Arrangement of genes TRP1 and TRP3 of Saccharomyces cerevisiae strains

The tryptophan biosynthetic genes TRP1 and TRP3 and partly also TRP2 and TRP4 have been compared by the technique of Southern hybridization and enzyme measurements in twelve wild isolates of Saccharomyces cerevisiae from natural sources of different continents, in the commonly used laboratory strain S. cerevisiae X2180-1A and in a Kluyveromyces marxianus strain. We could classify these strains into four groups, which did not correlate with their geographical distribution. In no case are the TRP3 and TRP1 genes fused as has been found in other ascomycetes. Two strains were found which, in contrast to strain X2180-1A, show derepression of gene TRP1. Two examples are discussed to demonstrate the usefulness of Southern hybridizations for the identification of closely related strains.Non-standard abbreviations InGP Indole-3-glycerolphosphate - PRA N(5-phosphoribosyl)-anthranilate     

酿酒酵母中过表达URA5及URA3基因催化合成UMP的初步研究

为提高乳清酸到尿嘧啶核苷酸(UMP)的转化效率,利用PCR方法扩增酿酒酵母乳清酸磷酸核糖转移酶基因URA5, 并将其连接到携带乳清苷酸脱羧酶基因URA3的表达载体pYX212中,构建了重组质粒pYX212-URA5,然后转化到酿酒酵母BJX12中进行表达,并进行转化乳清酸到UMP的初步研究。试验结果表明: pYX212-URA5/ BJX12发酵培养40h后以32 mM乳清酸为底物催化产生UMP的量约为7 mM。明显高于同等条件下pYX212/ BJX12的UMP产量2.7 mM和对照组野生型BJX12的UMP产量2.4 mM。     

System of centromeric, episomal, and integrative vectors based on drug resistance markers for Saccharomyces cerevisiae

Integrative, centromeric, and episomal plasmids are essential for easy, fast, and reliable genetic manipulation of yeast. We constructed a system of shuttle vectors based on the widely used plasmids of the pRS series. We used genes conferring resistance to Geneticin (kanMX4), nourseothricin (natNT2), and hygromycin B (hphNT1) as markers. The centromeric and episomal plasmids that we constructed can be used the same way as the traditional auxotrophic marker-based shuttle vectors (pRS41x and pRS42x series). Additionally, we created a set of nine yeast integrative vectors with the three dominant markers. These plasmids allow for direct integration in the LEU2, URA3, and HIS3 locus of any yeast strain and the concomitant partial deletion of the gene. This prevents multiple integrations and allows for the rapid identification of correct integrants. The set of new vectors considerably enhances the flexibility of genetic manipulations and gene expression in yeast. Most notably, the new vectors allow one to work with natural yeast isolates, which do not contain auxotrophic markers.     

Nucleotide sequence of Saccharomyces cerevisiae genes TRP2 and TRP3 encoding bifunctional anthranilate synthase: indole-3-glycerol phosphate synthase

Saccharomyces cerevisiae anthranilate synthase:indole-3-glycerol phosphate synthase is a multifunctional hetero-oligomeric enzyme encoded by genes TRP2 and TRP3. TRP2, encoding anthranilate synthase Component I, was cloned by complementation of a yeast trp2 mutant. The nucleotide sequence of TRP2 as well as that of TRP3 were determined. The deduced anthranilate synthase Component I primary structure from yeast exhibits only limited similarity to that of the corresponding Escherichia coli subunit encoded by trpE. On the other hand, yeast anthranilate synthase Component II and indole-3-glycerol phosphate synthase amino acid sequences from TRP3 are clearly homologous with the corresponding sequences of the E. coli trpG and trpC polypeptide segments and thereby establish the bifunctional structure of TRP3 protein. Based on comparisons of TRP3 amino acid sequence with homologous sequences from E. coli and Neurospora crassa, an 11-amino acid residue connecting segment was identified which fuses the trpG and trpC functions of the bifunctional TRP3 protein chain. These comparisons support the conclusion that the amino acid sequence of connectors in homologous multifunctional enzymes need not be conserved. Connector function is thus not dependent on a specific sequence. Nuclease S1 mapping was used to identify mRNA 5' termini. Heterogeneous 5' termini were found for both TRP2 and TRP3 mRNA. TRP2 and TRP3 5'-flanking regions were analyzed for sequences that might function in regulation of these genes by the S. cerevisiae general amino acid control system. The 9 base pair direct repeat (Hinnebusch, A.G., and Fink, G.R. (1983) J. Biol. Chem. 258, 5238-5247) and inverted repeats were identified in the 5'-flanking sequences of TRP2 and TRP3.     

Cloning of the LEU2 gene of Saccharomyces cerevisiae by in vivo recombination

We describe a convenient method for the in vivo construction of large plasmids that possess a multitude of restriction sites. A large (23 kbases) circular self-replicating plasmid carrying a partial LEU2-d gene was cotransformed with a circular non-replicating plasmid carrying the entire LEU2 gene. In vivo recombination results preferentially in a plasmid that carries both the LEU2-d and the entire LEU2 gene. In addition we also found one plasmid with a tandem LEU2 insertion and one plasmid where the LEU2-d gene was replaced by the entire LEU2 gene.     

