatly affect the production of ethyl acetate and isoamyl acetate. GC-MS analysis revealed that Atf1p and Atf2p are also responsible for the formation of a broad range of less volatile esters, such as propyl acetate, isobutyl acetate, pentyl acetate, hexyl acetate, heptyl acetate, octyl acetate, and phenyl ethyl acetate. With respect to the esters analyzed in this study, Atf2p seemed to play only a minor role compared to Atf1p. The atf1Δ atf2Δ double deletion strain did not form any isoamyl acetate, showing that together, Atf1p and Atf2p are responsible for the total cellular isoamyl alcohol acetyltransferase activity. However, the double deletion strain still produced considerable amounts of certain other esters, such as ethyl acetate (50% of the wild-type strain), propyl acetate (50%), and isobutyl acetate (40%), which provides evidence for the existence of additional, as-yet-unknown ester synthases in the yeast proteome. Interestingly, overexpression of different alleles of ATF1 and ATF2 led to different ester production rates, indicating that differences in the aroma profiles of yeast strains may be partially due to mutations in their ATF genes.     

Applicability of CoA/acetyl-CoA manipulation system to enhance isoamyl acetate production in Escherichia coli

Coenzyme A (CoA) and its thioester derivatives are important precursor molecules for many industrially useful compounds such as esters, PHBs, lycopene and polyketides. Previously, in our lab we could increase the intracellular levels of CoA and acetyl-Coenzyme A (acetyl-CoA) by overexpressing one of the upstream rate-controlling enzymes pantothenate kinase with a concomitant supplementation of the precursor pantothenic acid to the cell culture medium. In this study, we showed that the CoA/acetyl-CoA manipulation system could be used to increase the productivity of industrially useful compounds derived from acetyl-CoA. We chose the production of isoamyl acetate as a model system. Isoamyl acetate is an important flavor component of sake yeast and holds a great commercial value. Alcohol acetyl transferase (AAT) condenses isoamyl alcohol and acetyl-CoA to produce isoamyl acetate. The gene ATF2, coding for this AAT was cloned and expressed in Escherichia coli. This genetic engineered E. coli produces isoamyl acetate, an ester, from intracellular acetyl-CoA when isoamyl alcohol is added externally to the cell culture medium. In the current study, we showed that in a strain bearing ATF2 gene, an increase in intracellular CoA/acetyl-CoA by overexpressing panK leads to an increase in isoamyl acetate production. Additionally, the cofactor manipulation technique was combined with more traditional approach of competing pathway deletions to further increase isoamyl acetate production. The acetate production pathway competes with isoamyl acetate production for the common intracellular metabolite acetyl-CoA. Earlier we have shown that acetate pathway deletion (ackA-pta) increases isoamyl acetate production. The acetate production pathway was inactivated under elevated CoA/acetyl-CoA conditions, which lead to a further increase in isoamyl acetate production.     

Large-Scale Selection and Breeding To Generate Industrial Yeasts with Superior Aroma Production

The concentrations and relative ratios of various aroma compounds produced by fermenting yeast cells are essential for the sensory quality of many fermented foods, including beer, bread, wine, and sake. Since the production of these aroma-active compounds varies highly among different yeast strains, careful selection of variants with optimal aromatic profiles is of crucial importance for a high-quality end product. This study evaluates the production of different aroma-active compounds in 301 different Saccharomyces cerevisiae, Saccharomyces paradoxus, and Saccharomyces pastorianus yeast strains. Our results show that the production of key aroma compounds like isoamyl acetate and ethyl acetate varies by an order of magnitude between natural yeasts, with the concentrations of some compounds showing significant positive correlation, whereas others vary independently. Targeted hybridization of some of the best aroma-producing strains yielded 46 intraspecific hybrids, of which some show a distinct heterosis (hybrid vigor) effect and produce up to 45% more isoamyl acetate than the best parental strains while retaining their overall fermentation performance. Together, our results demonstrate the potential of large-scale outbreeding to obtain superior industrial yeasts that are directly applicable for commercial use.     

