scholarly journals Identification of Novel Alleles Conferring Superior Production of Rose Flavor Phenylethyl Acetate Using Polygenic Analysis in Yeast

mBio ◽  
2017 ◽  
Vol 8 (6) ◽  
Author(s):  
Bruna Trindade de Carvalho ◽  
Sylvester Holt ◽  
Ben Souffriau ◽  
Rogelio Lopes Brandão ◽  
Maria R. Foulquié-Moreno ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Fatty Acid ◽  
Genetic Mapping ◽  
Quantitative Trait ◽  
Mutant Allele ◽  
Fatty Acid Synthetase ◽  
Alcoholic Beverages ◽  
Industrial Yeast ◽  
Flavor Compound ◽  
Yeast Strains

ABSTRACT Flavor compound metabolism is one of the last areas in metabolism where multiple genes encoding biosynthetic enzymes are still unknown. A major challenge is the involvement of side activities of enzymes having their main function in other areas of metabolism. We have applied pooled-segregant whole-genome sequence analysis to identify novel Saccharomyces cerevisiae genes affecting production of phenylethyl acetate (2-PEAc). This is a desirable flavor compound of major importance in alcoholic beverages imparting rose- and honey-like aromas, with production of high 2-PEAc levels considered a superior trait. Four quantitative trait loci (QTLs) responsible for high 2-PEAc production were identified, with two loci each showing linkage to the genomes of the BTC.1D and ER18 parents. The first two loci were investigated further. The causative genes were identified by reciprocal allele swapping into both parents using clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9. The superior allele of the first major causative gene, FAS2, was dominant and contained two unique single nucleotide polymorphisms (SNPs) responsible for high 2-PEAc production that were not present in other sequenced yeast strains. FAS2 encodes the alpha subunit of the fatty acid synthetase complex. Surprisingly, the second causative gene was a mutant allele of TOR1, a gene involved in nitrogen regulation. Exchange of both superior alleles in the ER18 parent strain increased 2-PEAc production 70%, nearly to the same level as in the best superior segregant. Our results show that polygenic analysis combined with CRISPR/Cas9-mediated allele exchange is a powerful tool for identification of genes encoding missing metabolic enzymes and for development of industrial yeast strains generating novel flavor profiles in alcoholic beverages. IMPORTANCE Multiple reactions in flavor metabolism appear to be catalyzed by side activities of other enzymes that have been difficult to identify. We have applied genetic mapping of quantitative trait loci in the yeast Saccharomyces cerevisiae to identify mutant alleles of genes determining the production of phenylethyl acetate, an important flavor compound imparting rose- and honey-like aromas to alcoholic beverages. We identified a unique, dominant allele of FAS2 that supports high production of phenylethyl acetate. FAS2 encodes a subunit of the fatty acid synthetase complex and apparently exerts an important side activity on one or more alternative substrates in flavor compound synthesis. The second mutant allele contained a nonsense mutation in TOR1, a gene involved in nitrogen regulation of growth. Together the two alleles strongly increased the level of phenylethyl acetate. Our work highlights the potential of genetic mapping of quantitative phenotypic traits to identify novel enzymes and regulatory components in yeast metabolism, including regular metabolic enzymes with unknown side activities responsible for biosynthesis of specific flavor compounds. The superior alleles identified can be used to develop industrial yeast strains generating novel flavor profiles in alcoholic beverages. IMPORTANCE Multiple reactions in flavor metabolism appear to be catalyzed by side activities of other enzymes that have been difficult to identify. We have applied genetic mapping of quantitative trait loci in the yeast Saccharomyces cerevisiae to identify mutant alleles of genes determining the production of phenylethyl acetate, an important flavor compound imparting rose- and honey-like aromas to alcoholic beverages. We identified a unique, dominant allele of FAS2 that supports high production of phenylethyl acetate. FAS2 encodes a subunit of the fatty acid synthetase complex and apparently exerts an important side activity on one or more alternative substrates in flavor compound synthesis. The second mutant allele contained a nonsense mutation in TOR1, a gene involved in nitrogen regulation of growth. Together the two alleles strongly increased the level of phenylethyl acetate. Our work highlights the potential of genetic mapping of quantitative phenotypic traits to identify novel enzymes and regulatory components in yeast metabolism, including regular metabolic enzymes with unknown side activities responsible for biosynthesis of specific flavor compounds. The superior alleles identified can be used to develop industrial yeast strains generating novel flavor profiles in alcoholic beverages.

