Systematic analysis of yeast strains with possible defects in lipid metabolism

Lipids are essential components of all living cells because they are obligate components of biological membranes, and serve as energy reserves and second messengers. Many but not all genes encoding enzymes involved in fatty acid, phospholipid, sterol or sphingolipid biosynthesis of the yeast Saccharomyces cerevisiae have been cloned and gene products have been functionally characterized. Less information is available about genes and gene products governing the transport of lipids between organelles and within membranes or the turnover and degradation of complex lipids. To obtain more insight into lipid metabolism, regulation of lipid biosynthesis and the role of lipids in organellar membranes, a group of five European laboratories established methods suitable to screen for novel genes of the yeast Saccharomyces cerevisiae involved in these processes. These investigations were performed within EUROFAN (European Function Analysis Network), a European initiative to identify the functions of unassigned open reading frames that had been detected during the Yeast Genome Sequencing Project. First, the methods required for the complete lipid analysis of yeast cells based on chromatographic techniques were established and standardized. The reliability of these methods was demonstrated using tester strains with established defects in lipid metabolism. During these investigations it was demonstrated that different wild‐type strains, among them FY1679, CEN.PK2‐1C and W303, exhibit marked differences in lipid content and lipid composition. Second, several candidate genes which were assumed to encode proteins involved in lipid metabolism were selected, based on their homology to genes of known function. Finally, lipid composition of mutant strains deleted of the respective open reading frames was determined. For some genes we found evidence suggesting a possible role in lipid metabolism. Copyright © 1999 John Wiley & Sons, Ltd.     

Physical map locations of the phospholipid biosynthetic structural and regulatory genes of Saccharomyces cerevisiae

Here we report the physical map locations of five genes required for phospholipid biosynthesis in Saccharomyces cerevisiae. These include four structural genes (INO1, CHO2, OP13 and PIS1) and one global negative regulatory gene (UME6). Collectively, this information completes the mapping of all phospholipid biosynthetic structural and regulatory genes identified to date.     

Molecular biology of Fusarium mycotoxins

As the 20th century ended, Fusarium mycotoxicology entered the age of genomics. With complete genomes of Fusarium graminearum and F. verticillioides and several Fusarium gene expression sequence databases on hand, researchers worldwide are working at a rapid pace to identify mycotoxin biosynthetic and regulatory genes. Seven classes of mycotoxin biosynthetic genes or gene clusters have been identified in Fusarium to date; four are polyketide synthase gene clusters for equisetin, fumonisins, fusarins, and zearalenones. Other Fusarium mycotoxin biosynthetic genes include a terpene cyclase gene cluster for trichothecenes, a cyclic peptide synthetase for enniatins, and a cytochrome P450 for butenolide. From the perspective of the United States Department of Agriculture, the ultimate goal of research on Fusarium molecular biology is to reduce mycotoxins in cereal grains. With this goal in mind, efforts have focused on identifying aspects of mycotoxin biosynthesis and regulation that can be exploited for mycotoxin control. New information on fungal and plant genomes and gene expression will continue to provide information on genes important for fungal-plant interactions and to facilitate the development of targeted approaches for breeding and engineering crops for resistance to Fusarium infection and mycotoxin contamination.     

Metabolism of phospholipids in the yeast Schizosaccharomyces pombe

The fission yeast Schizosaccharomyces pombe is an important model organism for the study of fundamental questions in eukaryotic cell and molecular biology. A plethora of cellular processes are membrane associated and/or dependent on the proper functioning of cellular membranes. Phospholipids are not only the basic building blocks of cellular membranes; they also serve as precursors to numerous signaling molecules. In this review, we describe the biosynthetic pathways leading to major S. pombe phospholipids, how these pathways are regulated, and what is known about degradation and turnover of fission yeast phospholipids. This review also addresses the synthesis, regulation and the role of water-soluble phospholipid precursors. The last chapter of the review is devoted to the use of S. pombe for the biotechnological production of value-added lipid molecules.     

