调控酿酒酵母类异戊二烯合成途径强化芳樟醇合成

芳樟醇是一种重要单萜,广泛应用于食品、医药、日化等工业领域.然而芳樟醇在植物中含量低且难提取,限制了其大规模生产.目前通常以酿酒酵母Saccharomyces cerevisiae作为单萜生物合成宿主,其内源类异戊二烯合成途径提供合成单萜物质的前体——香叶基二磷酸(GPP).由于该途径代谢通量较低,导致GPP供应不足,极大地降低了异源单萜的合成效率.为了调节该途径的代谢通量,构建酿酒酵母整合表达载体pRS305-tHMG1和游离表达载体pYLIS-IDI1,并分别转入酿酒酵母CEN.PK2-lC中,获得酿酒酵母工程菌LS01和LS02.同时将载体pYLIS-IDIl转入酿酒酵母工程菌LS01中,构建酿酒酵母工程菌LS03.GC-MS检测结果显示,通过提高异戊二烯二磷酸异构酶(IDIl)和羟甲基戊二酸单酰辅酶A(HMG-CoA)还原酶活性区域(tHMGl)的表达水平,最终使芳樟醇产量提高1.3倍至(127.71±7.68) tg/L.结果表明,通过调控类异戊二烯合成途径,强化GPP合成前体供给,可以显著提高酿酒酵母中芳樟醇的产量.     

The SRD2 gene is involved in Saccharomyces cerevisiae morphogenesis

Saccharomyces cerevisiaepresents two alternative vegetative forms of growth, switching between yeast forms to pseudohyphal forms depending on the specific environmental conditions. To identify genes involved in cell wall morphogenesis, a haploid S. cerevisiae monomorphic mutant, W27, which exhibits pseudohyphal growth in the absence of the normal external signals that induce the formation of filamentous forms, was characterized. S. cerevisiaeW27 did not demonstrate agar-invasive growth, a characteristic of most filamentous strains. The mutant wall had no obvious alterations with respect to mannan and glucan content, but had three times more chitin than the parental strain. This produced an increase in the amount of proteins linked covalently to chitin. The same protein species, however, were released from the cell walls of the mutant and the parental strain. The W27 mutation was complemented with a genomic library and the SRD2/ECM23 gene was identified as the complementing ORF. Transformation of S. cerevisiaeW27 with the Ycplac33 vector carrying the SRD2 gene produced the original phenotype. These results suggest that the SRD2gene acts as a negative regulator of pseudohyphal growth.     

The glutathione-dependent glyoxalase pathway in the yeast Saccharomyces cerevisiae

Glyoxalase I (EC 4.4.1.5), which catalyzes the reaction methylglyoxal + GSH leads to S-lactoylglutathione, is a ubiquitous enzyme for which no clear physiological function has been shown. In the yeast Saccharomyces cerevisiae, methylglyoxal may derive from the spontaneous decay of intracellular glyceraldehyde-3-P, which may accumulate during growth on glycerol as the carbon source. The half-life time for the triose phosphate was found to be 4.6 h under physiological conditions (pH 6.2, 0.05 M phosphate at 30 degrees C). Glyoxalase I is induced by growth on glycerol or by the addition of methylglyoxal to the growth medium. The enzyme is also subject to carbon catabolite repression. A mutant strain, fully defective in glyoxalase I and bearing only one nuclear mutation, was obtained. The strain, which is killed by exposure to glycerol, excretes methylglyoxal into the medium. Growth of the mutant on glucose as carbon source appears to be similar to that of the wild type strain. This investigation has clearly demonstrated a physiological role of glyoxalase I in a eucaryotic cell.     

Reconstruction of a bacterial isoprenoid biosynthetic pathway in Saccharomyces cerevisiae

A eukaryotic mevalonate pathway transferred and expressed in Escherichia coli, and a mammalian hydrocortisone biosynthetic pathway rebuilt in Saccharomyces cerevisiae are examples showing that transferring metabolic pathways from one organism to another can have a powerful impact on cell properties. In this study, we reconstructed the E. coli isoprenoid biosynthetic pathway in S. cerevisiae. Genes encoding the seven enzymatic steps of the pathway were cloned and expressed in S. cerevisiae. mRNA from the seven genes was detected, and the pathway was shown able to sustain growth of yeast in conditions of inhibition of its constitutive isoprenoid biosynthetic pathway.     

