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1.
J Agric Food Chem ; 69(5): 1637-1646, 2021 Feb 10.
Artigo em Inglês | MEDLINE | ID: mdl-33502852

RESUMO

The n-propanol produced by Saccharomyces cerevisiae has a remarkable effect on the taste and flavor of Chinese Baijiu. The n-propanol metabolism-related genes were deleted to evaluate the role in the synthesis of n-propanol to ascertain the key genes and pathways for the production of n-propanol by S. cerevisiae. The results showed that CYS3, GLY1, ALD6, PDC1, ADH5, and YML082W were the key genes affecting the n-propanol metabolism in yeast. The n-propanol concentrations of α5ΔGLY1, α5ΔCYS3, and α5ΔALD6 increased by 121.75, 22.75, and 17.78%, respectively, compared with α5. The n-propanol content of α5ΔPDC1, α5ΔADH5, and α5ΔYML082W decreased by 24.98, 8.35, and 8.44%, respectively, compared with α5. The contents of intermediate metabolites were measured, and results showed that the mutual transformation of glycine and threonine in the threonine pathway and the formation of propanal from 2-ketobutyrate were the core pathways for the formation of n-propanol. Additionally, YML082W played important role in the synthesis of n-propanol by directly producing 2-ketobutyric acid through l-homoserine. This study provided valuable insights into the n-propanol synthesis in S. cerevisiae and the theoretical basis for future optimization of yeast strains in Baijiu making.

2.
Food Microbiol ; 95: 103713, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33397627

RESUMO

Higher alcohols are important flavor substance in alcoholic beverages. The content of α-amino nitrogen (α-AN) in the fermentation system affects the formation of higher alcohols by Saccharomyces cerevisiae. In this study, the effect of α-AN concentration on the higher alcohol productivity of yeast was explored, and the mechanism of this effect was investigated through metabolite and transcription sequence analyses. We screened 12 most likely genes and constructed the recombinant strain to evaluate the effect of each gene on high alcohol formation. Results showed that the AGP1, GDH1, and THR6 genes were important regulators of higher alcohol metabolism in S. cerevisiae. This study provided knowledge about the metabolic pathways of higher alcohols and gave an important reference for the breeding of S. cerevisiae with low-yield higher alcohols to deal with the fermentation system with different α-AN concentrations in the brewing industry.

3.
J Agric Food Chem ; 68(47): 13863-13870, 2020 Nov 25.
Artigo em Inglês | MEDLINE | ID: mdl-33166457

RESUMO

Ethyl lactate is an important flavor substance in baijiu, and it is also one of the common raw materials in the production of flavors and spices. In this study, we first established the ethyl lactate biosynthesis pathway in Saccharomyces cerevisiae α(L) by introducing propionyl coenzyme A transferase (Pct) and alcohol acyltransferase (AAT), and the results showed that strain α(L)-CP-Ae produced the most ethyl lactate 239.53 ± 5.45 mg/L. Subsequently, the copy number of the Pctcp gene and AeAT9 gene was increased, and the modified strain α(L)-tCP-tAe produced 346.39 ± 3.99 mg/L ethyl lactate. Finally, the porin gene (por2) and the mitochondrial pyruvate carrier gene (MPC2) were knocked to impede mitochondrial transport of pyruvate, and the final modified strain α(L)-tCP-tAeΔpor2 produced ethyl lactate 420.48 ± 6.03 mg/L.

4.
Biomed Res Int ; 2020: 6802512, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33204707

RESUMO

The higher alcohols produced by Saccharomyces cerevisiae exert remarkable influence on the taste and flavour of Chinese Baijiu. In order to study the regulation mechanism of amino acid metabolism genes on higher alcohol production, eight recombinant strains with amino acid metabolism gene deletion were constructed. The growth, fermentation performance, higher alcohol production, and expression level of genes in recombinant and original α5 strains were determined. Results displayed that the total higher alcohol concentration in α5ΔGDH1 strain decreased by 27.31% to 348.68 mg/L compared with that of α5. The total content of higher alcohols in α5ΔCAN1 and α5ΔGAT1 strains increased by 211.44% and 28.36% to 1493.96 and 615.73 mg/L, respectively, compared with that of α5. This study is the first to report that the CAN1 and GAT1 genes have great influence on the generation of higher alcohols. The results demonstrated that amino acid metabolism plays a substantial role in the metabolism of higher alcohols by S. cerevisiae. Interestingly, we also found that gene knockout downregulated the expression levels of the knocked out gene and other genes in the recombinant strain and thus affected the formation of higher alcohols by S. cerevisiae. This study provides worthy insights for comprehending the metabolic mechanism of higher alcohols in S. cerevisiae for Baijiu fermentation.