scholarly journals Polygenic Analysis in Absence of Major EffectorATF1Unveils Novel Components in Yeast Flavor Ester Biosynthesis

mBio ◽  
2018 ◽  
Vol 9 (4) ◽  
Author(s):  
Sylvester Holt ◽  
Bruna Trindade de Carvalho ◽  
María R. Foulquié-Moreno ◽  
Johan M. Thevelein
Keyword(s):  
Ethyl Acetate ◽  
Alcoholic Beverages ◽  
Industrial Yeast ◽  
Acetyl Coa ◽  
Flavor Compound ◽  
Effector Genes ◽  
Yeast Strains ◽  
Coa Transferase ◽  
Ester Biosynthesis ◽  
Polygenic Analysis

ABSTRACTFlavor production in yeast fermentation is of paramount importance for industrial production of alcoholic beverages. Although major enzymes of flavor compound biosynthesis have been identified, few specific mutations responsible for strain diversity in flavor production are known. TheATF1-encoded alcohol acetyl coenzyme A (acetyl-CoA) transferase (AATase) is responsible for the majority of acetate ester biosynthesis, but other components affecting strain diversity remain unknown. We have performed parallel polygenic analysis of low production of ethyl acetate, a compound with an undesirable solvent-like off-flavor, in strains with and without deletion ofATF1. We identified two unique causative mutations,eat1K179fsandsnf8E148*, not present in any other sequenced yeast strain and responsible for most ethyl acetate produced in absence ofATF1.EAT1encodes a putative mitochondrial ethanol acetyl-CoA transferase (EATase) and its overexpression, but not that ofEAT1K179fs, and strongly increases ethyl acetate without affecting other flavor acetate esters. Unexpectedly, a higher level of acetate esters (including ethyl acetate) was produced wheneat1K179fswas present together withATF1in the same strain, suggesting that the Eat1 and Atf1 enzymes are intertwined. On the other hand, introduction ofsnf8E148* lowered ethyl acetate levels also in the presence ofATF1, and it affected other aroma compounds, growth, and fermentation as well. Engineering ofsnf8E148* in three industrial yeast strains (for production of wine, saké, and ale beer) and fermentation in an application-relevant medium showed a high but strain-dependent potential for flavor enhancement. Our work has identifiedEAT1andSNF8as new genetic elements determining ethyl acetate production diversity in yeast strains.IMPORTANCEBasic research with laboratory strains of the yeastSaccharomyces cerevisiaehas identified the structural genes of most metabolic enzymes, as well as genes encoding major regulators of metabolism. On the other hand, more recent work on polygenic analysis of yeast biodiversity in natural and industrial yeast strains is revealing novel components of yeast metabolism. A major example is the metabolism of flavor compounds, a particularly important property of industrial yeast strains used for the production of alcoholic beverages. In this work, we have performed polygenic analysis of production of ethyl acetate, an important off-flavor compound in beer and other alcoholic beverages. To increase the chances of identifying novel components, we have used in parallel a wild-type strain and a strain with a deletion ofATF1encoding the main enzyme of acetate ester biosynthesis. This revealed a new structural gene,EAT1, encoding a putative mitochondrial enzyme, which was recently identified as an ethanol acetyl-CoA transferase in another yeast species. We also identified a novel regulatory gene,SNF8, which has not previously been linked to flavor production. Our results show that polygenic analysis of metabolic traits in the absence of major effector genes can reveal novel structural and regulatory genes. The mutant alleles identified can be used to affect the flavor profile in industrial yeast strains for production of alcoholic beverages in more subtle ways than by deletion or overexpression of the already known major effector genes and without significantly altering other industrially important traits. The effect of the novel variants was dependent on the genetic background, with a highly desirable outcome in the flavor profile of an ale brewing yeast.

scholarly journals Rapid Colorimetric Detection of Genome Evolution in SCRaMbLEd Synthetic Saccharomyces cerevisiae Strains

2020 ◽  
Vol 8 (12) ◽  
pp. 1914
Author(s):  
Elizabeth L. I. Wightman ◽  
Heinrich Kroukamp ◽  
Isak S. Pretorius ◽  
Ian T. Paulsen ◽  
Helena K. M. Nevalainen
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Genome Evolution ◽  
Colorimetric Detection ◽  
Cre Recombinase ◽  
Control Measures ◽  
Genomic Rearrangements ◽  
Industrial Yeast ◽  
Poor Growth ◽  
Yeast Strains ◽  
Industrial Strains

