Construction of an autonomously replicating plasmid in n-alkane-assimilating yeast, Candida tropicalis

A transformation system using the autonomously replicating plasmid in the n-alkane-assimilating and asporogenic diploid yeast, Candida tropicalis, was developed. For the cloning of a DNA fragment containing a potential autonomously replicating sequence (ARS) from the genomic DNA of C. tropicalis, the ura3 mutant obtained using ethylmethane sulfonate as the host and the URA3 gene amplified by PCR using the C. tropicalis genomic DNA as a selectable marker were prepared. Comparison of ARSs among yeasts revealed that the consensus sequence found in S. cerevisiae was also present in C. tropicalis. The autonomously replicating plasmid containing the putative ARS as the shuttle vector, capable of replicating in both E. coli and C. tropicalis, was first constructed. The transformation system using this plasmid, in addition to the integrative transformation system, will be applicable to genetic studies of C. tropicalis.     

Molecular cloning and analysis of autonomous replicating sequence of Candida maltosa.

A Candida maltosa chromosomal DNA fragment which confers high frequency transformation of C. maltosa and autonomous replication of recombinant plasmids was cloned and sequenced. Analysis of the nucleotide sequence of the cloned DNA revealed a sequence homologous for C. maltosa autonomously replicating sequence (ARS) elements. Vector pRJ1 for C. maltosa was constructed, which contained a 1.3 kb ARS sequence, pICEM-19H and the ADE1 gene of C. maltosa. Southern blot analysis suggested that the copy number of pRJ1 in C. maltosa was approximately 20 per genome. The sequence analysis also revealed an open reading frame, encoding a polypeptide with high homology (70%) to the RS15 protein of Brugia pagangi. This open reading frame has an intron with canonical sites for correct splicing in Saccharomyces cerevisiae.     

Construction of an autonomously replicating plasmid in n-alkane-assimilating yeast, Candida tropicalis

A transformation system using the autonomously replicating plasmid in the n-alkane-assimilating and asporogenic diploid yeast, Candida tropicalis, was developed. For the cloning of a DNA fragment containing a potential autonomously replicating sequence (ARS) from the genomic DNA of C. tropicalis, the ura3 mutant obtained using ethylmethane sulfonate as the host and the URA3 gene amplified by PCR using the C. tropicalis genomic DNA as a selectable marker were prepared. Comparison of ARSs among yeasts revealed that the consensus sequence found in S. cerevisiae was also present in C. tropicalis. The autonomously replicating plasmid containing the putative ARS as the shuttle vector, capable of replicating in both E. coli and C. tropicalis, was first constructed. The transformation system using this plasmid, in addition to the integrative transformation system, will be applicable to genetic studies of C. tropicalis.     

Development of a transformation system for the multinuclear yeast Dipodascus (Endomyces) magnusii

We have developed the first system for genetic transformation of the multinuclear yeast Dipodascus magnusii. The system is based on a dominant selectable marker and an autonomously replicating sequence. We have constructed a plasmid vector which contains a marker conferring resistance to zeocin and the segment of non-transcribed spacer of D. magnusii ribosomal DNA which supports the autonomous replication of plasmid DNA in yeast cells. Plasmid DNA has been transferred into D. magnusii cells by electroporation. The DNA sequence which is described in this article has been deposited in the EMBL data library under Accession Number Y14587. © 1998 John Wiley & Sons, Ltd.     

A strong carbon source effect is mediated by pUC plasmid sequences in a series of classical yeast vectors designed for promoter characterization

While using YIp356 and YEp356R lacZ reporter plasmids, we found lacZ expression driven by the ARG2 promoter to be much higher in cells grown on a non‐glucose carbon source than in glucose‐grown cells (5–10‐fold higher on galactose and up to 40‐fold higher on ethanol). Furthermore, expression increased 30‐fold upon shifting from a high‐glucose to a low‐glucose medium. This carbon source regulation requires Snf1p and possibly Ssn6p. It appears, however, to be artefactually mediated by plasmid sequences located upstream from the multicloning site. This emerged from the following observations: (a) the derepressive effect disappears if any extra piece of DNA is inserted upstream from the ARG2 promoter; and (b) similar derepression on low glucose is observed with another yeast promoter (ARG11), provided that the flanking 5′ region is short. We determined that the cis‐elements responsible for this physiologically irrelevant glucose regulation are located between positions 636 and 879 of the pUC18 DNA sequence. Copyright © 1999 John Wiley & Sons, Ltd.     