5.
Iran J Biotechnol ; 17(2): e1990, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31457054

RESUMO

Background: Enhancing the industrial yeast strains ethyl acetate yield through a precise and seamless genetic manipulation strategy without any extraneous DNA sequences is an essential requisite and significant demand. Objectives: For increasing the ethyl acetate yield of industrial brewer's yeast strain, all the ATF1 alleles were overexpressed through "self-cloning" integration strategy. Material and Methods: Escherichia coli strain DH5α was utilized for plasmid construction. ATF1 alleles were overexpressed through a precise and seamless insertion of the PGK1 promoter in industrial brewer's yeast strain S6. In addition, growth rates, ATF1 mRNA levels, AATase activity, the fermentation performance of the engineered strains, and gas chromatography (GC) analysis was conducted. Results: The two engineered strains (S6-P-12 and S6-P-30) overexpressed all ATF1 alleles but unaffected normal growth. The ATF1 mRNA levels of the S6-P-12 and S6-P-30 were all 4-fold higher than that of S6. The AATase (Alcohol acetyl transferases, encoded by ATF1 gene) activity of the two engineered strains was all 3-fold higher than that of the parent strain. In the beer fermentation at 10 ℃, the concentrations of ethyl acetate produced by the engineered strains S6-P-12 and S6-P-30 was increased to 23.98 and 24.00 mg L-1, respectively, about 20.44% and 20.54% higher than that of S6. Conclusions: These results verify that the ethyl acetate yield could be enhanced by the overexpressed of ATF1 in the polyploid industrial brewer's yeast strains via "self-cloning" integration strategy. The present study provides a reference for target gene modification in the diploid or polyploid industrial yeast strains.

6.
J Food Biochem ; 43(7): e12846, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31353733

RESUMO

Cyclic adenosine monophosphate (cAMP) plays an important role in modulating the activity of microbe cell. In this study, PKA (protein kinase A) activity was weakened through truncation of TPK2 promoter (-150 bp and -300 bp) and gene deletion of BCY1 (encodes the regulatory subunit of PKA), TPK1 and TPK3, generating strains BY9a-T2-150 and BY9a-T2-300, respectively. High-performance liquid chromatography analysis showed cAMP levels in BY9a-T2-150 and BY9a-T2-300 were increased by 5- and 18-fold, respectively, compared with that of parent strain, BY9a. The expression levels of TPK2 gene in two engineered strains were decreased by 95% and 97% compared with that of BY9a, respectively. The PKA activity reflected by heat resistance of engineered strains enhanced compared with parent strain BY9a. This study show a new method to increase the intracellular cAMP concentration in industrial yeast by fine-tuning of PKA activity, without influence in growth and fermentation properties. PRACTICAL APPLICATIONS: cAMP as the "second messenger," is essential for plant, animal, and microorganisms and human life. But its synthesis is still limited by expensive cost and time-consuming method. We constructed the industrial baker's yeast with high level of cAMP and desired to be used to produce functional food for relaxing smooth muscle, expanding blood vessels, improving liver function, and promoting nerve regeneration and as a food additive for treating hyperthyreosis and hepatopathy. The methods of two step homologous recombination and backcross operated in this study eliminate the exogenous gene in engineered strains, made it safety to be used in food production. Fine-tuning of PKA activity in engineered strains ensure produce high level of cAMP and exhibit normal growth performance in engineering strains. Therefore, this work is significant in functional foods product and has the potential to be used in practical application.