Genome-scale engineering and custom synthetic genomes are reshaping the next generation of industrial yeast strains. The Cre-recombinase-mediated chromosomal rearrangement mechanism of designer synthetic Saccharomyces cerevisiae chromosomes, known as SCRaMbLE, is a powerful tool which allows rapid genome evolution upon command. This system is able to generate millions of novel genomes with potential valuable phenotypes, but the excessive loss of essential genes often results in poor growth or even the death of cells with useful phenotypes. In this study we expanded the versatility of SCRaMbLE to industrial strains, and evaluated different control measures to optimize genomic rearrangement, whilst limiting cell death. To achieve this, we have developed RED (rapid evolution detection), a simple colorimetric plate-assay procedure to rapidly quantify the degree of genomic rearrangements within a post-SCRaMbLE yeast population. RED-enabled semi-synthetic strains were mated with the haploid progeny of industrial yeast strains to produce stress-tolerant heterozygous diploid strains. Analysis of these heterozygous strains with the RED-assay, genome sequencing and custom bioinformatics scripts demonstrated a correlation between RED-assay frequencies and physical genomic rearrangements. Here we show that RED is a fast and effective method to evaluate the optimal SCRaMbLE induction times of different Cre-recombinase expression systems for the development of industrial strains.

Fatty-Acid Elongation in a Mutant of Saccharomyces cerevisiae Deficient in Fatty-Acid Synthetase

1972 ◽  
Vol 24 (3) ◽  
pp. 407-415 ◽  
Author(s):  
Thomas W. Orme ◽  
Feodor Lynen ◽  
Jean Mcintyre ◽  
Lothar Kuhn ◽  
Eckhart Schweizer
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Fatty Acid ◽  
Fatty Acid Synthetase ◽  
Fatty Acid Elongation

scholarly journals Saccharomyces cerevisiae contains four fatty acid activation (FAA) genes: an assessment of their role in regulating protein N-myristoylation and cellular lipid metabolism.

1994 ◽  
Vol 127 (3) ◽  
pp. 751-762 ◽  
Author(s):  
D R Johnson ◽  
L J Knoll ◽  
D E Levin ◽  
J I Gordon
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Fatty Acids ◽  
Fatty Acid ◽  
Carbon Source ◽  
Vegetative Growth ◽  
Fatty Acid Synthetase ◽  
Acid Activation ◽  
Beta Oxidation ◽  
Fatty Acid Activation ◽  
Long Chain

Saccharomyces cerevisiae has been used as a model for studying the regulation of protein N-myristoylation. MyristoylCoA:protein N-myristoyl-transferase (Nmt1p), is essential for vegetative growth and uses myristoylCoA as its substrate. MyristoylCoA is produced by the fatty acid synthetase (Fas) complex and by cellular acylCoA synthetases. We have recently isolated three unlinked Fatty Acid Activation (FAA) genes encoding long chain acylCoA synthetases and have now recovered a fourth by genetic complementation. When Fas is active and NMT1 cells are grown on media containing a fermentable carbon source, none of the FAA genes is required for vegetative growth. When Fas is inactivated by a specific inhibitor (cerulenin), NMT1 cells are not viable unless the media is supplemented with long chain fatty acids. Supplementation of cellular myristoylCoA pools through activation of imported myristate (C14:0) is predominantly a function of Faa1p, although Faa4p contributes to this process. Cells with nmt181p need larger pools of myristoylCoA because of the mutant enzyme's reduced affinity for this substrate. Faa1p and Faa4p are required for maintaining the viability of nmt1-181 strains even when Fas is active. Overexpression of Faa2p can rescue nmt1-181 cells due to activation of an endogenous pool of C14:0. This pool appears to be derived in part from membrane phospholipids since overexpression of Plb1p, a nonessential lysophospholipase/phospholipase B, suppresses the temperature-sensitive growth arrest and C14:0 auxotrophy produced by nmt1-181. None of the four known FAAs is exclusively responsible for targeting imported fatty acids to peroxisomal beta-oxidation pathways. Introduction of a peroxisomal assembly mutation, pas1 delta, into isogenic NMT1 and nmt1-181 strains with wild type FAA alleles revealed that when Fas is inhibited, peroxisomes contribute to myristoylCoA pools used by Nmt1p. When Fas is active, a fraction of cellular myristoylCoA is targeted to peroxisomes. A NMT1 strain with deletions of all four FAAs is still viable at 30 degrees C on media containing myristate, palmitate, or oleate as the sole carbon source--indicating that S. cerevisiae contains at least one other FAA which directs fatty acids to beta-oxidation pathways.

scholarly journals Syringomycin E Inhibition of Saccharomyces cerevisiae: Requirement for Biosynthesis of Sphingolipids with Very-Long-Chain Fatty Acids and Mannose- and Phosphoinositol-Containing Head Groups

