Saccharomyces cerevisiae Atf1p is an alcohol acetyltransferase and a thioesterase in vitro

The alcohol‐O‐acyltransferases are bisubstrate enzymes that catalyse the transfer of acyl chains from an acyl‐coenzyme A (CoA) donor to an acceptor alcohol. In the industrial yeast Saccharomyces cerevisiae this reaction produces acyl esters that are an important influence on the flavour of fermented beverages and foods. There is also a growing interest in using acyltransferases to produce bulk quantities of acyl esters in engineered microbial cell factories. However, the structure and function of the alcohol‐O‐acyltransferases remain only partly understood. Here, we recombinantly express, purify and characterize Atf1p, the major alcohol acetyltransferase from S. cerevisiae. We find that Atf1p is promiscuous with regard to the alcohol cosubstrate but that the acyltransfer activity is specific for acetyl‐CoA. Additionally, we find that Atf1p is an efficient thioesterase in vitro with specificity towards medium‐chain‐length acyl‐CoAs. Unexpectedly, we also find that mutating the supposed catalytic histidine (H191) within the conserved HXXXDG active site motif only moderately reduces the thioesterase activity of Atf1p. Our results imply a role for Atf1p in CoA homeostasis and suggest that engineering Atf1p to reduce the thioesterase activity could improve product yields of acetate esters from cellular factories. © 2017 The Authors. Yeast published by John Wiley & Sons, Ltd.     

构建具有大麦脂转移蛋白1分泌能力的酿酒酵母

为了增强纯生啤酒的泡沫性能,从酿酒酵母表达质粒YEplac181出发,将大麦脂转移蛋白1(LTP1)成熟肽的编码序列置于酿酒酵母ADH1启动子(alcohol dehydrogenase promoter)和CYC1终止子(cytochrome C terminator)的调控下,构建大麦脂转移蛋白1的酿酒酵母表达质粒YEp181-KAMLC。通过酿酒酵母α-信息素信号肽的引导分泌,酿酒酵母表达的成熟大麦LTP1被分泌到发酵液中。对发酵液的检测表明,在发酵132h后LTP1的产量可达到29.45mg/L。     

本土酿酒酵母的筛选及其低醇葡萄酒特性研究

以天津宝坻产区的三种葡萄为原料,分别从自然发酵的葡萄汁中筛选出340株酵母菌,经生理生化,产酸、酯、尿酶、H2S,发酵能力测定及26SrRNA测序,确定4株为酿酒酵母,用于贵人香低醇白葡萄酒的酿造。以商用酵母为对照,定量分析了不同酵母所酿酒中的香气成分、氨基甲酸乙酯含量,并进行了理化指标和感官测定,发现酵母C508和G611酿造的低醇酒具有更独特的香气特点,氨基甲酸乙酯的含量较低,微生物稳定性较高,更适合低醇酒的酿造。     

Ady2p is essential for the acetate permease activity in the yeast Saccharomyces cerevisiae

To identify new genes involved in acetate uptake in Saccharomyces cerevisiae, an analysis of the gene expression profiles of cells shifted from glucose to acetic acid was performed. The gene expression reprogramming of yeast adapting to a poor non-fermentable carbon source was observed, including dramatic metabolic changes, global activation of translation machinery, mitochondria biogenesis and the induction of known or putative transporters. Among them, the gene ADY2/YCR010c was identified as a new key element for acetate transport, being homologous to the Yarrowia lipolytica GPR1 gene, which has a role in acetic acid sensitivity. Disruption of ADY2 in S. cerevisiae abolished the active transport of acetate. Microarray analyses of ady2Delta strains showed that this gene is not a critical regulator of acetate response and that its role is directly connected to acetate transport. Ady2p is predicted to be a membrane protein and is a valuable acetate transporter candidate.     

Schizosaccharomyces pombe Pmf1p is structurally and functionally related to Mmf1p of Saccharomyces cerevisiae

A novel family of small proteins, termed p14.5 or YERO57c/YJGFc, has been identified. Independent studies indicate that p14.5 family members are multifunctional proteins involved in several pathways, e.g. regulation of translation or activation of the protease mu-calpain. We have previously shown that Mmf1p, a p14.5 of the budding yeast Saccharomyces cerevisiae, is localized in the mitochondria and influences mitochondrial DNA stability. In addition, we have demonstrated that Mmf1p is functionally related to p14.5 of mammalian cells. To explore further the evolutionary conservation of the mitochondrial function(s) of the p14.5s we have extended our study to the fission yeast, Schizosaccharomyces pombe. In this organism two p14.5 homologous proteins are present: Pmf1p (pombe mitochondrial factor 1) and Hpm1p (homologous Pmf1p factor 1). We have generated a specific Pmf1p antibody, which recognizes a single band of approximately 15 kDa in total cellular extracts. Cellular fractionation experiments indicate that Pmf1p localizes in the mitochondria as well as in the cytoplasm. We also show that Pmf1p shares several properties of S. cerevisiae Mmf1p. Indeed, Pmf1p restores the wild-type phenotype when expressed in delta mmf1 S. cerevisiae cells. Deletion of the leader sequence of Pmf1p abrogates its ability to localize in mitochondria and to functionally replace Mmf1p. Thus, these data together with our previous study show that the mitochondrial function(s) of the p14.5 family members are highly conserved in eukaryotic cells.     

