The fission yeast MAPK Spc1 senses perturbations in Cdc25 and Wee1 activities and targets Rad24 to restore this balance

Mitogen‐activated protein kinases (MAPKs) play vital roles in multiple cellular processes and represent prominently pursued targets for development of therapeutic regimes. The MAPK Spc1 (p38 homologue) is known to be very important for both mitotic promotion and delay in Schizosaccharomyces pombe. However, the mechanism responsible for mitotic inhibition has remained elusive. Cdc25 (Cdc2 activator) and Wee1 (Cdc2 inhibtor) are important determinants of mitotic timing in all eukaryotes. Our results show that Spc1 can sense the perturbations in the balance of Cdc25 and Wee1 activities in S. pombe and that its function as a mitotic inhibitor is very important for controlling the same. An Spc1–Srk1–Rad24‐dependent pathway for mitotic inhibition has been reported earlier.Here we report the presence of an alternative mechanism wherein Spc1 targets the 14–3–3 protein, Rad24, independently of Srk1, leading to relocalization of Cdc25 and mitotic inhibition. Our observations suggest that this pathway can serve as a backup mechanism for Cdc2 inactivation in the absence of Wee1.     

发酵过程中啤酒酵母生长代谢活性的电化学分析

首次报道了电化学分析方法在 4 80t啤酒酵母发酵过程中对酵母细胞生长代谢的连续检测。从 0代到 4代 ,从满罐到降温结束 ,跟踪了 5代酵母发酵的全过程。研究表明 ,电化学方法可以描述细胞生长代谢的过程 ,反应细胞的代谢活性 ,在相应地细胞生长代谢阶段 ,细胞的电化学活性与细胞数有良好的线形关系。因此细胞电化学方法为酵母发酵过程提供了一个快速、准确的细胞代谢过程及代谢活性的分析方法     

Genetic/genomic evidence for a key role of polarized endocytosis in filamentous differentiation of S. cerevisiae

Unicellular S. cerevisiae cells switch from the yeast form to pseudohyphal or filamentous form in response to environmental cues. We report that wild-type BY diploids (in which yeast ORFs have been systematically deleted) undergo normal HU-induced filamentous growth and discernable nitrogen starvation-induced filamentous growth, despite their perceived filamentation-deficient S288C genetic background. This finding allowed us to perform a genome-wide survey for non-essential genes that are required for filamentous growth with the homozygous deletion strains. We report that genes involved in endocytosis are required for both HU-induced and nitrogen starvation-induced filamentous growth. Surprisingly, no known genes involved in exocytosis are required. Despite the fact that polarized growth involves transport of vesicles to the site of growth, we failed to obtain genetic/genomic evidence that exocytosis plays an essential role in filamentous growth. A possible key role of polarized endocytosis (from the growth tip) is consistent with the proposed biological function of filamentous growth as a foraging behaviour. In addition, BUD8 that encodes the distal landmark in yeast-form bipolar budding is required for nitrogen starvation-induced but not HU-induced filamentous growth. Moreover, BUD5, SPA2, PEA2 and BUD6 that regulate bipolar bud site selection do not regulate the unipolar distal budding pattern in HU-induced filamentous growth.     

酿酒酵母菌株发酵过程中硫化氢产率动态变化研究

利用3,5-二硝基水杨酸（DNS）法和硫化氢检测管检测法分析了高／低产硫化氢酿酒酵母菌株的发酵特性及其硫化氢产生特性。结果显示：高／低产硫化氢酿酒酵母茵株在发酵过程中的硫化氢产率变化趋势同为“双峰”趋势,且发酵前期峰值出现时间相同;高／低产硫化氢酿酒酵母在发酵速率和硫化氢产生比率上存在一定差异,且不同菌株的发酵速率与硫化氢产生比率之间没有必然联系。     

Screening of native S. cerevisiae strains in the production of Pajarete wine: a tradition of Atacama Region,Chile

Pajarete is a Chilean wine with an appellation of origin. Although it has organoleptic properties, intensive utilization of Saccharomyces cerevisiae commercial yeast through the years has presumably produced the loss of native strains that may be associated with Pajarete oenologic uniqueness. In order to evaluate the effect of re-incorporation of indigenous strains into Pajarete winemaking, native S. cerevisiae strains were isolated and selected based on their properties shown during small and large laboratory scale fermentation, and then evaluated in industrial bioreactors. From an initial set of 312 isolates, a single native strain was selected based on taxonomy, fermentation performance, aroma, residual sugars, and production of alcohol for incorporation into market scale.     

Comparison of three expression systems for heterologous xylanase production by S. cerevisiae in defined medium

The influence of the auxotrophic deficiencies of the host strain and expression vector selection on the production of a heterologous protein was investigated. Heterologous xylanase production by two prototrophic S. cerevisiae transformants, containing either a plasmid-based, YEp-type expression system or an integrative, YIp-type expression system, were compared with production by an auxotrophic transformant, containing an identical YEp-type expression system, in batch and continuous cultivation, using a chemically defined medium. Heterologous xylanase production by the auxotrophic strains in defined medium was critically dependent on the availability of amino acids, as extracellular xylanase production increased dramatically when amino acids were over-consumed from the medium to the point of saturating the cell. Saturation with amino acids, indicated by an increased leakage of amino acids from the cell, was thus a prerequisite for high level of heterologous protein production by the auxotrophic strain. Maximal xylanase production levels by the auxotrophic strain corresponded to the levels obtained with a similar prototrophic strain during cultivation in defined medium without amino acids. Superfluous auxotrophic markers thus had a strong deleterious effect on heterologous protein production by recombinant yeasts, and the use of such strains should be limited to initial exploratory investigations. The increased copy number and foreign gene dosage of the YEp-based expression vector, stabilized by the ura3 fur1 autoselection system, significantly improved production levels of heterologous xylanase, compared to the YIp system, which is based on a single integration into the yeast genome. No evidence was found of the possible saturation of the host secretory capacity by multicopy overexpression. Stable production of heterologous xylanase at high levels by the prototrophic YEp-based recombinant strain, compared to the YIp system, was demonstrated.     

