Michael Ciriacy

Xylose fermentation by Saccharomyces cerevisiae

We have performed a comparative study of xylose utilization in Saccharomyces cerevisiae transformants expressing two key enzymes in xylose metabolism, xylose reductase (XR) and xylitol dehydrogenase (XDH), and in a prototypic xylose-utilizing yeast, Pichia stipitis. In the absence of respiration (see text), bakers yeast cells convert half of the xylose to xylitol and ethanol, whereas P. stipilis cells display rather a homofermentative conversion of xylose to ethanol. Xylitol production by bakers yeast is interpreted as a result of the dual cofactor dependence of the XR and the generation of NADPH by the pentose phosphate pathway. Further limitations of xylose utilization in S. cerevisiae cells are very likely caused by an insufficient capacity of the non-oxidative pentose phosphate pathway, as indicated by accumulation of sedoheptulose-7-phosphate and the absence of fructose-1,6-bisphosphate and pyruvate accumulation. By contrast, uptake at high substrate concentrations probably does not limit xylose conversion in S. cerevisiae XYL1/XYL2 transformants.

Isolation and characterization of the Pichia stipitis xylitol dehydrogenase gene, XYL2, and construction of a xylose-utilizing Saccharomyces cerevisiae transformant.

SummaryA P. stipitis cDNA library in λgt11 was screened using antisera against P. stipitis xylose reductase and xylitol dehydrogenase, respectively. The resulting cDNA clones served as probes for screening a P. stipitis genomic library. The genomic XYL2 gene was isolated and the nucleotide sequence of the 1089 bp structural gene, and of adjacent non-coding regions, was determined. The XYL2 open-reading frame codes for a protein of 363 amino acids with a predicted molecular mass of 38.5 kDa. The XYL2 gene is actively expressed in S. cerevisiae transformants. S. cerevisiae cells transformed with a plasmid, pRD1, containing both the xylose reductase gene (XYL1) and the xylitol dehydrogenase gene (XYL2), were able to grow on xylose as a sole carbon source. In contrast to aerobic glucose metabolism, S. cerevisiae XYL1-XYL2 transformants utilize xylose almost entirely oxidatively.

Identification of novel HXT genes in Saccharomyces cerevisiae reveals the impact of individual hexose transporters on qlycolytic flux

In Saccharomyces cerevisiae, hexose uptake is mediated by HXT proteins which belong to a superfamily of monosaccharide facilitators. We have identified three more genes that encode hexose transporters (HXT5, 6, 7). Genes HXT6 and HXT7 are almost identical and located in tandem 3′ adjacent to HXT3 on chromosome IV. We have constructed a set of congenic strains expressing none or any one of the seven known HXT genes and followed growth and flux rates for glucose utilization. The hxt null strain does not grow on glucose, fructose or mannose, and both glucose uptake and flux rate were below the detection level. Expression of either HXT1, 2, 3, 4, 6 or 7 is basically sufficient for aerobic growth on these sugars. In most of the constructs, glucose was the preferred substrate compared to fructose or mannose. There is a considerable variation in flux and growth rates with 1% glucose, dependent on the expression of the individual HXT genes. Expression of either HXT2, 6 or 7 in the null background is sufficient for growth on 0.1% glucose, while growth of strains with only HXT1, 3 or 4 requires higher (≥1%) glucose concentrations. These results demonstrate that individual HXT proteins can function independently as hexose transporters, and that most of the metabolically relevant HXT transporters from S. cerevisiae have been identified.

Characterization of trans-acting mutations affecting Ty and Ty-mediated transcription in Saccharomyces cerevisiae.