Assuntos
AMP Cíclico/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Bioengenharia , Proteínas Quinases Dependentes de AMP Cíclico/genética , Deleção de Genes , Regiões Promotoras Genéticas , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento
7.
Appl Microbiol Biotechnol ; 103(12): 4917-4929, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31073877

RESUMO

Undesirable flavor caused by excessive higher alcohols restrains the development of the wheat beer industry. To clarify the regulation mechanism of the metabolism of higher alcohols in wheat beer brewing by the top-fermenting yeast Saccharomyces cerevisiae S17, the effect of temperature on the fermentation performance and transcriptional levels of relevant genes was investigated. The strain S17 produced 297.85 mg/L of higher alcohols at 20 °C, and the production did not increase at 25 °C, reaching about 297.43 mg/L. Metabolite analysis and transcriptome sequencing showed that the metabolic pathways of branched-chain amino acids, pyruvate, phenylalanine, and proline were the decisive factors that affected the formation of higher alcohols. Fourteen most promising genes were selected to evaluate the effects of single-gene deletions on the synthesis of higher alcohols. The total production of higher alcohols by the mutants Δtir1 and Δgap1 was reduced by 23.5 and 19.66% compared with the parent strain S17, respectively. The results confirmed that TIR1 and GAP1 are crucial regulatory genes in the metabolism of higher alcohols in the top-fermenting yeast. This study provides valuable knowledge on the metabolic pathways of higher alcohols and new strategies for reducing the amounts of higher alcohols in wheat beer.


Assuntos
Álcoois/metabolismo , Cerveja/microbiologia , Fermentação , Genes Reguladores , Saccharomyces cerevisiae/genética , Temperatura , Reatores Biológicos , Aromatizantes , Deleção de Genes , Perfilação da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Redes e Vias Metabólicas , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Paladar
8.
J Ind Microbiol Biotechnol ; 46(7): 1003-1011, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30969383

RESUMO

Flavor production by esters or by higher alcohols play a key role in the sensorial quality of fermented alcoholic beverages. In Saccharomyces cerevisiae cells, the syntheses of esters and higher alcohols are considerably influenced by intracellular CoA levels catalyzed by pantothenate kinase. In this work, we examined the effects of cofactor CoA and acetyl-CoA synthesis on the metabolism of esters and higher alcohols. Strains 12α-BAP2 and 12α+ATF1 where generated by deleting and overexpressing BAP2 (encoded branched-chain amino acid permease) and ATF1 (encoded alcohol acetyl transferases), respectively, in the parent 12α strains. Then, 12α-BAP2+CAB1 and 12α-BAP2+CAB3 strains were obtained by overexpressing CAB1 (encoded pantothenate kinase Cab1) and CAB3 (encoded pantothenate kinase Cab3) in the 12α-BAP2 strain, and 12α-BAP2+CAB1+ATF1 and 12α-BAP2+CAB3+ATF1 were generated by overexpressing ATF1 in the pantothenate kinase overexpression strains. The acetate ester level in 12α-BAP2 was slightly changed relative to that in the control strain 12α, whereas the acetate ester levels in 12α-BAP2+CAB1, 12α-BAP2+CAB3, 12α-BAP2+CAB1+ATF1, and 12α-BAP2+CAB3+ATF1 were distinctly increased (44-118% for ethyl acetate and 18-57% for isoamyl acetate). The levels of n-propanol, methyl-1-butanol, isopentanol, isobutanol, and phenethylol levels were changed and varied among the six engineered strains. The levels of acetate esters and higher alcohols can be modulated by changing the CoA and acetyl-CoA levels. The method proposed in this work supplies a practical means of breeding yeast strains by modulating acetate ester and higher alcohol production.


