Yeast metallothionein function in metal ion detoxification

A genetic approach was taken to test the function of yeast metallothionein in metal ion detoxification. A yeast strain was constructed in which the metallothionein locus was deleted (cup1 delta). The cup1 delta strain was complemented with normal or mutant metallothionein genes under normal or constitutive regulatory control on high copy episomal plasmids. Metal resistance of the cup1 delta strain with and without the metallothionein-expressing vectors was analyzed. The normally regulated metallothionein gene conferred resistance only to copper (1000-fold); constitutively expressed metallothionein conferred resistance to both copper (500-fold) and cadmium (1000-fold), but not to mercury, zinc, silver, cobalt, nickel, gold, platinum, lanthanum, uranium, or tin. Two mutant versions of the metallothionein gene were constructed and tested for their ability to confer metal resistance in the cup1 delta background. The first had a deletion of a highly conserved amino acid sequence (Lys-Lys-Ser-Cys-Cys-Ser). The second was a hybrid gene consisting of the sequences coding for the first 20 amino acids of the yeast protein fused to the monkey metallothionein gene. Expression of these genes under the CUP1 promoter provided significant protection from copper, but none of the other metals tested. These results demonstrate that there is significant flexibility in the structural requirements for metallothionein to function in copper detoxification and that yeast metallothionein is also capable of detoxifying cadmium under conditions of constitutive expression.     

The CUP2 gene product regulates the expression of the CUP1 gene, coding for yeast metallothionein.

The yeast CUP1 gene codes for a copper-binding protein similar to metallothionein. Copper sensitive cup1s strains contain a single copy of the CUP1 locus. Resistant strains (CUP1r) carry 12 or more multiple tandem copies. We isolated 12 ethyl methane sulfonate-induced copper sensitive mutants in a wild-type CUP1r parental strain, X2180-1A. Most mutants reduce the copper resistance phenotype only slightly. However, the mutant cup2 lowers resistance by nearly two orders of magnitude. We cloned CUP2 by molecular complementation. The smallest subcloned fragment conferring function was approximately 2.1 kb. We show that CUP2, which is on chromosome VII, codes for or controls the synthesis or activity of a protein which binds the upstream control region of the CUP1 gene on chromosome VIII. Mutant cup2 cells produced extremely low levels of CUP1-specific mRNA, with or without added copper ions and lacked a factor which binds to the CUP1 promoter. Integrated at the cup2 site, the CUP2 plasmid restored the basal level and inducibility of CUP1 expression and led to reappearance of the CUP1-promoter binding factor. Taken collectively, our data establish CUP2 as a regulatory gene for expression of the CUP1 metallothionein gene product.     

Regulation of the yeast metallothionein gene

To study regulation of the yeast CUP1 gene, we have employed plasmids containing the CUP1 regulatory sequences fused to the Escherichia coli galK gene. A comparison of galK expression from low- and high-copy-number CUP1/galK fusion plasmids demonstrated that both basal and induced levels of galactokinase (GalK) increase proportionately with plasmid copy number. Host strains with an amplified, single or deleted CUP1 locus were compared to look for effects of chromosomal CUP1 gene dosage on expression from the episomal CUP1 promoter. Basal GalK levels are similar in CUP1R and cupls hosts, but can be induced to higher levels in the cup1s than the CUP1R host. In contrast, in a strain deleted for the chromosomal copy of CUP1, synthesis of GalK is constitutive but can be induced to yet higher levels by copper. A hybrid vector, placing the CUP1 coding sequence under the control of a constitutive promoter, was constructed. Introduction of this hybrid CUP1 gene into the deletion host containing the CUP1/galK plasmid restores regulation. Thus, metallothionein, in trans, can effect repression of the CUP1 promoter. The possible roles of metallothionein and free copper in CUP1 regulation are discussed.     

Chemical synthesis and expression of a cassette adapted ubiquitin gene

A gene encoding the yeast ubiquitin was chemically synthesized and expressed in yeast under regulatory control of the copper metallothionein (CUP1) promoter. The gene was assembled in a one-step ligation reaction from eight oligonucleotide fragments ranging in length from 50 to 64 nucleotides. To facilitate mutagenesis and gene fusion studies, eight unique 6-base-cutting restriction enzyme sites were placed in the reading frame which did not alter the encoded protein sequence or force the utilization of rare codons. In a copper-resistant yeast strain (CUP1r), expression of the gene was induced by copper to approximately 5% of the total yeast proteins, as determined by Coomassie-stained polyacrylamide gels. The protein, purified from yeast, reacted with ubiquitin-specific antibodies and was found to be biologically active in supporting ubiquitin-dependent protein degradation in vitro.     