酿酒酵母26SrDNAD1/D2区域序列分析及其系统发育研究

利用26SrDNAD1/D2区序列分析法对中国工业微生物菌种保藏管理中心(CICC)保藏的22株酿酒酵母(Saccharomycescerevisiae)和1株威尔酵母(S.willianus)进行了复核鉴定,通过序列比对及构建系统发育树分析后,结果显示:21株菌株与原名称一致,与酿酒酵母CBS1171T序列相似性在99.0%以上;CICC1313原定名为威尔酵母,此次鉴定结果为酿酒酵母,与酿酒酵母CBS1171T序列相似性为99.8%;CICC1859与异常毕赤酵母(Pichiaanomala)CBS5759T相似性为100%,鉴定为异常毕赤酵母.进一步对酿酒酵母与酵母属内其他种之间的发育关系进行了分析,通过构建酵母属内各菌种模式株的26SrDNAD1/D2区系统发育树,发现酿酒酵母与属内其他菌种间差异均大于1%,表明26SrDNAD1/D2区域序列分析方法能够应用于酿酒酵母的分子生物学鉴定.     

Production of Beer with a Genetically Engineered Strain of S. cerevisiae with Modified Beta Glucanase Expression

This study used a recombinant Saccharomyces cerevisiae strain, which expressed both β‐glucanase enzyme and reduced Pro‐teinase A expression during wort fermentations. The genetic stability and fermentation features of the strain were examined. The recombinant strain's proteinase A activity was reduced compared to the parent strain; β‐glucanase was produced throughout the fermentation. The fermentation with the recombinant S. cerevisiae strain exhibited a larger reduction in β‐glucan content than what was observed with the control strain, with β‐glucan degradation above 80%. The foam stability period was reduced when the beer produced by the recombinant S. cerevisiae was stored for 3 months. SDS‐PAGE analysis of the beer proteins indicated that lipid transfer protein 1 had disappeared. Fermentation studies indicated that based on the parameters examined, this recombinant strain was suitable for industrial beer production.     

A PMR1-like calcium ATPase of Aspergillus fumigatus: cloning, identification and functional expression in S. cerevisiae

The understanding of the controlling factors of calcium homeostasis in Aspergillus fumigatus is very poor, although this ion is involved in several important events of these particular cells. We have cloned, identified and expressed for functional complementation a PMR1-like Ca(2+)-ATPase gene from A. fumigatus. The Afpmr1 gene encodes a protein of 1061 deduced amino acids, containing all the conserved subdomains found in other P-type ATPases: the phosphatase region, phosphorylation site, FITC labelling site, ATP binding domain; E(386), N871, D875 amino acid residues for calcium ion interaction and Q880, a residue that alters ion selectivity in PMR1. The expressed AfPMR1 in S. cerevisiae K616 strain functionally complemented the deficient growth in EGTA (5-20 mM)- and MnCl2 (4 mM)-containing medium. These results demonstrate the first evidence of a Ca(2+)-ATPase in A. fumigatus and strongly suggest a role for this enzyme in calcium and manganese homeostasis.     

Expression,purification, and characterization of N-acetylglucosaminylphosphatidylinositol de-N-acetylase (ScGpi12), the enzyme that catalyses the second step of GPI biosynthesis in S. cerevisiae

ScGpi12 is a 304 amino residue long endoplasmic reticulum membrane protein, which participates in the de-N-acetylation of N-acetylglucosaminyl phosphatidylinositol to produce glucosaminyl phosphatidylinositol in the second step of GPI anchor biosynthesis pathway in Saccharomyces cerevisiae. ScGpi12 was cloned in a pMAL-c2x vector and expressed heterologously in Rosetta-gami (DE3) strain of E. coli. Affinity purification of the protein yielded low amounts of the MBP-tagged enzyme, which was active. To the best of our knowledge, this is the first successful purification of full-length Gpi12 enzyme, without the accompanying GroEL that was seen in other studies. The presence of the tag did not greatly alter the activity of the enzyme. ScGpi12 was optimally active in the pH range of 6.5–8.5 and at 30 °C. It was not sensitive to treatment with EDTA but was stimulated by multiple divalent cations. The divalent cation did not alter the pH profile of the enzyme, suggesting no role of the divalent metal in creating a nucleophile for catalysis. Divalent cations did, however, enhance the turnover number of the enzyme for its substrate, suggesting that they are probably required for the production of a catalytically competent active site by bringing the active site residues within optimum distance of the substrate for catalysis.     

“珠研”2~＃菌株缩短发酵周期的生产性试验研究（初报）

经诱变、筛选处理的“珠研”２~＃菌株经小试、中试证明其啤酒发酵性能比原引进的菌株有较大的优越性。为验证此菌株对麦汁及扩大生产的适应性，我们对此菌株进行生产性扩大试验。实践证明，“珠研”２~＃菌株对麦汁及扩大生产适应性较强。该菌株具有发酵温度高、降糖快、双乙酰还原能力强、酵母凝集性好、发酵周期短、生产的啤酒质量优越等优点。取得与小试、中试基本一致的结果。我们认为“珠研”２~＃菌株啤酒快速发酵新工艺，可望有良好的推广应用前景。