Assuntos
Álcoois/metabolismo , Ésteres/metabolismo , Saccharomyces cerevisiae/metabolismo , Acetatos/metabolismo , Ácido Acético/metabolismo , Acetilcoenzima A/metabolismo , Fermentação
9.
Biotechnol Biofuels ; 11: 307, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30455736

RESUMO

Background: The biological production of 2,3-butanediol from xylose-rich raw materials from Klebsiella pneumoniae is a low-cost process. RpoD, an encoding gene of the sigma factor, is the key element in global transcription machinery engineering and has been successfully used to improve the fermentation with Escherichia coli. However, whether it can regulate the tolerance in K. pneumoniae remains unclear. Results: In this study, the kpC mutant strain was constructed by altering the expression quantity and genotype of the rpoD gene, and this exhibited high xylose tolerance and 2,3-butanediol production. The xylose tolerance of kpC strain was increased from 75 to 125 g/L, and the yield of 2,3-butanediol increased by 228.5% compared with the parent strain kpG, reaching 38.6 g/L at 62 h. The RNA sequencing results showed an upregulated expression level of 500 genes and downregulated expression level of 174 genes in the kpC mutant strain. The pathway analysis further showed that the differentially expressed genes were mainly related to signal transduction, membrane transport, carbohydrate metabolism, and energy metabolism. The nine most-promising genes were selected based on transcriptome sequencing, and were evaluated for their effects on xylose tolerance. The overexpression of the tktA encoding transketolase, pntA encoding NAD(P) transhydrogenase subunit alpha, and nuoF encoding NADH dehydrogenase subunit F conferred increased xylose consumption and increased 2,3-butanediol production to K. pneumoniae. Conclusions: These results suggest that the xylose tolerance and 2,3-butanediol production of K. pneumoniae can be greatly improved by the directed evolution of rpoD. By applying transcriptomic analysis, the upregulation of tktA, pntA, and nuoF that were coded are essential for the xylose consumption and 2,3-butanediol production. This study will provide reference for further research on improving the fermentation abilities by means of other organisms.

10.
J Agric Food Chem ; 66(28): 7417-7427, 2018 Jul 18.
Artigo em Inglês | MEDLINE | ID: mdl-29939025

RESUMO

Appropriate concentrations and proportion of acetate esters and higher alcohols improve the quality of Chinese Baijiu. To regulate the concentrations of acetate esters in Chinese Baijiu, we constructed a PGK1 promoter library through error-prone PCR. Then, we used an enhanced green fluorescent protein as a reporter to characterize the activities of PGK1p mutants. The PGK1p library contained 28 PGK1p mutants and spanned an activity that ranged between 0.1% and 141% of wild-type PGK1p. Seven PGK1p mutants were characterized by an additional reporter ß-galactosidase and then used for the overexpression of ATF1 with BAT2 deletion in Saccharomyces cerevisiae a45. The production of ethyl acetate in strains A8, A17, A18, A27, A22, A25, A28, and AWT were 1.66-, 3.09-, 10.59-, 13.07-, 15.99-, 22.67-, 24.06-, and 27.22-fold higher than that of the parental strain. The results on alcohol acetyltransferase (AATase) activity showed that the PGK1p library precisely controlled ATF1 expression and regulated the acetate esters production.


Assuntos
Ácido Acético/metabolismo , Ésteres/metabolismo , Fosfoglicerato Quinase/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Vinho/microbiologia , Ácido Acético/análise , Ésteres/análise , Etanol/análise , Etanol/metabolismo , Fermentação , Biblioteca Gênica , Fosfoglicerato Quinase/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Vinho/análise
11.
J Ind Microbiol Biotechnol ; 45(9): 827-838, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-29936578

RESUMO

Maltose metabolism of baker's yeast (Saccharomyces cerevisiae) in lean dough is suppressed by the glucose effect, which negatively affects dough fermentation. In this study, differences and interactions among SNF4 (encoding for the regulatory subunit of Snf1 kinase) overexpression and REG1 and REG2 (which encodes for the regulatory subunits of the type I protein phosphatase) deletions in maltose metabolism of baker's yeast were investigated using various mutants. Results revealed that SNF4 overexpression and REG1 and REG2 deletions effectively alleviated glucose repression at different levels, thereby enhancing maltose metabolism and leavening ability to varying degrees. SNF4 overexpression combined with REG1/REG2 deletions further enhanced the increases in glucose derepression and maltose metabolism. The overexpressed SNF4 with deleted REG1 and REG2 mutant ΔREG1ΔREG2 + SNF4 displayed the highest maltose metabolism and strongest leavening ability under the test conditions. Such baker's yeast strains had excellent potential applications.


