Localization of the dominant flocculation genes FLO5 and FLO8 of Saccharomyces cerevisiae

In the yeast Saccharomyces cerevisiae three dominant flocculation genes, FLO1, FLO5 and FLO8 have been described. Until now only the FLO1 gene, which is located at chromosome I, has been cloned and sequenced. FLO5 and FLO8 were previously localized at chromosomes I and VIII respectively (Vezinhet, F., Blondin, B. and Barre, P. (1991). Mapping of the FLO5 gene of Saccharomyces cerevisiae by transfer of a chromosome during cytoduction. Biotechnol. Lett. 13 , 47–52; Yamashita, I. and Fukui, S. (1983). Mating signals control expression of both starch fermentation genes and a novel flocculation gene FLO8 in the yeast Saccharomyces. Agric. Biol. Chem. 47 , 2889–2896). This was not in agreement with our results. Here, we report the location of FLO5 and FLO8 on chromosomes VIII and I respectively. By induced chromosome loss and genetic mapping, the FLO5 gene was localized at the right end of chromosome VIII approximately 34 cM centromere distal of PET3. This is part of the region that is present both at chromosome I and chromosome VIII. The location of FLO5 in this area of chromosome VIII made it necessary to re-evaluate the localization of FLO8, which was previously thought to occur in this region. Both genetic and physical mapping showed that FLO8 is allelic to FLO1. Hence, there are only two known dominant flocculation genes, FLO1 and FLO5. Analysis of the nucleotide sequence of chromosome VIII of a non-flocculent strain revealed an open reading frame encoding a putative protein that is approximately 96% identical to the Flo1 protein. This suggests that both dominant flocculation genes encode similar, cell wall-associated, proteins with the same function in the flocculation mechanism.     

Genetic and physical localization of the acetyl-coenzyme A synthetase gene ACS1 on chromosome I of Saccharomyces cerevisiae

The ACS1 gene, encoding acetyl-coenzyme A synthetase, was mapped genetically at the left arm of chromosome I between pURA3 and PYK1 at 19 and 28 cM respectively. Comparison with the physical map defined a recombinational ‘hot-spot’ in this region in addition to the one between CDC24 and PYK1.     

Sequence of the open reading frame of the FL01 gene from Saccharomyces cerevisiae

The cloned part of the flocculation gene FLO1 of Saccharomyces cerevisiae (Teunissen, A.W.R.H., van den Berg, J.A. and Steensma, H.Y. (1993). Physical localization of the flocculation gene FLO1 on chromosome I of Saccharomyces cerevisiae, Yeast, in press) has been sequenced. The sequence contains a large open reading frame of 2685 bp. The amino acid sequence of the putative protein reveals a serine- and threonine-rich C-terminus (46%), the presence of repeated sequences and a possible secretion signal at the N-terminus. Although the sequence is not complete (we assume the missing fragment consists of repeat units), these data strongly suggest that the protein is located in the cell wall, and thus may be directly involved in the flocculation process.     

Molecular cloning of chromosome I DNA from Saccharomyces cerevisiae: Isolation of the MAK16 gene and analysis of an adjacent gene essential for growth at low temperatures

MAK16 is an essential gene on chromosome I defined by the thermosensitive lethal mak16–1 mutation. MAK16 is also necessary for M double-stranded RNA replication at the permissive temperature for cell growth. As part of an effort to clone all the DNA from chromosome I, plasmids that complemented both the temperature-sensitive growth defect, and the M₁ replication defects of mak16–1 strains were isolated from a plasmid YCp50: Saccharomyces cerevisiae recombinant DNA library. The two plasmids analysed contained overlapping inserts that hybridized proportionally to strains carrying different dosages of chromosome I. Furthermore, integration of a fragment of one of these clones occurred at a site linked to ade1, confirming that this clone was derived from the appropriate region of chromosome I. An open reading frame adjacent to MAK16 potentially coding for a 468 amino acid protein was defined by sequence analysis. 185 amino acids of this open reading frame were replaced with a 1·2 kb fragment carrying the S. cerevisiae URA3 gene by a one-step gene disruption. The resulting strains grew at a rate indistinguishable from the wild type at 20°C, 30°C, or 37°C, but could not grow at 8°C. The deleted region is thus essential only at 8°C, and we name this gene LTE1 (low temperature essential).     

TOR1基因缺失对酿酒酵母耐受性的影响

酿酒酵母是最常见且应用最为广泛的酵母菌种,是以糖质和淀粉质为原料的乙醇发酵最经典的菌株.在发酵过程中,有很多不可避免的胁迫环境如高温条件、高渗条件等出现,这些胁迫会阻碍细胞生长并降低细胞的发酵能力,给发酵行业带来一定的经济损失.因此,为改善菌种的耐受性,该研究主要以实验室现有菌株AY12a为亲本菌株,URA3基因作筛选...     