2000 ◽  
Vol 44 (5) ◽  
pp. 1174-1180 ◽  
Author(s):  
Stephen D. Stock ◽  
Hiroko Hama ◽  
Jeffrey A. Radding ◽  
Debra A. Young ◽  
Jon Y. Takemoto
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Fatty Acids ◽  
Fatty Acid ◽  
Mutant Allele ◽  
Polar Head Group ◽  
Long Chain Fatty Acids ◽  
Syringomycin E ◽  
Yeast Genes ◽  
Long Chain ◽  
Chain Fatty Acids

ABSTRACT Syringomycin E is an antifungal cyclic lipodepsinonapeptide that inhibits the growth of Saccharomyces cerevisiae by interaction with the plasma membrane. A screen conducted to find the yeast genes necessary for its fungicidal action identified two novel syringomycin E response genes, SYR3 and SYR4. Asyr3 mutant allele was complemented by ELO2 andELO3. These genes encode enzymes that catalyze the elongation of sphingolipid very long chain fatty acids. Tetrad analysis showed that SYR3 was ELO2. Strains with deletions of SYR3/ELO2 and ELO3 were resistant to syringomycin E, and lipid analyses of both mutants revealed shortened fatty acid chains and lower levels of sphingolipids.SYR4 was identified by Tn5 inactivation of genomic library plasmids that complemented a syr4 mutant allele. SYR4 was found to be identical to IPT1, which encodes the terminal sphingolipid biosynthetic enzyme, mannosyl-diinositolphosphoryl-ceramide synthase. Deletion Δsyr4/ipt1 strains were viable, were resistant to syringomycin E, did not produce mannosyl-diinositolphosphoryl-ceramide, and accumulated mannosyl-inositolphosphoryl-ceramide. Accumulation of mannosyl-inositolphosphoryl-ceramide was not responsible for resistance since a temperature-sensitive secretory pathway mutant (sec14-3ts ) accumulated this sphingolipid and was sensitive to syringomycin E. Finally, Δcsg1/sur1 and Δcsg2 strains defective in the transfer of mannose to inositolphosphoryl-ceramide were resistant to syringomycin E. These findings show that syringomycin E growth inhibition of yeast is promoted by the production of sphingolipids with fully elongated fatty acid chains and the mannosyl and terminal phosphorylinositol moieties of the polar head group.

scholarly journals Isolation of a Saccharomyces cerevisiae long chain fatty acyl:CoA synthetase gene (FAA1) and assessment of its role in protein N-myristoylation.

1992 ◽  
Vol 117 (3) ◽  
pp. 515-529 ◽  
Author(s):  
R J Duronio ◽  
L J Knoll ◽  
J I Gordon
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Fatty Acid ◽  
De Novo ◽  
Fatty Acid Synthetase ◽  
Acid Synthesis ◽  
Single Copy ◽  
Synthetase Activity ◽  
Acid Activation ◽  
The Right ◽  
Long Chain

Regulation of myristoylCoA pools in Saccharomyces cerevisiae plays an important role in modulating the activity of myristoylCoA:protein N-myristoyltransferase (NMT), an essential enzyme with an ordered Bi Bi reaction that catalyzes the transfer of myristate from myristoylCoA to greater than or equal to 12 cellular proteins. At least two pathways are available for generating myristoylCoA: de novo synthesis by the multifunctional, multisubunit fatty acid synthetase complex (FAS) and activation of exogenous myristate by acylCoA synthetase. The FAA1 (fatty acid activation) gene has been isolated by genetic complementation of a faal mutant. This single copy gene, which maps to the right arm of chromosome XV, specifies a long chain acylCoA synthetase of 700 amino acids. Analyses of strains containing NMT1 and a faal null mutation indicated that FAA1 is not essential for vegetative growth when an active de novo pathway for fatty acid synthesis is present. The role of FAA1 in cellular lipid metabolism and protein N-myristoylation was therefore assessed in strains subjected to biochemical or genetic blockade of FAS. At 36 degrees C, FAA1 is required for the utilization of exogenous myristate by NMT and for the synthesis of several phospholipid species. This requirement is not apparent at 24 or 30 degrees C, suggesting that S. cerevisiae contains another acylCoA synthetase activity whose chain length and/or temperature optima may differ from Faalp.

scholarly journals Molecular cloning of fatty acid synthetase genes from Saccharomyces cerevisiae.

1983 ◽  
Vol 258 (19) ◽  
pp. 11648-11653 ◽  
Author(s):  
M A Kuziora ◽  
J H Chalmers ◽  
M G Douglas ◽  
R A Hitzeman ◽  
J S Mattick ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Fatty Acid ◽  
Molecular Cloning ◽  
Fatty Acid Synthetase
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