Enhanced production of isoamyl alcohol and isoamyl acetate by ubiquitination-deficient Saccharomyces cerevisiae mutants

Aromatic compounds are an important element in the flavor of yeast-fermented alcohol. We isolated mutants of Saccharomyces cerevisiae capable of growth at high levels of hydrostatic pressure. Among them, the HPG1 mutants, with a defect in their Rsp5 ubiquitin ligase, were found to produce high amounts of aromatics due to enhanced leucine uptake, with isoamyl alcohol production 2- to 3-fold and isoamyl acetate production 4- to 8-fold that of the wild-type strain. The result suggests that the HPG1/RSP5 mutant alleles could be new resources for producing these flavoring compounds for yeast-fermented alcoholic beverages.     

Genetic and physiological analysis of UV-sensitive mutants of Saccharomyces cerevisiae

The use of tetrad analysis and complementation tests indicates that the groups of UV-sensitive mutants assigned the labels radI and rad3 are alleles of two single genes involved in the process of cellular repair of UV-induced damage in the yeast Saccharomyces cerevisiae.     

Increase ethyl acetate production in Saccharomyces cerevisiae by genetic engineering of ethyl acetate metabolic pathway

Journal of Industrial Microbiology & Biotechnology - Ethyl acetate has attracted much attention as an important chemical raw material and a flavor component of alcoholic beverages. In this...     

Engineering high-level production of fatty alcohols by Saccharomyces cerevisiae from lignocellulosic feedstocks

Fatty alcohols in the C12-C18 range are used in personal care products, lubricants, and potentially biofuels. These compounds can be produced from the fatty acid pathway by a fatty acid reductase (FAR), yet yields from the preferred industrial host Saccharomyces cerevisiae remain under 2% of the theoretical maximum from glucose. Here we improved titer and yield of fatty alcohols using an approach involving quantitative analysis of protein levels and metabolic flux, engineering enzyme level and localization, pull-push-block engineering of carbon flux, and cofactor balancing. We compared four heterologous FARs, finding highest activity and endoplasmic reticulum localization from a Mus musculus FAR. After screening an additional twenty-one single-gene edits, we identified increasing FAR expression; deleting competing reactions encoded by DGA1, HFD1, and ADH6; overexpressing a mutant acetyl-CoA carboxylase; limiting NADPH and carbon usage by the glutamate dehydrogenase encoded by GDH1; and overexpressing the Δ9-desaturase encoded by OLE1 as successful strategies to improve titer. Our final strain produced 1.2 g/L fatty alcohols in shake flasks, and 6.0 g/L in fed-batch fermentation, corresponding to ~ 20% of the maximum theoretical yield from glucose, the highest titers and yields reported to date in S. cerevisiae. We further demonstrate high-level production from lignocellulosic feedstocks derived from ionic-liquid treated switchgrass and sorghum, reaching 0.7 g/L in shake flasks. Altogether, our work represents progress towards efficient and renewable microbial production of fatty acid-derived products.     

Recombinant Saccharomyces cerevisiae strain triggers acetate production to fuel biosynthetic pathways

Although the metabolism and physiology of the growth of yeast strains has been extensively studied, many questions remain unanswered when the induced production of a recombinant protein is concerned. This work addresses the production of a Fusarium solani pisi cutinase by a recombinant Saccharomyces cerevisiae strain induced through the use of a galactose promoter. It was observed that whenever the strain needed to activate biosynthetic pathways, either for cutinase synthesis, or for the synthesis of the enzymes required for galactose intake, acetate production occurred. The on-line detection of acetate in the medium might prove useful for the control and the supervision of recombinant protein production processes using yeast. The volumes of acid and base added to control the pH throughout the time course of the cultivations were used to calculate an on-line estimator for acetate concentration.     

Genetic analysis of the metabolic pathways responsible for aroma metabolite production by Saccharomyces cerevisiae

During alcoholic fermentation, higher alcohols, esters, and acids are formed from amino acids via the Ehrlich pathway by yeast, but many of the genes encoding the enzymes have not yet been identified. When the BAT1/2 genes, encoding transaminases that deaminate amino acids in the first step of the Ehrlich pathway are deleted, higher metabolite formation is significantly decreased. Screening yeast strains with deletions of genes encoding decarboxylases, dehydrogenases, and reductases revealed nine genes whose absence had the most significant impact on higher alcohol production. The seven most promising genes (AAD6, BAT2, HOM2, PAD1, PRO2, SPE1, and THI3) were further investigated by constructing double- and triple-deletion mutants. All double-deletion strains showed a greater decrease in isobutanol, isoamyl alcohol, isobutyric, and isovaleric acid production than the corresponding single deletion strains with the double-deletion strains in combination with ?bat2 and the ?hom2-?aad6 strain revealing the greatest impact. BAT2 is the dominant gene in these deletion strains and this suggests the initial transaminase step of the Ehrlich pathway is rate-limiting. The triple-deletion strains in combination with BAT2 (?bat2-?thi3-?aad6 and ?bat2-?thi3-?hom2) had the greatest impact on the end metabolite production with the exception of isoamyl alcohol and isovaleric acid. The strain deleted for two dehydrogenases and a reductase (?hom2-?pro2-?aad6) had a greater effect on the levels of these two compounds. This study contributes to the elucidation of the Ehrlich pathway and its significance for aroma production by fermenting yeast cells.     

Genetic and physiological relationships between L-asparaginase I and asparaginase II in Saccharomyces cerevisiae.