CO2背压啤酒发酵中乙酰辅酶A与酿酒酵母生长和酯类生成的相关性分析

该研究探讨CO2背压发酵中乙酰辅酶A（acetyl-CoA）与酿酒酵母生长和酯类生成的相关性。结果表明：常压和CO2背压发酵条件下,acetyl-CoA含量在发酵初期（0～20 h）急速增长,并在20 h时达到峰值,在100 h之后均趋于稳定一致;酵母细胞数量分别在100 h、140 h时达到峰值,在180 h之后趋于一致;总酯含量分别在100 h、180 h时达到峰值,在180 h之后趋于稳定,常压发酵条件下总酯含量始终高于CO2背压发酵。常压条件下,啤酒发酵中acetyl-CoA含量与酵母数量呈极显著正相关（P＜0.01）,与总酯含量呈极显著负相关（P＜0.01）,相关系数R分别为0.937 17、-0.833 96;CO2背压条件下,啤酒发酵中acetyl-CoA含量与酵母数量呈极显著正相关（P＜0.01）,与总酯含量呈极显著负相关（P＜0.01）,相关系数R分别为0.905 66、-0.750 42。     

The effect of distillation conditions and alcohol content in ‘heart’ fractions on the concentration of aroma volatiles and undesirable compounds in plum brandies

This study investigates the effect of double‐ or single‐stage distillation and different alcohol content in ‘hearts’ (middle fractions) on the distribution of aroma volatiles and undesirable compounds (methanol, hydrocyanic acid, ethyl carbamate) during distillation of plum brandies. Irrespective of the distillation method used, the first fractions (‘heads’) included mainly aliphatic aldehydes, acetals and esters as well as higher alcohols (1‐propanol, 2‐methyl‐1‐propanol, 1‐butanol, 2‐methyl‐1‐butanol and 3‐methyl‐1‐butanol). Furfural, 1‐hexanol, benzyl alcohol, 2‐phenylethanol and ethyl carbamate occurred in relatively high concentrations in the ‘tail’ fractions. Increasing the concentration of alcohol in the heart fractions from 70 to 90% v /v resulted in a gradual decrease in the concentration of all detected volatile compounds. Compared with single‐stage distillation, double distillation produced heart fractions with lower concentration of acetaldehyde and benzaldehyde and with higher contents of furfural and esters, such as isobutyl acetate and isoamyl acetate. There was a statistically significant increase in the amounts of methanol and ethyl carbamate obtained from double distillation compared with similar fractions derived from the single‐stage process. However, in all fractions these compounds occurred in concentrations much lower than the limits specified by EU regulations. The heart fraction from the double‐stage process with 83% v /v alcohol content received the best scores for aroma and flavour. Copyright © 2017 The Institute of Brewing & Distilling     

Saccharomyces cerevisiae QNS1 codes for NAD(+) synthetase that is functionally conserved in mammals

NAD(+), an essential molecule involved in a variety of cellular processes, is synthesized through de novo and salvage pathways. NAD(+) synthetase catalyses the final step in both pathways. Here we show that this enzyme is encoded by the QNS1 gene in Saccharomyces cerevisiae. Expression of Escherichia coli or Bacillus subtilis NAD(+) synthetases was able to suppress the lethality of a qns1 deletion, while a B. subtilis NAD(+) synthetase mutant with lowered catalytic activity was not. Overexpression of QNS1 tagged with HA led to elevated levels of NAD(+) synthetase activity in yeast extracts, and this activity can be recovered by immunoprecipitation using anti-HA antibody. An allele of QNS1 was constructed that carries a point mutation predicted to reduce the catalytic activity. Overexpression of this allele, qns1(G521E), failed to elevate NAD(+) synthetase levels and qns1(G521E) could not rescue the lethality caused by the depletion of Qns1p. These results demonstrate that NAD(+) synthetase activity is essential for cell viability. A GFP-tagged version of Qns1p displayed a diffuse localization in both the nucleus and the cytosol. Finally, the rat homologue of QNS1 was cloned and shown to functionally replace yeast QNS1, indicating that NAD(+) synthetase is functionally conserved from bacteria to yeast and mammals.     