Assuntos
Proteínas Quinases Ativadas por AMP/genética , Proteínas de Transporte/genética , Deleção de Genes , Maltose/metabolismo , Proteína Fosfatase 1/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Fatores de Transcrição/genética , Proteínas Quinases Ativadas por AMP/metabolismo , Fermentação , Regulação Fúngica da Expressão Gênica , Glucose/metabolismo , Proteínas de Transporte de Monossacarídeos/genética , Proteínas de Transporte de Monossacarídeos/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Simportadores/genética , Simportadores/metabolismo , Fatores de Transcrição/metabolismo , alfa-Glucosidases/genética , alfa-Glucosidases/metabolismo
12.
Biotechnol Prog ; 34(2): 328-336, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29314788

RESUMO

As content and proportion of ethyl acetate is critical to the flavor and quality of beverages, the concise regulation of the ethyl acetate metabolism is a major issue in beverage fermentations. In this study, for ethyl acetate yield regulation, we finely modulated the expression of ATF1 through precise and seamless insertion of serially truncated PGK1 promoter from the 3' end by 100bp steps in the Chinese liquor yeast, CLy12a. The three engineered promoters carrying 100-, 200-, and 300-bp truncations exhibited reduced promoter strength but unaffected growth. These three promoters were integrated into the CLy12a strain, generating strains CLy12a-P-100, CLy12a-P-200, and CLy12a-P-300, respectively. The transcription levels of CLy12a-P-100, CLy12a-P-200, and CLy12a-P-300 were 20%, 17%, and 10% of that of CLy12a-P, respectively. The AATase (alcohol acetyl transferases, encoded by the ATF1 gene) activity of three engineered strains were 36%, 56%, and 62% of that of CLy12a-P. In the liquid fermentation of corn hydrolysate at 30°C, the concentration of ethyl acetate in CLy12a-P-100, CLy12a-P-200, and CLy12a-P-300 were reduced by 28%, 30%, and 42%, respectively, compared to CLy12a-P. These results verifying that the ethyl acetate yield could be gradually enhanced by finely modulating the expression of ATF1. The engineered strain CLy12a-P-200 produced the ethyl acetate concentration with the best sensorial quality compared to the other engineered yeast strains. The method proposed in this work supplies a practical proposal for breeding Chinese liquor yeast strains with finely modulated ethyl acetate yield. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:328-336, 2018.


Assuntos
Acetatos/metabolismo , Engenharia Genética/métodos , Proteínas/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Bebidas Alcoólicas/microbiologia , Escherichia coli/genética , Fermentação , Regulação Fúngica da Expressão Gênica , Microrganismos Geneticamente Modificados , Fosfoglicerato Quinase/genética , Regiões Promotoras Genéticas , Proteínas/metabolismo , Zea mays
13.
Appl Microbiol Biotechnol ; 102(4): 1783-1795, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-29305698

RESUMO

Higher alcohols significantly influence the quality and flavor profiles of Chinese Baijiu. ILV1-encoded threonine deaminase, LEU1-encoded α-isopropylmalate dehydrogenase, and LEU2-encoded ß-isopropylmalate dehydrogenase are involved in the production of higher alcohols. In this work, ILV1, LEU1, and LEU2 deletions in α-type haploid, a-type haploid, and diploid Saccharomyces cerevisiae strains and ILV1, LEU1, and LEU2 single-allele deletions in diploid strains were constructed to examine the effects of these alterations on the metabolism of higher alcohols. Results showed that different genetic engineering strategies influence carbon flux and higher alcohol metabolism in different manners. Compared with the parental diploid strain, the ILV1 double-allele-deletion diploid mutant produced lower concentrations of n-propanol, active amyl alcohol, and 2-phenylethanol by 30.33, 35.58, and 11.71%, respectively. Moreover, the production of isobutanol and isoamyl alcohol increased by 326.39 and 57.6%, respectively. The LEU1 double-allele-deletion diploid mutant exhibited 14.09% increased n-propanol, 33.74% decreased isoamyl alcohol, and 13.21% decreased 2-phenylethanol production, which were similar to those of the LEU2 mutant. Furthermore, the LEU1 and LEU2 double-allele-deletion diploid mutants exhibited 41.72 and 52.18% increased isobutanol production, respectively. The effects of ILV1, LEU1, and LEU2 deletions on the production of higher alcohols by α-type and a-type haploid strains were similar to those of double-allele deletion in diploid strains. Moreover, the isobutanol production of the ILV1 single-allele-deletion diploid strain increased by 27.76%. Variations in higher alcohol production by the mutants are due to the carbon flux changes in yeast metabolism. This study could provide a valuable reference for further research on higher alcohol metabolism and future optimization of yeast strains for alcoholic beverages.