Two-Dimensional protein map of Saccharomyces cerevisiae: Construction of a gene–protein index

This publication marks the beginning of the construction of a gene–protein index that relates proteins which are resolved on the two-dimensional protein map of Saccharomyces cerevisiae with their corresponding genes. We report the identification of 36 novel polypeptide spots on the yeast protein map. They correspond to the products of 26 genes. Together with the polypeptide spots previously identified, this raises to 41 the number of genes whose products have been identified on the protein map. The proteins identified here are concerned with four major areas of yeast cellular physiology: carbon metabolism, heat shock, amino acid biosynthesis and purine biosynthesis. Given the molecular weight and isoelectric point of the identified proteins, and the codon-usage bias of the corresponding genes, it can be estimated that 25 to 35% of all the soluble yeast proteins are detectable under the labelling and running gel conditions used in this study.     

Molecular,functional and evolutionary characterization of the gene encoding HMG-CoA reductase in the fission yeast,Schizosaccharomyces pombe

The synthesis of mevalonate, a molecule required for both sterol and isoprene biosynthesis in eukaryotes, is catalysed by 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase. Using a gene dosage approach, we have isolated the gene encoding HMG-CoA reductase, hmg1+, from the fission yeast Schizosaccharomyces pombe (Accession Number L76979). Specifically, hmg1+ was isolated on the basis of its ability to confer resistance to lovastatin, a competitive inhibitor of HMG-CoA reductase. Gene disruption analysis showed that hmg1+ was an essential gene. This result provided evidence that, unlike Saccharomyces cerevisiae, S. pombe contained only a single functional HMG-CoA reductase gene. The presence of a single HMG-CoA reductase gene was confirmed by genomic hybridization analysis. As observed for the S. cerevisiae HMG1p, the hmg1+ protein induced membrane proliferations known as karmellae. A previously undescribed ‘feed-forward’ regulation was observed in which elevated levels of HMG-CoA synthase, the enzyme catalysing the synthesis of the HMG-CoA reductase substrate, induced elevated levels of hmg1+ protein in the cell and conferred partial resistance to lovastatin. The amino acid sequences of yeast and human HMG-CoA reductase were highly divergent in the membrane domains, but were extensively conserved in the catalytic domains. We tested whether the gene duplication that produced the two functional genes in S. cerevisiae occurred before or after S. pombe and S. cerevisiae diverged by comparing the log likelihoods of trees specified by these hypotheses. We found that the tree specifying post-divergence duplication had significantly higher likelihood. Moreover, phylogenetic analyses of available HMG-CoA reductase sequences also suggested that the lineages of S. pombe and S. cerevisiae diverged approximately 420 million years ago but that the duplication event that produced two HMG-CoA reductase genes in the budding yeast occurred only approximately 56 million years ago. To date, S. pombe is the only unicellular eukaryote that has been found to contain a single HMG-CoA reductase gene. Consequently, S. pombe may provide important opportunities to study aspects of the regulation of sterol biosynthesis that have been difficult to address in other organisms and serve as a test organism to identify novel therapies for modulating cholesterol synthesis.     

Complementation analysis reveals a potential role of human ARV1 in GPI anchor biosynthesis

ARV1 is involved in regulating lipid homeostasis but also in the biosynthesis of glycosylphosphatidylinositol (GPI) in Saccharomyces cerevisiae. Here, we examined whether human ARV1 can complement the role of yeast ARV1 in GPI biosynthesis. Overexpression of human ARV1 could rescue the phenotypes associated with GPI anchor synthesis defect in the yeast arv1Δ mutant. The results suggest that Arv1 function in GPI biosynthesis may be conserved in all eukaryotes, from yeast to humans. Copyright © 2015 John Wiley & Sons, Ltd.     