The Hsp90/Cdc37p chaperone system is a determinant of molybdate resistance in Saccharomyces cerevisiae

Saccharomyces cerevisiae lacks enzymes that contain the molybdopterin co‐factor and therefore any requirement for molybdenum as a trace mineral supplement. Instead, high molybdate levels are inhibitory to its growth. Low cellular levels of heat shock protein 90 (Hsp90), an essential chaperone, were found to enhance this sensitivity to molybdate. Certain Hsp90 point mutations and co‐chaperone protein defects that partially compromise the function of the Hsp90/Cdc37p chaperone system also rendered S. cerevisiae hypersensitive to high molybdate levels. Sensitivity was especially apparent with mutations close to the Hsp90 nucleotide binding site, with the loss of the non‐essential co‐chaperone Sti1p (the equivalent of mammalian Hop), and with the abolition of residue Ser14 phosphorylation on the essential co‐chaperone Cdc37p. While it remains to be proved that these effects reflect direct inhibition of the Hsp90 of the cell by the MoO₄²⁺ oxyanion in vivo; this possibility is suggested by molybdate sensitivity arising with a mutation in the Hsp90 nucleotide binding site that does not generate stress sensitivity or an impaired stress response. Molybdate sensitivity may therefore be a useful phenotype to score when studying mutations in this chaperone system. Copyright © 2009 John Wiley & Sons, Ltd.     

The centromere-binding factor Cbf1p from Candida albicans complements the methionine auxotrophic phenotype of Saccharomyces cerevisiae

A gene encoding the centromere binding factor 1 (Cbf1p) of the human pathogenic yeast Candida albicans was cloned and characterized. An open reading-frame was detected which encoded a 223 amino acid protein with a calculated molecular weight of 25.8 kDa and a relative isoelectric point of 5.55. It shares 39% overall amino acid sequence identity with Saccharomyces cerevisiae Cbf1p. We localized the CaCBF1 gene on chromosome 4. Southern analysis indicated that CaCBF1 is probably present as a single copy gene per haploid genome. The CaCBF1 gene under the control of its own promoter was able to complement the methionine auxotrophic growth, the increased mitotic instability of CEN plasmids and the slow growth of a Saccharomyces cerevisiae cbf1Delta mutant strain.     

Amino acid residues important for substrate specificity of the amino acid permeases Can1p and Gnp1p in Saccharomyces cerevisiae.

Deletion of the general amino acid permease gene GAP1 abolishes uptake of L-citrulline in Saccharomyces cerevisiae, resulting in the inability to grow on L-citrulline as sole nitrogen source. Selection for suppressor mutants that restored growth on L-citrulline led to isolation of 21 mutations in the arginine permease gene CAN1. One similar mutation was found in the glutamine-asparagine permease gene GNP1. L-[(14)C]citrulline uptake measurements confirmed that suppressor mutations in CAN1 conferred uptake of this amino acid, while none of the mutant permeases had lost the ability to transport L-[(14)C]arginine. Substrate specificity seemed to remain narrow in most cases, and broad substrate specificity was only observed in the cases where mutations affect two proline residues (P148 and P313) that are both conserved in the amino acid-polyamine-choline (APC) transporter superfamily. We found mutations affecting six predicted domains (helices III and X, and loops 1, 2, 6 and 7) of the permeases. Helix III and loop 7 are candidates for domains in direct contact with thetransported amino acid. Helix III was affected in both CAN1 (Y173H, Y173D) and GNP1 (W239C) mutants and has previously been found to be important for substrate preference in other members of the family. Furthermore, the mutations affecting loop 7 (residue T354, S355, Y356) are close to a glutamate side chain (E367) potentially interacting with the positively charged substrate, a notion supported by conservation of the side chain in permeases for cationic substrates.     

The role of two putative nitroreductases,Frm2p and Hbn1p,in the oxidative stress response in Saccharomyces cerevisiae

The nitroreductase family is comprised of a group of FMN‐ or FAD‐dependent enzymes that are able to metabolize nitrosubstituted compounds using the reducing power of NAD(P)H. These nitroreductases can be found in bacterial species and, to a lesser extent, in eukaryotes. There is little information on the biochemical functions of nitroreductases. Some studies suggest their possible involvement in the oxidative stress response. In the yeast Saccharomyces cerevisiae, two nitroreductase proteins, Frm2p and Hbn1p, have been described. While Frm2p appears to act in the lipid signalling pathway, the function of Hbn1p is completely unknown. In order to elucidate the functions of Frm2p and Hbn1p, we evaluated the sensitivity of yeast strains, proficient and deficient in both oxidative stress proteins, for respiratory competence, antioxidant‐enzyme activities, intracellular reactive oxygen species (ROS) production and lipid peroxidation. We found reduced basal activity of superoxide dismutase (SOD), ROS production, lipid peroxidation and petite induction and higher sensitivity to 4‐nitroquinoline‐oxide (4‐NQO) and N‐nitrosodiethylamine (NDEA), as well as higher basal activity of catalase (CAT) and glutathione peroxidase (GPx) and reduced glutathione (GSH) content in the single and double mutant strains frm2Δ and frm2Δ hbn1Δ. These strains exhibited less ROS accumulation and lipid peroxidation when exposed to peroxides, H₂O₂ and t‐BOOH. In summary, the Frm1p and Hbn1p nitroreductases influence the response to oxidative stress in S. cerevisae yeast by modulating the GSH contents and antioxidant enzymatic activities, such as SOD, CAT and GPx. Copyright © 2009 John Wiley & Sons, Ltd.