The cistron that codes for L-asparaginase I in Saccharomyces cerevisiae (aspl) is not genetically linked to either of the cistrons coding for expression of asparaginase II (asp2 and asp3). Cells containing different combinations of theses enzymes grow at different rates in media in which L-asparagine or D-asparagine is the only source of nitrogen for cell replication. Cells lacking L-asparaginase I but possessing asparaginase II grow more rapidly in medium containing D-asparagine as a nitrogen source than cells containing both enzymes, even though D-asparagine is not a substrate of L-asparaginase I. These results indicate that L-asparaginase I and asparaginase II interact in some way to regulate the utilization of asparagine as a nitrogen source for cell growth.     

Modulating acetate ester and higher alcohol production in Saccharomyces cerevisiae through the cofactor engineering

Journal of Industrial Microbiology & Biotechnology - Flavor production by esters or by higher alcohols play a key role in the sensorial quality of fermented alcoholic beverages. In...     

Genetic determination of polygalacturonase production in wild-type and laboratory strains of Saccharomyces cerevisiae

The genetic determination of polygalacturonase (PG) production in Saccharomyces cerevisiae was studied by biochemical and classical genetic techniques. Crosses of PG⁺ strains with PG^– strains showed that in the haploid wild-type-derived strain, two structural genes were involved in the production of a hydrolysis halo on plates with polygalacturonic acid. However, in the case of PG⁺ laboratory strain IM1-8b, the phenotype was controlled by only one structural gene although the analysis of PG^– IM1-8b mutants demonstrated the existence of at least two complementation groups. All these genetic results were assessed biochemically by means of cation-exchange chromatography. Two enzymes were separated in the wild-type strain, and only one in the laboratory strain. The three enzymes had different K _m values, molecular masses, and optimal pHs for activity. Received: 24 October 1996 / Accepted: 17 December 1996     

Molecular and physiological comparisons between Saccharomyces cerevisiae and Saccharomyces boulardii

In this paper, comparative molecular studies between authentic Saccharomyces cerevisiae strains, related species, and the strain described as Saccharomyces boulardii were performed. The response of a S. boulardii strain and a S. cerevisiae strain (W303) to different stress conditions was also evaluated. The results obtained in this study show that S. boulardii is genetically very close or nearly identical to S. cerevisiae. Metabolically and physiologically, however, it shows a very different behavior, particularly in relation to growth yield and resistance to temperature and acidic stresses, which are important characteristics for a microorganism to be used as a probiotic.     

酿酒酵母类丙酮酸脱羧酶基因缺失对高级醇生成量的影响

【目的】通过构建酿酒酵母类丙酮酸脱羧酶基因(YDL080C)缺失的工程菌株,研究该基因对酿酒酵母浓醪发酵产高级醇特别是异戊醇的影响。【方法】以酿酒酵母工业菌株AY-15的单倍体a-8或α-22的基因组DNA为模板,PCR分别扩增YDL080C上下游非编码区片段YA和YB;以pUG6质粒为模板,PCR扩增KanMX抗性基因片段。分别将YA、YB和KanMX片段连入pUC19载体,构建重组质粒pUC-YABK;并以其为模板,PCR扩增YA-KanMX-YB重组盒,分别电转化单倍体a-8和α-22。将转化子和亲本分别进行酒精浓醪发酵,发酵结束后测定其发酵性能和高级醇的生成量。【结果】筛选获得了YDL080C基因缺失突变株。酒精发酵后发酵性能和高级醇测定结果显示,转化子的异戊醇及总高级醇生成量与对应的单倍体亲本相比没有明显变化,但酒精度分别比亲本提高了0.6(%,v/v)和0.4(%,v/v)。【结论】YDL080C基因缺失对降低酿酒酵母发酵产高级醇特别是异戊醇没有明显作用,但会使酒精度有所提高。     

Genetic fine-structural analysis of the Saccharomyces cerevisiae alpha-pheromone receptor.

The alpha-pheromone receptor encoded by the STE2 gene contains seven potential transmembrane domains. Its ability to transduce the pheromone signal is thought to require the action of a G protein. As an initial step toward defining the structural features of the receptor required for its activity, we examined the phenotypic consequences of linker insertion mutations (12 bp) at 10 different sites in the STE2 gene. Three mutant classes, which correspond to three different regions of the receptor protein, were observed. 1) The two mutants affecting the C-terminal region (C-terminal mutants) were essentially wild type for mating efficiency, pheromone binding, and pheromone sensitivity. 2) The three mutants in the N-terminus mated with reduced efficiency, showed reduced pheromone binding capacity, and were partially defective in pheromone induction of agglutinin production and cell division arrest. Increased gene dosage of these N-terminal alleles suppressed their mutant phenotypes, whereas the sst2-1 mutation, which blocks adaptation to pheromone, did not result in suppression. Thus, the N-terminal mutants were apparently limited by receptor production, but not by the adaptation function SST2. 3) The five mutants in the central region containing the seven transmembrane segments (central mutants) were completely defective for mating and did not respond to pheromone, but could be distinguished by their ability to bind pheromone. Inserts in or near transmembrane domains 2 and 4 blocked pheromone binding, whereas inserts into transmembrane domains 1, 5, and 6 retained partial pheromone binding activity even though they failed to transduce a signal. The central mutants were not suppressed by increased gene dosage, and one mutant (ste2-/101) was partially suppressed by sst2-1. Furthermore, the central core mutants were also distinguished from one another in that three of the five mutants were able to partially complement the temperature sensitivity of ste2-3.