Structural and functional studies of the amino terminus of yeast metallothionein

Purified yeast copper-metallothionein lacks 8 amino-terminal residues that are predicted from the DNA sequence of its gene. The removed sequence is unusual for metallothionein in its high content of hydrophobic and aromatic residues and its similarity to mitochondrial leader sequences. To study the significance of this amino-terminal cleavage, several mutations were introduced into the metallothionein coding gene, CUP1. One mutant, which deletes amino acid residues 2-8, had a minor effect on the ability of the molecule to confer copper resistance to yeast but did not affect CUP1 gene regulation. A second mutation, which changes two amino acids adjacent to the cleavage site, blocked removal of the extension peptide but had no effect on copper detoxification or gene regulation. Immunofluorescence studies showed that both the wild-type and these two mutant proteins are predominantly cytoplasmic with no evidence for mitochondrial localization. The cleavage site mutation allowed isolation and structural characterization of a full length metallothionein polypeptide. The copper content and luminescent properties of this molecule were identical to those of the truncated wild-type protein indicating a homologous cluster structure. Moreover, the amino-terminal peptide was selectively removed by various endopeptidases and an exopeptidase suggesting that it does not participate in the tertiary fold. These results argue that the amino-terminal peptide is not required for either the structural integrity or biological function of yeast metallothionein.     

Isolation and characterization of a Saccharomyces cerevisiae mutant deficient in pyruvate kinase activity.

A mutant of the yeast Saccharomyces cerevisiae that is deficient in pyruvate kinase activity has been isolated. The mutant strain is capable of growth when supplied with lactate as the carbon source but not capable of growth when supplied with dextrose or other fermentable sugars or glycerol as the carbon source. Genetic analysis demonstrated that the phenotype of the pyruvate kinase-deficient strain was due to a single nuclear mutation, which was designated pyk1, and preliminary genetic mapping experiments located the pyk1 locus on chromosome I, 30 centimorgans from the ade1 locus. Adenine nucleotide levels in the mutant and parental strains were compared when the cells were subjected to various growth and starvation conditions. When carbon supply and energy production were dissociated by supplying the mutant strain with dextrose, adenine nucleotide levels fell dramatically. This result suggests that the initial reactions of glycolysis are not rate limiting, nor are they readily inhibited by feedback controls.     

Versatile cassettes designed for the copper inducible expression of proteins in yeast

A series of yeast expression vectors and cassettes utilizing the CUP1 gene of Saccharomyces cerevisiae have been constructed. The cassettes contain multiple cloning sites for gene fusions and were created by inserting a 27-bp polylinker at the +14 position of the CUP1 gene. The cassettes are portable as restriction fragments and enable copper-regulated expression of foreign proteins in S. cerevisiae. In copper sensitive yeast, multiple copies of the CUP1 cassettes confer copper resistance due to the production of the copper metallothionein. Genes cloned into the CUP1 cassettes, however, usually prevent translation of the metallothionein leading to a loss of resistance. This could be useful for one-step cloning into yeast.     

Extracellular production of palmitoleic triglycerides by a yeast,Trichosporon

A yeast belonging to Trichosporon which produces triglycerides extracellularly was isolated. This strain accumulated palmitoleic triglycerides from ethyl palmitate used as a sole carbon source. To increase the level of extracellular palmitoleic triglycerides, mutant strains which supported growth of unsaturated-fatty-acid-auxotrophic cells (Saccharomyces cerevisiae KD115) layered on the mutant colonies were screened. The mutant strain excreted palmitoleic acid as triglyceride form at a significantly high level, corresponding to about double level of the parental strain.     

Mutational Analysis of Pre-mRNA Splicing in Saccharomyces Cerevisiae Using a Sensitive New Reporter Gene, Cup1

We have developed a new reporter gene fusion to monitor mRNA splicing in yeast. An intron-containing fragment from the Saccharomyces cerevisiae ACT1 gene has been fused to CUP1, the yeast metallothionein homolog. CUP1 is a nonessential gene that allows cells to grow in the presence of copper in a dosage-dependent manner. By inserting previously characterized intron mutations into the fusion construct, we have established that the efficiency of splicing correlates with the level of copper resistance of these strains. A highly sensitive assay for 5' splice site usage was designed by engineering an ACT1-CUP1 construct with duplicated 5' splice sites; mutations were introduced into the upstream splice site in order to evaluate the roles of these highly conserved nucleotides in intron recognition. Almost all mutations in the intron portion of the 5' consensus sequence abolish recognition of the mutated site, while mutations in the exon portion of the consensus sequence have variable affects on cleavage at the mutated site. Interestingly, mutations at intron position 4 demonstrate that this nucleotide plays a role in 5' splice site recognition other than by base pairing with U1 snRNA. The use of CUP1 as a reporter gene may be generally applicable for monitoring cellular processes in yeast.     