Para-aminobenzoate synthase gene of Saccharomyces cerevisiae encodes a bifunctional enzyme

The Saccharomyces cerevisiae gene for para-aminobenzoate (PABA) synthase has been identified based upon its ability to confer sulfonamide resistance when present on a multicopy episomal vector. The 3840 bp DNA sequence fragment reported here contains a 2199 bp open reading frame encoding a 733 amino acid protein with similarity to the two components of PABA synthase described for prokaryotes (Escherichia coli PabA and PabB), suggesting that PABA synthase is bifunctional in yeast. The cloned sequence was confirmed to be PABA synthase by gene disruption. Chromosome gel analysis places the gene for PABA synthase on chromosome XIV.     

乙醇脱氢酶I基因敲除的酿酒酵母重组菌构建的初步研究

本实验根据酿酒酵母乙醇代谢途径,构建一株低乙醇产量的酿酒酵母基因工程菌株,以满足人们对低醇啤酒的需要。利用抗性基因筛选基因敲除突变体的方法,通过引物L1和L2扩增潮霉素B基因(两翼与酿酒酵母同源),按常规醋酸锂法转化酵母细胞后,筛选标记与酵母adhI基因发生同源重组,得到一株ADHI酶活性降低的工程菌株。发酵实验结果表明,转化菌株乙醇含量平均值为1.8%(V/V),较原始菌株低了65%。说明转化菌株体内乙醇生成途径受到干扰。     

Low-Affinity glucose carrier gene LGT1 of Saccharomyces cerevisiae,a homologue of the Kluyveromyces lactis RAG1 gene

A low-affinity glucose transporter gene of Saccharomyces cerevisiae was cloned by complementation of the rag1 mutation in a strain of Kluyveromyces lactis defective in low-affinity glucose transport. Gene sequence and effects of null mutation in S. cerevisiae were described. Data indicated that there are multiple genes for low-affinity glucose transport.     

酿酒酵母细胞表面电位与絮凝相关性分析

为探究酿酒酵母细胞表面所带电荷与絮凝的相关性,以两种液态发酵酵母和两种固态发酵酵母为研究对象,并对液态发酵酵母FFC2144和固态发酵酵母L2Y进行连续传代培养,用zeta电位仪测定其zeta电位,以细胞沉降速率的方法考察酵母的絮凝性。结果显示,离子强度为0时,固态发酵酵母zeta电位(绝对值)均小于30 m V,液态发酵酵母均大于30 m V,培养72 h后的固态发酵酵母絮凝率达到了80.2%,液态发酵酵母絮凝率最大仅为14.4%。在发酵过程中不同酿酒酵母电位无明显变化。随着传代次数的增加酿酒酵母FFC2144 zeta电位无明显变化,絮凝性能却逐渐增强,到第20代时絮凝率达到了94.8%。不同酿酒酵母具有不同的zeta电位,其大小客观地反映出酿酒酵母絮凝性的强弱。酿酒酵母在衰老过程中细胞的絮凝性与zeta电位无关。      

Saccharomyces cerevisiae SCY1发酵kefir的工艺研究

本文采用Saccharomyces cerevisiaeSCY1和乳酸菌混合发酵牛乳制备kefir,分别研究了接种量、灭菌条件、发酵温度和加糖量对kefir风味的影响,最终确定最佳工艺条件为:XPL-1接种量为0.0400g/L,SCY1接种量为103个/mL;灭菌条件为80~85℃下灭菌10min;发酵温度为32℃;加糖量为4%。通过此工艺条件制备的kefir,具有独特的风味和较高的营养价值。     

Identification and functional analysis of the Saccharomyces cerevisiae nicotinamidase gene, PNC1

Nicotinamidase (NAMase) from the budding yeast, Saccharomyces cerevisiae, was purified by Ni(2+) affinity chromatography and gel filtration. N-terminal microsequencing revealed sequence identity with a hypothetical polypeptide encoded by the yeast YGL037C open reading frame sharing 30% sequence identity with Escherichia coli pyrazinamidase/nicotinamidase. A yeast strain in which the NAMase gene, hereafter named PNC1, was deleted shows a decreased intracellular NAD(+) concentration, consistent with the loss of NAMase activity in the null mutant. In wild-type strains, NAMase activity is stimulated during the stationary phase of growth, by various hyperosmotic shocks or by ethanol treatment. Using a P(PNC1)::lacZ gene fusion, we have shown that this stimulation of NAMase activity results from increased levels of the protein and requires stress response elements in the 5'non-coding region of PNC1. These results suggest that NAMase helps yeast cells to adapt to various stress conditions and nutrient depletion, most likely via the activation of NAD-dependent biological processes.