虾青素化学和生物合成研究进展

虾青素是一种重要的次级类胡萝卜素,具有极强的抗氧化性能,在食品、化工、医疗、水产养殖等方面具有广泛应用。虾青素的合成方法有化学合成及生物合成,化学合成是目前商业化虾青素来源,但生物合成的虾青素更安全,在食品、保健品行业更受欢迎。研究发现生物体内虾青素的代谢合成与油脂代谢路径间存在着一定的联系。本文综述了虾青素的化学合成法和生物合成法,重点综述了微生物中参与虾青素生物合成的关键基因及其代谢调控网络。概述了利用随机诱变、代谢工程、酶工程等手段提高细菌、酵母、海洋真核微生物等虾青素合成积累的研究进展。本文可为虾青素的高效合成研究提供理论指导。     

Green fluorescent protein as a marker for gene expression and subcellular localization in budding yeast

The green fluorescent protein (GFP) from the jellyfish Aequorea victoria has attracted much attention as a tool to study a number of biological processes. This study describes the use of GFP as a vital reporter molecule for localization and expression studies in Saccharomyces cerevisiae. Construction of GFP expression vectors which allow N- or C-terminal fusion of the gfp gene to a gene of interest allowed the generation of fusion proteins whose subcellular localization was followed by fluorescence microscopy in living yeast cells. Analysis of three unknown open reading frames obtained from the budding yeast chromosome XIV resulted in distinct staining patterns, allowing prediction of the cellular localization of these unknown proteins. Furthermore, GFP was used to construct a gene replacement cassette which, after homologous integration into the genomic locus, placed the gfp gene behind a promoter of interest. The amount of GFP produced from this promoter was then quantified in living yeast cells by flow cytometry. With this novel replacement cassette a gene of interest can be deleted and at the same time its expression level studied under various growth conditions. The experiments presented here suggest that GFP represents a convenient fluorescent marker for localization studies as well as gene expression studies in budding yeast. Systematic studies of a large number of genes should benefit from such assays.     

FEN2: A gene implicated in the catabolite repression-mediated regulation of ergosterol biosynthesis in yeast

We have isolated and characterized a pleiotropic recessive mutation, fen2-1, that causes resistance to fenpropimorph and a low level of ergosterol in Saccharomyces cerevisiae. Ergosterol synthesis in the mutant strain was 5·5-fold slower than in the wild type; however, in vitro assays of the enzymes involved in ergosterol biosynthesis could not account for this low rate in the mutant. The mutant phenotype was expressed only in media exerting both carbon and nitrogen catabolite repression. To our knowledge, this is the first locus in yeast that reveals a concerted regulation between different pathways (carbon and nitrogen catabolite repression and/or general control of amino acid biosynthesis and ergosterol biosynthesis). The yeast gene FEN2 has been isolated and contains an open reading frame (ORF) of 512 codons. This ORF was found to be identical to YCR28C of chromosome III. A possible function of the FEN2 gene product in yeast is discussed.     

Function and regulation of yeast genes involved in higher alcohol and ester metabolism during beverage fermentation

The biochemical formation of yeast-derived sensory-active metabolites like higher alcohols and esters determines the different characteristics of aroma and taste in fermented beverages. In yeast fermentation process, a large number of environmental factors affecting the production of volatile aroma compounds are abundant. Factors like substrate composition in fermentation media as well as process parameters influencing these flavor-active metabolites have already been described. These factors can act on the expression of yeast genes involved in aroma metabolism resulting in concentration differences in esters and higher alcohols important for flavor and taste. The understanding of the function of genes involved in biosynthetic pathways of aroma-active substances as well as their regulatory mechanisms is needed to control the production of ester and higher alcohol synthesis to create specific aroma profiles in fermented beverages. This review discusses the known regulation and function of several individual genes (ATF1, ATF2, EEB1, EHT1, BAT1, BAT2 and BAP2) described in fusel alcohol and ester synthesis mainly in S. cerevisiae and S. pastorianus var. carlsbergensis. Also, different factors like oxygen and temperature that allow ester and higher alcohol synthesis to be controlled during yeast fermentation are described.     