Selection of yeast cells with a higher plasmid copy number in a Saccharomyces cerevisiae autoselection system

Autoselection systems allow the selection of a genetically engineered population independently of the growth medium composition. The structure of a Saccharomyces cerevisiae population transformed with an autoselection plasmid, in which a carbon-source-dependent modulation of the plasmid copy number occurs, was analysed. By means of flow cytometric procedures we tested the cell viability, dynamics of growth and heterologous protein production at single cell level. Such analyses allow the identification and the tracking of a specific cellular sub-population with a higher plasmid copy number which arises after the carbon source shift. The effects of the cellular plasmid distribution on the dynamics of growth are also discussed.     

Isolation and sequence analysis of the gene URA3 encoding the orotidine-5'-phosphate decarboxylase from Candida glycerinogenes WL2002-5, an industrial glycerol producer

The URA3 gene of Candida glycerinogenes WL2002-5, an industrial glycerol producer encoding orotidine-5'-phosphate decarboxylase enzyme, was isolated by complementation cloning in Saccharomyces cerevisiae. DNA sequence analysis revealed the presence of an open reading frame (ORF) of 786 bp, encoding a 262 amino acid protein, which shares 71.65% amino acid sequence similarity to the S. cerevisiae URA3 protein. Furthermore, the cloned ORF fully complemented the ura3 mutation of S. cerevisiae, confirming that it encodes for the C. glycerinogenes Ura3 (CgUra3) protein.     

高渗工业菌株Candida glycerinogenes整合型表达载体的构建及验证

构建应用于工业用产甘油假丝酵母(Candida glycerinogenes)CGMCC NO.6830的整合型表达载体,建立一种可利用底物形成的渗透压调控表达外源基因的体系。以p UC19质粒为基本骨架,C.glycerinogenes CGMCC NO.6830的18S r DNA为整合位点,运用其3-磷酸甘油脱氢酶基因启动子PCggpd启动外源基因表达,腐草霉素抗性基因ble作为重组酵母转化子筛选标记,获得应用于产甘油假丝酵母的稳定的表达型整合载体;以绿色荧光蛋白基因gfp作为报告基因考察该表达质粒的性能,实现了外源基因gfp的渗透压调控表达。     

Production of phytase in a low phosphate medium by a novel yeast Candida krusei

A yeast strain producing high levels of phytase was isolated from soil and identified as Candida krusei. The phytase was located on the yeast cell wall and was a glucanase-extractable protein. The phytase production was controlled by the phosphate concentration in the medium used. The maximum production of phytase occurred in a medium containing 0.5 mg of phosphorus per 100 ml, and most of the cells were ellipsoid-shaped and did not exhibit budding. Increasing the concentration of phosphorus in the medium to more than 5 mg of phosphorus per 100 ml caused inhibition of phytase production and 90% of the cells exhibited budding. On the other hand, transferring cells grown in the high-phosphate medium into a phosphate-free one derepressed the phytase production. For example, transferring cells grown in 2 mg of phosphorus per 100 ml into the phosphate-free medium, enhanced the total phytase activity up to 5.5-fold that in the medium containing 0.5 mg of phosphorus per 100 ml. The phytase showed two optimum pHs of 2.5 and 5.5, an optimum temperature of 40 degrees C and the K(m) value for Na-phytate was 0.03 mM. Using in vitro experiments that simulated the conditions of the digestive tract, 50-80% phosphorus was liberated from different plant samples (wheat bran, rice bran and feeds) by the strain.     