The heparan and heparin metabolism pathway is involved in regulation of fatty acid composition

Six genes involved in the heparan sulfate and heparin metabolism pathway, DSEL (dermatan sulfate epimerase-like), EXTL1 (exostoses (multiple)-like 1), HS6ST1 (heparan sulfate 6-O-sulfotransferase 1), HS6ST3 (heparan sulfate 6-O-sulfotransferase 3), NDST3 (N-deacetylase/N-sulfotransferase (heparan glucosaminyl) 3), and SULT1A1 (sulfotransferase family, cytosolic, 1A, phenol-preferring, member 1), were investigated for their associations with muscle lipid composition using cattle as a model organism. Nineteen single nucleotide polymorphisms (SNPs)/multiple nucleotide length polymorphisms (MNLPs) were identified in five of these six genes. Six of these mutations were then genotyped on 246 Wagyu x Limousin F(2) animals, which were measured for 5 carcass, 6 eating quality and 8 fatty acid composition traits. Association analysis revealed that DSEL, EXTL1 and HS6ST1 significantly affected two stearoyl-CoA desaturase activity indices, the amount of conjugated linoleic acid (CLA), and the relative amount of saturated fatty acids (SFA) and monounsaturated fatty acids (MUFA) in skeletal muscle (P<0.05). In particular, HS6ST1 joined our previously reported SCD1 and UQCRC1 genes to form a three gene network for one of the stearoyl-CoA desaturase activity indices. These results provide evidence that genes involved in heparan sulfate and heparin metabolism are also involved in regulation of lipid metabolism in bovine muscle. Whether the SNPs affected heparan sulfate proteoglycan structure is unknown and warrants further investigation.     

The sexual agglutination substance is secreted through the yeast secretory pathway in Saccharomyces cerevisiae

Molecular Genetics and Genomics - A Saccharomyces cerevisiae a strain carrying the secretory mutation sec1, sec7 or sec18 showed no sexual agglutination ability when treated with α pheromone...     

G-protein-coupled receptor Gpr1 and G-protein Gpa2 of cAMP-dependent signaling pathway are involved in glucose-induced pexophagy in the yeast Saccharomyces cerevisiae

In yeast cell, glucose induces various changes of cellular metabolism on genetic and metabolic levels. One of such changes is autophagic degradation of dispensable peroxisomes (pexophagy) which occurs in vacuoles. We have found that in Saccharomyces cerevisiae, defect of G-protein-coupled receptor Gpr1 and G-protein Gpa2, both the components of cAMP-signaling pathway, strongly suppressed glucose-induced degradation of matrix peroxisomal protein thiolase. We conclude that proteins Gpr1 and Gpa2 are involved in glucose sensing and signal transduction during pexophagy process in yeast.     

Comprehensive evaluation of isoprenoid biosynthesis regulation in Saccharomyces cerevisiae utilizing the Genome Reporter Matrix

Gene expression profiling is rapidly becoming a mainstay of functional genomic studies. However, there have been relatively few studies of how the data from expression profiles integrate with more classic approaches to examine gene expression. This study used gene expression profiling of a portion of the genome of Saccharomyces cerevisiae to explore the impact of blocks in the isoprenoid biosynthetic pathway on the expression of genes and the regulation of this pathway. Approximately 50% of the genes whose expression was altered by blocks in isoprenoid biosynthesis were genes previously known to participate in the pathway. In contrast to this simple correspondence, the regulatory patterns revealed by different blocks, and in particular by antifungal azoles, was complex in a manner not anticipated by earlier studies.     

LCD1: an essential gene involved in checkpoint control and regulation of the MEC1 signalling pathway in Saccharomyces cerevisiae

We identified YDR499W as a Saccharomyces cerevisiae open reading frame with homology to several checkpoint proteins, including S. cerevisiae Rfc5p and Schizosaccharomyces pombe Rad26. Disruption of YDR499W (termed LCD1) results in lethality that is rescued by increasing cellular deoxyribonucleotide levels. Cells lacking LCD1 are very sensitive to a range of DNA-damaging agents, including UV irradiation, and to the inhibition of DNA replication. LCD1 is necessary for the phosphorylation and activation of Rad53p in response to DNA damage or DNA replication blocks, and for Chk1p activation in response to DNA damage. LCD1 is also required for efficient DNA damage-induced phosphorylation of Rad9p and for the association of Rad9p with the FHA2 domain of Rad53p after DNA damage. In addition, cells lacking LCD1 are completely defective in the G(1)/S and G(2)/M DNA damage checkpoints. Finally, we reveal that endogenous Mec1p co-immunoprecipitates with Lcd1p both before and after treatment with DNA-damaging agents. These results indicate that Lcd1p is a pivotal checkpoint regulator, involved in both the essential and checkpoint functions of the Mec1p pathway.