Lipase of <Emphasis Type="Italic">Aspergillus niger</Emphasis> NCIM 1207: A Potential Biocatalyst for Synthesis of Isoamyl Acetate

Commercial lipase preparations and mycelium bound lipase from Aspergillus niger NCIM 1207 were used for esterification of acetic acid with isoamyl alcohol to obtain isoamyl acetate. The esterification reaction was carried out at 30°C in n-hexane with shaking at 120 rpm. Initial reaction rates, conversion efficiency and isoamyl acetate concentration obtained using Novozyme 435 were the highest. Mycelium bound lipase of A. niger NCIM 1207 produced maximal isoamyl acetate formation at an alcohol/acid ratio of 1.6. Acetic acid at higher concentrations than required for the critical alcohol/acid ratio lower than 1.3 and higher than 1.6 resulted in decreased yields of isoamyl acetate probably owing to lowering of micro-aqueous environmental pH around the enzyme leading to inhibition of enzyme activity. Mycelium bound A. niger lipase produced 80 g/l of isoamyl acetate within 96 h even though extremely less amount of enzyme activity was used for esterification. The presence of sodium sulphate during esterification reaction at higher substrate concentration resulted in increased conversion efficiency when we used mycelium bound enzyme preparations of A. niger NCIM 1207. This could be due to removal of excess water released during esterification reaction by sodium sulphate. High ester concentration (286.5 g/l) and conversion (73.5%) were obtained within 24 h using Novozyme 435 under these conditions.     

Isolation of Auxotrophic Mutants of Diploid Industrial Yeast Strains after UV Mutagenesis

Auxotrophic mutants of the yeast Saccharomyces cerevisiae are usually isolated in haploid strains because the isolation of recessive mutations in diploids is thought to be difficult due to the presence of two sets of genes. We show here that auxotrophic mutants of diploid industrial sake yeast strains were routinely obtained by a standard mutant selection procedure following UV mutagenesis. We isolated His⁻, Met⁻, Lys⁻, Trp⁻, Leu⁻, Arg⁻, and Ura⁻ auxotrophic mutants of five sake strains, Kyokai no. 7, no. 9, no. 10, no. 701, and no. 901, by screening only 1,700 to 3,400 colonies from each treated strain. Wild-type alleles were cloned and used as markers for transformation. With HIS3 as a selectable marker, the yeast TDH3 overexpression promoter was inserted upstream of ATF1, encoding alcohol acetyltransferase, by one-step gene replacement in a his3 mutant of Kyokai no. 7. The resulting strain contained exclusively yeast DNA, making it acceptable for commercial use, and produced a larger amount of isoamyl acetate, a banana-like flavor. We argue that the generally recognized difficulty of isolating auxotrophic mutants of diploid industrial yeast strains is misleading and that genetic techniques used for haploid laboratory strains are applicable for this purpose.     

Purification and characterization of isoamyl acetate-hydrolyzing esterase encoded by the IAH1 gene of Saccharomyces cerevisiae from a recombinant Escherichia coli

The IAH1 gene of Saccharomyces cerevisiae encodes an esterase that preferentially acts on isoamyl acetate; however, the enzyme has not yet been completely purified from the yeast S. cerevisiae. We constructed the IAH1 gene expression system in Escherichia coli, and purified the IAH1 gene product (Iah1p). The amount of Iah1p produced by recombinant E. coli was more than 40% of total cellular proteins. The molecular size of Iah1p was 28 kDa by SDS-polyacrylamide gel electrophoresis. Judging from the molecular weight estimation by gel filtration of purified Iah1p, the enzyme was thought to be a homodimer. The K _m values for isoamyl acetate and isobutyl acetate were 40.3 mM and 15.3 mM, respectively. The enzyme activity was inhibited by Hg²⁺, p-chloromercuribenzoate, and diisopropylfluorophosphate. Received: 23 May 1999 / Received revision: 27 October 1999 / Accepted: 5 November 1999     