Assuntos
Bebidas Alcoólicas/microbiologia , Ciclo do Carbono/genética , Etanol/metabolismo , Microbiologia de Alimentos/métodos , Hidroliases/genética , Engenharia Metabólica/métodos , Redes e Vias Metabólicas/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Treonina Desidratase/genética , 3-Isopropilmalato Desidrogenase/genética , 3-Isopropilmalato Desidrogenase/metabolismo , China , Fermentação , Deleção de Genes , Humanos , Hidroliases/metabolismo , Saccharomyces cerevisiae/enzimologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Treonina Desidratase/metabolismo
14.
Microb Cell Fact ; 16(1): 194, 2017 Nov 09.
Artigo em Inglês | MEDLINE | ID: mdl-29121937

RESUMO

BACKGROUND: Tup1 is a general transcriptional repressor of diverse gene families coordinately controlled by glucose repression, mating type, and other mechanisms in Saccharomyces cerevisiae. Several functional domains of Tup1 have been identified, each of which has differing effects on transcriptional repression. In this study, we aim to investigate the role of Tup1 and its domains in maltose metabolism of industrial baker's yeast. To this end, a battery of in-frame truncations in the TUP1 gene coding region were performed in the industrial baker's yeasts with different genetic background, and the maltose metabolism, leavening ability, MAL gene expression levels, and growth characteristics were investigated. RESULTS: The results suggest that the TUP1 gene is essential to maltose metabolism in industrial baker's yeast. Importantly, different domains of Tup1 play different roles in glucose repression and maltose metabolism of industrial baker's yeast cells. The Ssn6 interaction, N-terminal repression and C-terminal repression domains might play roles in the regulation of MAL transcription by Tup1 for maltose metabolism of baker's yeast. The WD region lacking the first repeat could influence the regulation of maltose metabolism directly, rather than indirectly through glucose repression. CONCLUSIONS: These findings lay a foundation for the optimization of industrial baker's yeast strains for accelerated maltose metabolism and facilitate future research on glucose repression in other sugar metabolism.


Assuntos
Maltose/metabolismo , Proteínas Nucleares/genética , Proteínas Repressoras/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Metabolismo dos Carboidratos , Fermentação , Deleção de Genes , Regulação Fúngica da Expressão Gênica , Proteínas Nucleares/metabolismo , Domínios e Motivos de Interação entre Proteínas/genética , Proteínas Repressoras/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo
15.
J Ind Microbiol Biotechnol ; 44(3): 397-405, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-28154948

RESUMO

Diacetyl causes an unwanted buttery off-flavor in lager beer. The production of diacetyl is reduced by modifying the metabolic pathway of yeast in the beer fermentation process. In this study, BDH2 and ILV5 genes, coding diacetyl reductase and acetohydroxy acid reductoisomerase, respectively, were expressed using a PGK1 promoter in Saccharomyces cerevisiae, which deleted one ILV2 allelic gene. Diacetyl contents and fermentation performances were examined and compared. Results showed that the diacetyl content in beer was remarkably reduced by 16.52% in QI2-KP (one ILV2 allelic gene deleted), 55.65% in QI2-B2Y (overexpressed BDH2 gene and one ILV2 allelic gene deleted), and 69.13% in QI2-I5Y (overexpressed ILV5 gene and one ILV2 allelic gene deleted) compared with the host strain S2. The fermentation ability of mutant strains was similar to that of S2. Results of the present study can lead to further advances in this technology and its broad application in scientific investigations and industrial beer production.