A Short Region from the LEU2 Gene of Saccharomyces cerevisiae Functions as an ARS in the Yeast Saccharomyces exiguus Yp74L-3

We examined the autonomously replicating sequence (ARS) activity of some fragments derived from the LEU2 region of Saccharomyces cerevisiae onto Saccharomyces exiguus Yp74L-3. A DNA fragment functioning as an ARS in S. exiguus, but not in S. cerevisiae, was shown to exist. The ARS activity for S. exiguus was reduced by the 2-μm plasmid origin of S. cerevisiae when both elements coexisted on a single circular plasmid. Analysis of ARS activity with the PCR products from the fragment revealed that the ARS-acting sequence was located in the 3′-terminal area of the transcribed region of the LEU2 gene of S. cerevisiae. It is suggested that the ARS recognition system in S. exiguus is significantly different from that of S. cerevisiae. Received: 16 June 1998 / Accepted: 3 August 1998     

Anaerobic growth and improved fermentation of Pichia stipitis bearing a URA1 gene from Saccharomyces cerevisiae

Respiratory and fermentative pathways co-exist to support growth and product formation in Pichia stipitis. This yeast grows rapidly without ethanol production under fully aerobic conditions, and it ferments glucose or xylose under oxygen-limited conditions, but it stops growing within one generation under anaerobic conditions. Expression of Saccharomyces cerevisiaeURA1 (ScURA1) in P. stipitis enabled rapid anaerobic growth in minimal defined medium containing glucose when essential lipids were present. ScURA1 encodes a dihydroorotate dehydrogenase that uses fumarate as an alternative electron acceptor to confer anaerobic growth. Initial P. stipitis transformants grew and produced 32 g/l ethanol from 78 g/l glucose. Cells produced even more ethanol faster following two anaerobic serial subcultures. Control strains without ScURA1 were incapable of growing anaerobically and showed only limited fermentation. P. stipitis cells bearing ScURA1 were viable in anaerobic xylose medium for long periods, and supplemental glucose allowed cell growth, but xylose alone could not support anaerobic growth even after serial anaerobic subculture on glucose. These data imply that P. stipitis can grow anaerobically using metabolic energy generated through fermentation but that it exhibits fundamental differences in cofactor selection and electron transport with glucose and xylose metabolism. This is the first report of genetic engineering to enable anaerobic growth of a eukaryote. Received: 6 January 1998 / Received revision: 9 April 1998 / Accepted: 19 April 1998     

Anaerobic xylose fermentation by recombinant Saccharomyces cerevisiae carrying XYL1, XYL2, and XKS1 in mineral medium chemostat cultures

For ethanol production from lignocellulose, the fermentation of xylose is an economic necessity. Saccharomyces cerevisiae has been metabolically engineered with a xylose-utilizing pathway. However, the high ethanol yield and productivity seen with glucose have not yet been achieved. To quantitatively analyze metabolic fluxes in recombinant S. cerevisiae during metabolism of xylose-glucose mixtures, we constructed a stable xylose-utilizing recombinant strain, TMB 3001. The XYL1 and XYL2 genes from Pichia stipitis, encoding xylose reductase (XR) and xylitol dehydrogenase (XDH), respectively, and the endogenous XKS1 gene, encoding xylulokinase (XK), under control of the PGK1 promoter were integrated into the chromosomal HIS3 locus of S. cerevisiae CEN.PK 113-7A. The strain expressed XR, XDH, and XK activities of 0.4 to 0.5, 2.7 to 3.4, and 1.5 to 1.7 U/mg, respectively, and was stable for more than 40 generations in continuous fermentations. Anaerobic ethanol formation from xylose by recombinant S. cerevisiae was demonstrated for the first time. However, the strain grew on xylose only in the presence of oxygen. Ethanol yields of 0.45 to 0.50 mmol of C/mmol of C (0.35 to 0.38 g/g) and productivities of 9.7 to 13.2 mmol of C h(-1) g (dry weight) of cells(-1) (0.24 to 0.30 g h(-1) g [dry weight] of cells(-1)) were obtained from xylose-glucose mixtures in anaerobic chemostat cultures, with a dilution rate of 0.06 h(-1). The anaerobic ethanol yield on xylose was estimated at 0.27 mol of C/(mol of C of xylose) (0.21 g/g), assuming a constant ethanol yield on glucose. The xylose uptake rate increased with increasing xylose concentration in the feed, from 3.3 mmol of C h(-1) g (dry weight) of cells(-1) when the xylose-to-glucose ratio in the feed was 1:3 to 6.8 mmol of C h(-1) g (dry weight) of cells(-1) when the feed ratio was 3:1. With a feed content of 15 g of xylose/liter and 5 g of glucose/liter, the xylose flux was 2.2 times lower than the glucose flux, indicating that transport limits the xylose flux.     

Fine structure of the URA2 locus in Saccharomyces cerevisiae. II. Meiotic and mitotic mapping studies.

Summary The URA2 locus codes for a multifunctional enzyme complex carrying aspartate transcarbamylase (ATCase) and carbamyl phosphate synthetase (CPSase) activities. Three different types of ura2 mutants were tested in meiotic and mitotic recombination experiments: ura2A mutants devoid of ATCase activity, ura2C mutants devoid of CPSase activity and ura2B mutants devoid of both activities. All the ura2C mutations were found to be clustered at one end of the URA2 locus, called zone A, while the ura2C mutations were localized in a region at the other end, called zone C. All but two ura2B mutations (most of them suppressible) were distributed throughout zone C; the two ura2B exceptions which are small deletions, mapped in zone A. On the meiotic as well as on the mitotic map an intermediary or dead-space zone is located between zones A and C. No mutation has yet been found to map in this zone. The relative lengths of the three zones A, intermediary and C are 1:2–3:3–4, respectively.These data are consistent with the hypothesis that the URA2 locus consisting of at least two cistrons: C (CPSase) and A (ATCase), is transcribed into a single polycistronic message in the direction C to A. However, alternative hypotheses in reference to Peterson and MacLaughlin's observations (1973) are discussed.This paper is dedicated to the memory of our friend and colleague Huguette de Robichon-Szulmajster