The Aspergillus parasiticus estA-Encoded Esterase Converts Versiconal Hemiacetal Acetate to Versiconal and Versiconol Acetate to Versiconol in Aflatoxin Biosynthesis

In aflatoxin biosynthesis, the pathway for the conversion of 1-hydroxyversicolorone to versiconal hemiacetal acetate (VHA) to versiconal (VHOH) is part of a metabolic grid. In the grid, the steps from VHA to VHOH and from versiconol acetate (VOAc) to versiconol (VOH) may be catalyzed by the same esterase. Several esterase activities are associated with the conversion of VHA to VHOH, but only one esterase gene (estA) is present in the complete aflatoxin gene cluster of Aspergillus parasiticus. We deleted the estA gene from A. parasiticus SRRC 2043, an O-methylsterigmatocystin (OMST)-accumulating strain. The estA-deleted mutants were pigmented and accumulated mainly VHA and versicolorin A (VA). A small amount of VOAc and other downstream aflatoxin intermediates, including VHOH, versicolorin B, and OMST, also were accumulated. In contrast, a VA-accumulating mutant, NIAH-9, accumulated VA exclusively and neither VHA nor VOAc were produced. Addition of the esterase inhibitor dichlorvos (dimethyl 2,2-dichlorovinylphosphate) to the transformation recipient strain RHN1, an estA-deleted mutant, or NIAH-9 resulted in the accumulation of only VHA and VOAc. In in vitro enzyme assays, the levels of the esterase activities catalyzing the conversion of VHA to VHOH in the cell extracts of two estA-deleted mutants were decreased to approximately 10% of that seen with RHN1. Similar decreases in the esterase activities catalyzing the conversion of VOAc to VOH were also obtained. Thus, the estA-encoded esterase catalyzes the conversion of both VHA to VHOH and VOAc to VOH during aflatoxin biosynthesis.     

Global Gene Expression Analysis of Yeast Cells during Sake Brewing

During the brewing of Japanese sake, Saccharomyces cerevisiae cells produce a high concentration of ethanol compared with other ethanol fermentation methods. We analyzed the gene expression profiles of yeast cells during sake brewing using DNA microarray analysis. This analysis revealed some characteristics of yeast gene expression during sake brewing and provided a scaffold for a molecular level understanding of the sake brewing process.     

Molecular breeding of a sake yeast with a mutated ARO4 gene which causes both resistance to o-fluoro-dl-phenylalanine and increased production of β-phenethyl alcohol

A sake yeast, Saccharomyces cerevisiae, was transformed with a YCp vector containing a mutated ARO4 gene (ARO4-OFP) derived from S. cerevisiae which encodes the 3-deoxy-d-arabino-heptulosonate-7-phosphate (DAHP) synthase released from feedback inhibition by tyrosine. The transformant showed feedback-insensitive DAHP synthase activity and simultaneously acquired both o-fluoro-dl-phenylalanine (OFP)-resistance and increased production of β-phenethyl alcohol and tyrosine. The greater production of the alcohol and its acetate, rose-like flavor components, improved the aroma and flavor of sake prepared experimentally with the transformant. The ARO4-OFP gene was also demonstrated to be available as a dominant selection marker in yeast vectors for the transformation of industrial strains of S. cerevisiae.     

Metabolic engineering of the anaerobic central metabolic pathway in Escherichia coli for the simultaneous anaerobic production of isoamyl acetate and succinic acid