Assuntos
Oxirredutases do Álcool/genética , Diacetil/metabolismo , Deleção de Genes , Proteínas Mitocondriais/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Oxirredutases do Álcool/metabolismo , Alelos , Cerveja/análise , Cerveja/microbiologia , Fermentação , Microbiologia de Alimentos , Proteínas Mitocondriais/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo
16.
J Ind Microbiol Biotechnol ; 44(6): 949-960, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-28176138

RESUMO

Acetate esters and higher alcohols greatly influence the quality and flavor profiles of Chinese Baijiu (Chinese liquor). Various mutants have been constructed to investigate the interactions of ATF1 overexpression, IAH1 deletion, and BAT2 deletion on the production of acetate esters and higher alcohols. The results showed that the overexpression of ATF1 under the control of the PGK1 promoter with BAT2 and IAH1 double-gene deletion led to a higher production of acetate esters and a lower production of higher alcohols than the overexpression of ATF1 with IAH1 deletion or overexpression of ATF1 with BAT2 deletion. Moreover, deletion of IAH1 in ATF1 overexpression strains effectively increased the production of isobutyl acetate and isoamyl acetate by reducing the hydrolysis of acetate esters. The decline in the production of higher alcohol by the ATF1 overexpression strains with BAT2 deletion is due to the interaction of ATF1 overexpression and BAT2 deletion. Mutants with varying abilities of producing acetate esters and higher alcohols were developed by genetic engineering. These strains have great potential for industrial application.


Assuntos
Bebidas Alcoólicas , Fermentação , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Acetatos/metabolismo , Álcoois/metabolismo , Hidrolases de Éster Carboxílico/genética , Ésteres/metabolismo , Deleção de Genes , Engenharia Genética , Pentanóis/metabolismo , Proteínas/genética , Proteínas/metabolismo , Saccharomyces cerevisiae/genética , Transaminases/genética
17.
Microb Cell Fact ; 15: 54, 2016 Apr 04.
Artigo em Inglês | MEDLINE | ID: mdl-27039899

RESUMO

BACKGROUND: Trehalose is related to several types of stress responses, especially freezing response in baker's yeast (Saccharomyces cerevisiae). It is desirable to manipulate trehalose-related genes to create yeast strains that better tolerate freezing-thaw stress with improved fermentation capacity, which are in high demand in the baking industry. RESULTS: The strain overexpressing MAL62 gene showed increased trehalose content and cell viability after prefermention-freezing and long-term frozen. Deletion of NTH1 in combination of MAL62 overexpression further strengthens freezing tolerance and improves the leavening ability after freezing-thaw stress. CONCLUSIONS: The mutants of the industrial baker's yeast with enhanced freezing tolerance and leavening ability in lean dough were developed by genetic engineering. These strains had excellent potential industrial applications.


Assuntos
Aclimatação/genética , Fermentação/genética , Congelamento , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Trealase/genética , alfa-Glucosidases/genética , Temperatura Baixa , Farinha/microbiologia , Deleção de Genes , Regulação Fúngica da Expressão Gênica , Organismos Geneticamente Modificados , Regulação para Cima/genética
18.
Appl Microbiol Biotechnol ; 100(14): 6375-6383, 2016 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-27041690

RESUMO

Leavening ability in sweet dough is required for the commercial applications of baker's yeast. This property depends on many factors, such as glycolytic activity, sucrase activity, and osmotolerance. This study explored the importance of sucrase level on the leavening ability of baker's yeast in sweet dough. Furthermore, the baker's yeast strains with varying sucrase activities were constructed by deleting SUC2, which encodes sucrase or replacing the SUC2 promoter with the VPS8/TEF1 promoter. The results verify that the sucrase activity negatively affects the leavening ability of baker's yeast strains under high-sucrose conditions. Based on a certain level of osmotolerance, sucrase level plays a significant role in the fermentation performance of baker's yeast, and appropriate sucrase activity is an important determinant for the leavening property of baker's yeast in sweet dough. Therefore, modification on sucrase activity is an effective method for improving the leavening properties of baker's yeast in sweet dough. This finding provides guidance for the breeding of industrial baker's yeast strains for sweet dough leavening. The transformants BS1 with deleted SUC2 genetic background provided decreased sucrase activity (a decrease of 39.3 %) and exhibited enhanced leavening property (an increase of 12.4 %). Such a strain could be useful for industrial applications.