转基因作物筛查用阳性质粒分子的构建及应用研究

通过分析全球商业化种植的转基因作物分子特征,选取常用的调控元件启动子CaMV35S、FMV35S和nos,终止子NOS、T-35S、g73′和E93′,标记基因bar、NPTII、hptII、pmi作为筛查靶标序列,通过重组PCR技术将这些元件克隆到双元载体pBI121上,构建成包含11个转基因元件的通用阳性质粒分子pBI121-ELEMENTS。质粒序列信息已提交至GenBank并获得序列号HM047294。该质粒分子对目前国内外批准的5类主要转基因作物（大豆、油菜、玉米、棉花和水稻）理论筛查覆盖率达到91.5%,对我国批准进口的27种转基因作物的理论筛查覆盖率达100%。以pBI121-ELEMENTS为阳性对照,筛查7个转基因品系,结果显示该分子能够有效用于转基因作物定性筛查。     

南通服装产业升级网络策略的思考

服装产业是南通地区传统产业和支柱产业,正面临产业升级调整。网络对服装业的整体发展起到了提升作用,且两者结合日益紧密。针对南通服装产业升级调整方向和网络利用的不足,提出了促进本地服装产业升级的网络发展策略。     

Vectors for rapid selection of integrants with different plasmid copy numbers in the yeast Hansenula polymorpha DL1

Plasmids with different selectable markers were constructed and used to transform the Hansenula polymorpha strain DL1. It was shown that, depending on the host mutant strain, the use of these plasmids enables rapid selection of transformants with plasmids integrated in low (1–2), moderate (6–9) or high (up to 100) copy numbers. The vectors and mutants described are potentially useful for the construction of efficient producers of heterologous proteins in H. polymorpha. Copyright © 1999 John Wiley & Sons, Ltd.     

Construction of a large plasmid lacking linearizing single restriction sites by simultaneous in vivo recombination and plasmid shuffling in yeast

Creation of large ( approximately 15 kb) recombinant plasmids can be done in a single step by co-transformation of yeast cells with a partial restriction digest of a plasmid vector and a linear insert whose ends overlap one of the vector restriction sites. This method is used to generate a plasmid expressing the Saccharomyces cerevisiae rRNA genes containing the Ca.LSU group I intron ribozyme from Candida albicans. This plasmid expresses functional rRNA and ribozyme.     

分阶段温度控制策略提高重组葡萄酒酵母合成β-胡萝卜素的研究

利用农副产品为发酵底物,生产高附加值的牛物产品具有良好的应用前景。本研究以模拟葡萄汁为培养基,在7L发酵罐水平L研究了不同发酵温度（25℃、27℃、30℃、33℃）重组葡萄酒酵母T73-63生长和合成外源B．胡萝b素的情况。结果发现,重组葡萄酒酵母最适牛长温度与β-胡萝b素最适合成温度是不一致的：较高的发酵温度（30℃）有利于细胞的生长,能够获得较高的牛物量;但较低的温度（25qc）却能够获得较高的比产物合成速率。根据细胞生长和产物合成动力学曲线,提出了两阶段温度控制策略：发酵前20h温度控制为30℃,20h后降低至25℃至发酵结束;在此调控策略下,最高β-胡萝b素产量和β-胡萝b素合成速率分别达到56．42mg／L和1．28rag／（L．h）,与单一温度发酵模式的最高水平（30℃）相比分别提高了25．7％和14．3％。     

Systematic mapping of autonomously replicating sequences on chromosome V of Saccharomyces cerevisiae using a novel strategy

We have developed a new procedure for easy and rapid identification of autonomously replicating sequences (ARSs) and have applied it to the analysis of chromosome V of Saccharomyces cerevisiae. The procedure makes use of the ordered λ phage clone bank of this chromosome that we have constructed, and includes transposition of a mini-transposon and selection of transposon-containing derivatives, isolation of their DNA and circularization at their cos-ends, transformation of yeast cells with the circularized DNA, and scoring transformation frequency. The transposon used was derived from Tn5supF, contained the yeast LEU2 gene, and was placed, together with the hyperactive transposase gene, on a mini-F plasmid for stable maintenance in Escherichia coli K-12. Sixteen regions of chromosome V showing ARS activity were identified, of which 12 were newly found in this work. Thus, the procedure will be useful for systematic genomic scale analysis of ARSs in yeast and related organisms in which ordered clone banks have been established. The average distance between adjacent ARS-containing regions was approximately 40 kb. Two-dimensional gel electrophoretic analysis of chromosome replication indicated that one of the newly identified ARSs was functional as an actual in situ replication origin, at least under the conditions employed.     