An in vivo method of producing isoamyl acetate and succinate simultaneously has been developed in Escherichia coli to maximize yields of both high value compounds as well as maintain the proper redox balance between NADH and NAD⁺. Previous attempts at producing the ester isoamyl acetate anaerobically did not produce the compound in high concentrations because of competing pathways and the need for NAD⁺ regeneration. The objective of this study is to produce succinate as an example of a reduced coproduct to balance the ratio of NADH/NAD⁺ as a way of maximizing isoamyl acetate production. Because the volatility of the two compounds differs greatly, the two could be easily separated in an industrial setting. An ldhA, adhE double mutant strain (SBS110MG) served as the control strain to test the effect of an additional ackA‐pta mutation as found in SBS990MG. Both strains overexpressed the two heterologous genes pyruvate carboxylase and alcohol acetyltransferase (for ester production). The triple mutant SBS990MG was found to produce higher levels of both isoamyl acetate and succinate. At the optimal condition of 25°C, the culture produced 9.4 mM isoamyl acetate and 45.5 mM succinate. SBS990MG produced 36% more ester and over 700% more succinate than SBS110MG. In addition, this study demonstrated that a significantly higher isoamyl acetate concentration can be attained by simultaneously balancing the carbon and cofactor flow; the isoamyl acetate concentration of 9.4 mM is more than seven times higher than an earlier report of about 1.2 mM. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Extremely Stable and Versatile Carboxylesterase from a Hyperthermophilic Archaeon

We have found that the hyperthermophilic archaeon Pyrobaculum calidifontis VA1 produced a thermostable esterase. We isolated and sequenced the esterase gene (est_Pc) from strain VA1. est_Pc consisted of 939 bp, corresponding to 313 amino acid residues with a molecular mass of 34,354 Da. As est_Pc showed significant identity (30%) to mammalian hormone-sensitive lipases (HSLs), esterase of P. calidifontis (Est) could be regarded as a new member of the HSL family. Activity levels of the enzyme were comparable or higher than those of previously reported enzymes not only at high temperature (6,410 U/mg at 90°C), but also at ambient temperature (1,050 U/mg at 30°C). The enzyme displayed extremely high thermostability and was also stable after incubation with various water-miscible organic solvents at a concentration of 80%. The enzyme also exhibited activity in the presence of organic solvents. Est of P. calidifontis showed higher hydrolytic activity towards esters with short to medium chains, with p-nitrophenyl caproate (C₆) the best substrate among the p-nitrophenyl esters examined. As for the alcoholic moiety, the enzyme displayed esterase activity towards esters with both straight- and branched-chain alcohols. Most surprisingly, we found that this Est enzyme hydrolyzed the tertiary alcohol ester tert-butyl acetate, a feature very rare among previously reported lipolytic enzymes. The extreme stability against heat and organic solvents, along with its activity towards a tertiary alcohol ester, indicates a high potential for the Est of P. calidifontis in future applications.     

Discovery of an Escherichia coli Esterase with High Activity and Enantioselectivity toward 1,2-O-Isopropylideneglycerol Esters

Escherichia coli has been widely used as an expression host for the identification of desired biocatalysts through screening or selection assays. We have previously used E. coli in growth selection and screening assays for identification of Bacillus subtilis lipase variants (located in the periplasm) with improved activity and enantioselectivity toward 1,2-O-isopropylideneglycerol (IPG) esters. In the course of these studies, we discovered that E. coli itself exhibits significant cytoplasmic esterase activity toward IPG esters. In order to identify the enzyme (or enzymes) responsible for this esterase activity, we analyzed eight E. coli knockout strains, in which single esterase genes were deleted, for their ability to hydrolyze IPG butyrate. This approach led to the identification of esterase YbfF as the major E. coli enzyme responsible for the hydrolytic activity toward IPG esters. The gene coding for YbfF was cloned and overexpressed in E. coli, and the corresponding protein was purified and characterized for its biocatalytic performance. YbfF displays a high level of activity toward IPG butyrate and IPG caprylate and prefers the R-enantiomer of these substrates, producing the S-enantiomer of the IPG product with high enantiomeric excess (72 to 94% ee). The enantioselectivity of YbfF for IPG caprylate (E = 40) could be significantly enhanced when using dimethylformamide (DMF) or dimethyl sulfoxide (DMSO) as cosolvents in kinetic resolution experiments. The enzyme also shows high enantioselectivity toward 1-phenylethyl acetate (E ≥ 200), giving the chiral product (R)-1-phenylethanol with >99% ee. The high activity and enantioselectivity of YbfF make it an attractive enzyme for organic synthesis.     