Assuntos
Pão/microbiologia , Manipulação de Alimentos , Microbiologia de Alimentos , Saccharomyces cerevisiae/metabolismo , Sacarase/metabolismo , Biomassa , Pão/análise , Meios de Cultura/química , Fermentação , Deleção de Genes , Glucose/análise , Glicerol/análise , Fator 1 de Elongação de Peptídeos/genética , Fator 1 de Elongação de Peptídeos/metabolismo , Plasmídeos/genética , Regiões Promotoras Genéticas , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Sacarose/análise , Trealose/análise , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo , beta-Frutofuranosidase/genética , beta-Frutofuranosidase/metabolismo
19.
J Ind Microbiol Biotechnol ; 43(5): 671-9, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-26831650

RESUMO

Ethyl carbamate (EC), a pluripotent carcinogen, is mainly formed by a spontaneous chemical reaction of ethanol with urea in wine. The arginine, one of the major amino acids in grape musts, is metabolized by arginase (encoded by CAR1) to ornithine and urea. To reduce the production of urea and EC, an arginase-deficient recombinant strain YZ22 (Δcarl/Δcarl) was constructed from a diploid wine yeast, WY1, by successive deletion of two CAR1 alleles to block the pathway of urea production. The RT-qPCR results indicated that the YZ22 almost did not express CAR1 gene and the specific arginase activity of strain YZ22 was 12.64 times lower than that of parent strain WY1. The fermentation results showed that the content of urea and EC in wine decreased by 77.89 and 73.78 %, respectively. Furthermore, EC was forming in a much lower speed with the lower urea during wine storage. Moreover, the two CAR1 allele deletion strain YZ22 was substantially equivalent to parental strain in terms of growth and fermentation characteristics. Our research also suggested that EC in wine originates mainly from urea that is produced by the arginine.


Assuntos
Arginase/genética , Fermentação , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Uretana/metabolismo , Vinho/análise , Vinho/microbiologia , Alelos , Arginase/metabolismo , Arginina/metabolismo , Carcinógenos/metabolismo , Etanol/metabolismo , Ornitina/metabolismo , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/metabolismo , Ureia/metabolismo
20.
J Food Sci ; 80(12): M2879-85, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26580148

RESUMO

Maltose metabolism of baker's yeast (Saccharomyces cerevisiae) in lean dough is negatively influenced by glucose repression, thereby delaying the dough fermentation. To improve maltose metabolism and leavening ability, it is necessary to alleviate glucose repression. The Snf1 protein kinase is well known to be essential for the response to glucose repression and required for transcription of glucose-repressed genes including the maltose-utilization genes (MAL). In this study, the SNF1 overexpression and deletion industrial baker's yeast strains were constructed and characterized in terms of maltose utilization, growth and fermentation characteristics, mRNA levels of MAL genes (MAL62 encoding the maltase and MAL61 encoding the maltose permease) and maltase and maltose permease activities. Our results suggest that overexpression of SNF1 was effective to glucose derepression for enhancing MAL expression levels and enzymes (maltase and maltose permease) activities. These enhancements could result in an 18% increase in maltose metabolism of industrial baker's yeast in LSMLD medium (the low sugar model liquid dough fermentation medium) containing glucose and maltose and a 15% increase in leavening ability in lean dough. These findings provide a valuable insight of breeding industrial baker's yeast for rapid fermentation.


Assuntos
Pão , Fermentação , Manipulação de Alimentos , Maltose/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Microbiologia de Alimentos , Genes Fúngicos , Glucose/metabolismo , Humanos , Proteínas de Transporte de Monossacarídeos/genética , Proteínas de Transporte de Monossacarídeos/metabolismo , Proteínas Serina-Treonina Quinases/genética , RNA Mensageiro/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Simportadores/genética , Simportadores/metabolismo , alfa-Glucosidases/genética , alfa-Glucosidases/metabolismo
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