Comparative proteomic analysis of Rhodosporidium toruloides during lipid accumulation

Intracellular lipid accumulation is a common biological process for some eukaryotic microorganisms under specific growth conditions, yet study on this phenomenon at an ‘‐omics’ level remains rare. In this study we induced lipid accumulation by the oleaginous yeast Rhodosporidium toruloides by transferring cells into a nitrogen‐limited medium and performed a comparative and semi‐quantitative proteomic analysis of cell samples obtained thereafter by a 2D‐LC–MS/MS approach. A total of 184 proteins were identified, based on the database of yeast Saccharomyces cerevisiae. Semi‐quantitative analysis suggested that 46 proteins were notably changed during the lipid production process. Among them, seven, three and four proteins were significantly upregulated only at the late stage, the early stage and both stages, respectively. There were 26 proteins drastically downregulated at both stages. The majority of the downregulated proteins are related to protein metabolism and carbohydrate metabolism, whereas the upregulated proteins are mainly involved in alternative nitrogen sources metabolism and lipid biosynthesis. Our data indicated that a nitrogen deficiency environment had a key impact on cellular metabolism that likely stimulated the lipid accumulation process by R. toruloides. This work provids valuable information for further exploration of the molecular mechanism of cellular lipid metabolism and should be of great interest in oleaginous microorganisms engineering. Copyright © 2009 John Wiley & Sons, Ltd.     

破囊壶菌属微生物中超长链多不饱和脂肪酸的生物合成及其代谢工程应用

超长链多不饱和脂肪酸（VLCPUFAs）包括ARA、EPA、DHA等不但能营养机体,又具有独特生理功能而受到广泛的关注。破囊壶菌属微生物作为VLCPUFAs主要生产者,是鱼类、贝类等海洋生物富集积累VLCPUFAs的重要来源。许多研究指出,破囊壶菌具有大规模工业化生产VLCPUFAs的潜力。但是,目前对其的生物合成途径和组装机理仍未清楚。该综述从破囊壶菌属微生物的生物合成途径、储藏性脂质的组装机理和提高VLCPUFAs产量的基因工程策略三个方面进行介绍,着重对其VLCPUFAs的生物合成途径,及其油脂组装机理开展详细的介绍,并整理、结合一些较为前沿的研究发现对这些途径研究的潜在应用进行探讨,提高人们对破囊壶菌的生物合成及甘油酯组装途径的认知。研究表明,通过异源表达参与VLCPUFAs合成的基因,能在微生物工程菌和油料作物中产生EPA、DPA和DHA等功能油脂,含量能达到总脂的5%~40%,而通过基因工程敲除或改造破囊壶菌微生物的脂质合成途径基因,能提高破囊壶菌DHA产量约3%~55%。这些实例为指导VLCPUFAs的工业化生产,提供理论依据。     

外源CO对采后枣果实链格孢菌浸染过程中蛋白组的影响

为了阐明外源CO处理对链格孢菌(Aternaria alternata)浸染过程中的枣果实蛋白组影响,采用同位素标记蛋白组分析技术测定链格孢菌入侵枣果实过程中枣果实的蛋白质表达水平,鉴定差异蛋白,并对差异蛋白进行基因本体(gene oncology,GO)及基因通路(kyoto encyclopedia of gene...     

胶原的生物合成过程及其调节

胶原的生物合成涉及一系列的复杂过程,主要包括胶原基因的转录、翻译、胶原分子的后加工、分泌及聚 集等。本文介绍了胶原的生物合成过程,生物合成中所涉及的酶,胶原分子的结构,聚集态结构,调节胶 原合成的多种因素,并对已确定的１８种胶原类型,３２种具有遗传特异性的多肽链以及它们在动物体内 的分布作了简要描述。     

食品中主要真菌毒素生物合成途径研究进展

真菌毒素是真菌重要的次生代谢产物。食品和饲料等农产品在收获、储藏、加工过程中广泛受其污染,造成品质下降、经济损失巨大,并严重威胁人身健康。本文对食品中5种常见真菌毒素(黄曲霉毒素、赭曲霉毒素、伏马菌素、玉米赤霉烯酮和脱氧雪腐镰刀菌烯醇)的生物合成途径分别进行阐释。采用克隆基因的传统分子生物学方法和基因组测序的现代测序技术研究发现,真菌毒素的生物合成基因大多成簇存在,该簇中包含控制合成基因表达的调控基因,并受多种应对外部环境的调控基因所控制。因此本文还结合5种真菌毒素的生物合成基因簇及其基因功能进行综合解析。真菌毒素生物合成的研究将为食品和饲料等农产品的防控、预警以及脱毒提供理论基础和指导方向。