Enhancement of Ethanol Fermentation in Saccharomyces cerevisiae Sake Yeast by Disrupting Mitophagy Function

Saccharomyces cerevisiae sake yeast strain Kyokai no. 7 has one of the highest fermentation rates among brewery yeasts used worldwide; therefore, it is assumed that it is not possible to enhance its fermentation rate. However, in this study, we found that fermentation by sake yeast can be enhanced by inhibiting mitophagy. We observed mitophagy in wild-type sake yeast during the brewing of Ginjo sake, but not when the mitophagy gene (ATG32) was disrupted. During sake brewing, the maximum rate of CO₂ production and final ethanol concentration generated by the atg32Δ laboratory yeast mutant were 7.50% and 2.12% higher than those of the parent strain, respectively. This mutant exhibited an improved fermentation profile when cultured under limiting nutrient concentrations such as those used during Ginjo sake brewing as well as in minimal synthetic medium. The mutant produced ethanol at a concentration that was 2.76% higher than the parent strain, which has significant implications for industrial bioethanol production. The ethanol yield of the atg32Δ mutant was increased, and its biomass yield was decreased relative to the parent sake yeast strain, indicating that the atg32Δ mutant has acquired a high fermentation capability at the cost of decreasing biomass. Because natural biomass resources often lack sufficient nutrient levels for optimal fermentation, mitophagy may serve as an important target for improving the fermentative capacity of brewery yeasts.     

Effect of alternative NAD<Superscript>+</Superscript>-regenerating pathways on the formation of primary and secondary aroma compounds in a <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> glycerol-defective mutant

Saccharomyces cerevisiae maintains a redox balance under fermentative growth conditions by re-oxidizing NADH formed during glycolysis through ethanol formation. Excess NADH stimulates the synthesis of mainly glycerol, but also of other compounds. Here, we investigated the production of primary and secondary metabolites in S. cerevisiae strains where the glycerol production pathway was inactivated through deletion of the two glycerol-3-phosphate dehydrogenases genes (GPD1/GPD2) and replaced with alternative NAD⁺-generating pathways. While these modifications decreased fermentative ability compared to the wild-type strain, all improved growth and/or fermentative ability of the gpd1Δgpd2Δ strain in self-generated anaerobic high sugar medium. The partial NAD⁺ regeneration ability of the mutants resulted in significant amounts of alternative products, but at lower yields than glycerol. Compared to the wild-type strain, pyruvate production increased in most genetically manipulated strains, whereas acetate and succinate production decreased in all strains. Malate production was similar in all strains. Isobutanol production increased substantially in all genetically manipulated strains compared to the wild-type strain, whereas only mutant strains expressing the sorbitol producing SOR1 and srlD genes showed increases in isoamyl alcohol and 2-phenyl alcohol. A marked reduction in ethyl acetate concentration was observed in the genetically manipulated strains, while isobutyric acid increased. The synthesis of some primary and secondary metabolites appears more readily influenced by the NAD⁺/NADH availability. The data provide an initial assessment of the impact of redox balance on the production of primary and secondary metabolites which play an essential role in the flavour and aroma character of beverages.     

Chromosomal Integration and Expression of Two Bacterial α-Acetolactate Decarboxylase Genes in Brewer's Yeast

A bacterial gene encoding α-acetolactate decarboxylase, isolated from Klebsiella terrigena or Enterobacter aerogenes, was expressed in brewer's yeast. The genes were expressed under either the yeast phosphoglycerokinase (PGK1) or the alcohol dehydrogenase (ADH1) promoter and were integrated by gene replacement by using cotransformation into the PGK1 or ADH1 locus, respectively, of a brewer's yeast. The expression level of the α-acetolactate decarboxylase gene of the PGK1 integrant strains was higher than that of the ADH1 integrants. Under pilot-scale brewing conditions, the α-acetolactate decarboxylase activity of the PGK1 integrant strains was sufficient to reduce the formation of diacetyl below the taste threshold value, and no lagering was needed. The brewing properties of the recombinant yeast strains were otherwise unaltered, and the quality (most importantly, the flavor) of the trial beers produced was as good as that of the control beer.