肉制品中毛皮动物源性成分掺假检测阳性质粒分子的构建与评价

目的 建立一种肉制品中毛皮动物源性成分掺假快速检测技术。方法 构建狐狸、貉、水貂三种检测用阳性质粒分子,利用实时荧光定量PCR方法对其特异性、灵敏度和方法适用性等关键指标进行分析。结果 构建好的狐狸、貉、水貂阳性质粒分子特异性强、灵敏度高,检验灵敏度均可达到10-4 ng/μL,标准曲线扩增效率分别为94.451%、117.461%、114.709%,且相关系数(R2)均在0.995以上。在混合肉样品和市售肉制品中均可检测,三种成分的检测限均低至1%,具有较好的可行性及适用性。结论 本方法构建的三种阳性质粒分子可以满足实际工作中肉类掺假检测的需求。     

The 2μm plasmid of laboratory yeast strains is a type-1/type-2 hybrid

Industrial yeast strains carry one of two homeologous 2μm plasmids designated as type-1 or type-2. The 2μm plasmid, Scp1, found in common laboratory strains of Saccharomyces cerevisiae is considered a type-2 plasmid, since the ori, STB, RAF and REP1 loci and intergenic sequences of the right-unique region of Scp1 are homologous to the corresponding loci in industrial strain type-2 plasmids. However, within both its 599 bp inverted repeats Scp1 has 142-bp sequences homologous to the bakers' yeast type-1 plasmid. DNA sequence analyses and oligonucleotide hybridizations indicate that the 142-bp insertion in Scp1 was probably due to homeologous recombination between type-1 and type-2 plasmids. These results suggest that some of the plasmid and chromosomal sequence polymorphisms seen in laboratory yeast strains result from homeologous recombination in their ancestral breeding stock.     

Reduced acetaldehyde production by genome shuffling of an industrial brewing yeast strain

High levels of acetaldehyde produced by yeast during fermentation can be of concern to product quality. A novel approach, based on genome shuffling, was applied to reduce the production of acetaldehyde by industrial brewing strain YS86. Four isolates with different impacts of acetaldehyde concentration were obtained from populations generated by ultraviolet irradiation and nitrosoguanidine mutagenesis. These yeast strains were then subjected to recursive pool‐wise protoplast fusion. A strain library that was likely to yield positive colonies was created by fusing the lethal protoplasts obtained from both UV irradiation and heat treatments. After two rounds of genome shuffling, a recombinant YSF2–9 strain produced less acetaldehyde than wild‐type strain YS86, by 64.5 and 66.2% in laboratory and pilot plant fermentations, respectively. The shuffled yeast strain YSF2–9 was genetically stable and may have a potential application in brewing industry for managing acetaldehyde in beer. Copyright © 2017 The Institute of Brewing & Distilling     

Isolation of a haploid from an industrial Chinese rice wine yeast for metabolic engineering manipulation

The complex metabolic processes of yeast influence wine fermentation and therefore the quality of wine. Wine yeasts, owing to their being typically prototrophic and often polyploid, have been restricted in terms of exploiting classical recombinant genetic techniques to improve their characteristics. To overcome this problem, haploids have been isolated from a commercial Chinese rice wine strain N85, by disruption of the HO gene. In this study, the Cre–loxP system and a removable G418^r marker were used to construct an HO disruption cassette. Most of the heterologous sequences of constructed disruption cassette were successfully excised from the genome of the haploids by loop‐out of the KanMX gene, through induced expression of the Cre recombinase. The removal of the resistant marker ensures the biological safety of the strains. As expected, no difference in fermentation capacity between the parental and the haploid strains was seen. The present work reports the construction of an HO disruption cassette by touchdown polymerase chain reaction and its application with a Chinese rice wine yeast for haploid isolation and to broaden physiological investigations and industrial applications. Copyright © 2013 The Institute of Brewing & Distilling