GFP工程酵母菌的构建及其对Cu2+的荧光响应

从酿酒酵母基因组DNA中克隆到金属硫蛋白启动子(PCUP1)片段,将绿色荧光蛋白(GFP)基因置于PCUP1的调控下,构建重组质粒pCUP9K-GFP,并通过氯化锂法转化毕赤酵母,获得工程菌株。工程菌细胞及其发酵液中可检出GFP荧光,表明PCUP1能启动外源基因GFP转录,使工程菌表达并分泌GFP。研究发现,工程菌培养液中分别加入10μmol/L的铜、铬、镉和砷离子后,铜处理组GFP荧光强度明显增加,其余三种离子对工程菌荧光强度影响不大;用铜离子诱导后,工程菌发酵上清液的荧光强度明显增强,并与铜离子浓度(0～1mmol/L)呈正相关。研究表明,该工程菌中启动子PCUP1受铜离子诱导,GFP的表达对铜离子具有剂量依赖性,在一定浓度范围内,GFP荧光强度与铜离子浓度呈正相关。     

Construction of self-cloning, indigenous wine strains of Saccharomyces cerevisiae with enhanced glycerol and glutathione production

To improve wine taste and flavor stability, a novel indigenous strain of Saccharomyces cerevisiae with enhanced glycerol and glutathione (GSH) production for winemaking was constructed. ALD6 encoding an aldehyde dehydrogenases of the indigenous yeast was replaced by a GPD1 and CUP1 gene cassette, which are responsible for NAD-dependent glycerol-3-phosphatase dehydrogenase and copper resistance, respectively. Furthermore, the α-acetohydroxyacid synthase gene ILV2 of the indigenous yeast was disrupted by integration of the GSH1 gene which encodes γ-glutamylcysteine synthetase and the CUP1 gene cassette. The fermentation capacity of the recombinant was similar to that of the wild-type strain, with an increase of 21 and 19?% in glycerol and GSH production. No heterologous DNA was harbored in the recombinant in this study.     

具有a-淀粉酶分泌活性以及低双乙酰产量的啤酒酵母工程菌的构建

扣囊复膜酵母(Saccharomycopsis fibuligera)因具有较强的a-淀粉酶以及葡聚糖酶活性, 使其在以淀粉为唯一碳源的培养基上能够良好的生长。从其基因组中克隆了a-淀粉酶的编码区, 构建了由酵母磷酸甘油酸激酶基因(PGK1)启动子、酿酒酵母a-因子信号序列以及扣囊复膜酵母a-淀粉酶基因编码序列组成的基因表达盒。将该表达盒插入到质粒pPLZ-2的ILV2基因序列内部, 使其两翼具有ILV2基因的同源区。将该表达盒通过同源重组的方式整合到啤酒酵母工业菌株YSF-5的a-乙酰乳酸合成酶(AHAS)基因ILV2内部。在以淀粉为唯一碳源的培养基上进行转化子的筛选。通过多对引物PCR、a-淀粉酶活性以及AHAS活性分析对转化子进行鉴定, 得到一株具有a-淀粉酶分泌表达活性、较低AHAS活性, 并且发酵液中双乙酰产量也相对较低的啤酒酵母工程菌。该菌株在非选择压力条件下连续培养50代后仍然保持其遗传稳定性。还对pH、温度以及金属离子对该转化菌株的a-淀粉酶活性的影响进行了研究。由于所构建的菌株不含有非酵母来源的DNA, 所以生物安全性相对较高, 对酵母育种以及啤酒生产工业都具有较为重要的意义。     

Citric acid production by 2-deoxyglucose-resistant mutant strains of Aspergillus niger

Summary Many mutant strains showing resistance to 2-deoxy-d-glucose (DG) on minimal medium containing glycerol as a carbon source were induced from Aspergillus niger WU-2223L, a citric acid-producing strain. The mutant strains were classifiable into two types according to their growth characteristics. On the agar plates containing glucose as a sole carbon source, mutant strains of the first type showed good growth irrespective of the presence or absence of DG. When cultivated in shake cultures, some strains of the first type, such as DGR1–2, showed faster glucose consumption and growth than strain WU-2223L. The period for citric acid production shortened from 9 days for strain WU-2223L to 6–7 days for these mutant strains. The levels and yields of citric acid production of the mutant strains were almost the same as those of strain WU-2223L. The mutant strains of the second type, however, showed very slow or no growth on both the agar plates containing glucose and fructose as sole carbon sources. In shake cultures, mutant strains such as DGR2-8 showed decreased glucose consumption rates, resulting in very low production of citric acid.     

Transport of acetate in mutants of Saccharomyces cerevisiae defective in monocarboxylate permeases

The strain Saccharomyces cerevisiae W303-1a, able to grow in a medium containing acetic acid as the sole carbon and energy source, was subjected to mutagenesis in order to obtain mutants deficient in monocarboxylate permeases. Two mutant clones exhibiting growth in ethanol, but unable to grow in a medium with acetic acid as the sole carbon and energy source, were isolated (mutants Ace12 and Ace8). In both mutants, the activity for the acetate carrier was strongly affected. The mutant Ace8 revealed not to be affected in the transport of lactate, while the mutant Ace12 did not display activity for that carrier. These results reinforced those previously found in the strain IGC 4072, where two distinct transport systems for monocarboxylates have been described, depending on the growth carbon source. It is tempting to postulate that the Ace8 mutant seems to be affected in the gene coding for an acetate permease. In contrast, the absence of activity for both monocarboxylate permeases in mutant Ace12 could be attributed to a mutation in a gene coding for a regulatory protein not detected before.