SummaryBy recessive mutations, we have identified five genes, TYE1-TYE5, that are required for Ty-mediated expression of ADH2. These tye mutations not only suppress transcription of ADH2 when associated with a Ty element but are also defective in transcription of all Ty1 and Ty2 elements. Moreover, some of these mutations cause growth defects on non-fermentable carbon sources as well as sporulation defects. tye mutations also strongly suppress ADH2 expression when controlled by a polyA/T insertion mutation. Genetic analysis revealed that genes TYE3 and TYE4 are allelic to the previously identified genes SNF2 and SNF5 which code for transcription factors. These findings suggest that TYE gene products influence transcription of many genes rather than specifically Ty and Ty-mediated transcrption. We have also found that null alleles of certain STE genes (ste7, ste11 and ste12), known to affect cell-type specific gene expression and expression of some Ty-adjacent genes, have a clear effect on Ty-controlled ADH2 expression depending on the carbon source. On the basis of ADH2 transcript levels in glucose-grown cells, all three ste alleles cause of five-fold reduction of ADH2 expression/transcription. In ethanol-grown cells, ste11 and ste12 mutations caused an almost complete loss of Ty-mediated ADH2 activation while ste7 has only a rather moderate effect. Surprisingly, ste11 and ste12 mutations lead to a significant increase in total Ty transcript levels. This would indicate that the STE12 protein, which is known to bind specifically to Ty1 sequences and thereby serve as an activator of a Ty-adjacent gene, can negatively modulate Ty transcription. The STE7 and STE11 genes which encode protein kinases apparently act in a different manner in both Ty and Ty-mediated transcription.

Isolation and characterization of xyl mutants in a xylose-utilizing yeast, Pichia stipitis

SummaryMutants of the xylose-utilizing yeast, Pichia stipitis, unable to grow on xylose as the sole carbon source were isolated and characterized. The mutants were deficient in either xylose reductase or xylitol dehydrogenase. By immunological means and upon analysis of revertants, both mutant types could be attributed to the structural genes XYL1 and XYL2, which code for xylose reductase and xylitol dehydrogenase, respectively. These data support previous assumptions that both NADH- and NADPH-dependent xylose reductase activity are due to one protein or gene, respectively. Revertant analysis of xyl1 mutants has revealed the existence of a second xylose reductase gene in P. stipitis. This gene is very likely cryptic. Its corresponding xylose reductase activity is NADPH-dependent.

Gene conversion and reciprocal exchange in a Ty-mediated translocation in yeast

SummaryA haploid yeast mutant carrying a reciprocal translocation was analyzed. Cloning and comparison of sequences involved in the translocation event in wildtype and mutant revealed that the crossover between non-homologous chromosomes has occured within Ty sequences. By DNA sequence analysis it could be demonstrated that the reciprocal recombination event is accompanied by a short segment of non-reciprocal exchange (gene conversion) in the immediate vicinity of the crossover. Analysis of the translocation mutant and revertant isolates also indicated that the regulatory effect of Ty elements on adjacent genes can be modified by discrete changes within a Ty element.

Isolation of the TYE2 gene reveals its identity to SW13 encoding a general transcription factor in Saccharomyces cerevisiae

The TYE2 gene was identified by recessive mutations which result in a significant reduction of Ty-mediated ADH2 expression. We cloned the TYE2 gene and analyzed its sequence. A large open reading frame of 825 codons was found encoding a rather hydrophilic, 93-kilodalton protein which contains a highly acidic region at its N-terminus. By sequence comparison we found that TYE2 is identical to gene SWI3 which has recently been shown to encode a nuclear protein which may function as a global transcription activation factor. The TYE2/SWI3 protein is necessary for the initiation of Ty1 transcription at its major initiation site in the δ element. Furthermore TYE2 function seems to be important for the expression of a variety of Ty-unrelated functions such as ADH1 expression, sporulation, growth on maltose, galactose, raffinose, and on non-fermentable carbon sources.

δ Sequences mediate DNA rearrangements in Saccharomyces cerevisiae

SummaryA solo δ sequence flanking the 5′ end of the ADHII structural gene, ADR2, can promote a number of DNA rearrangements some of which were investigated in detail. In a selective system haploid mutants were screened in which a solo S sequence flanking ADR2 had been joined to a Ty element. Three different types of events can create such a structure: Reintegration of a Ty sequence at the δ-ADR2 site, inversion of ADR2 and flanking material, and transposition of ADR2 along with 3′ flanking material. The involvement of reciprocal or non-reciprocal exchange mechanisms in creating such events are discussed.

Function of the Genetic Material: Transposable Elements in Lower Eukaryotes

In the past 10 years increasing evidence has been provided that eukaryotic genomes are inhabited by a variety of mobile genetic elements. The occurrence of such elements in forms of plasmids or transposons has changed our view of the eukaryotic genome in terms of its stability and its evolutionary origins quite considerably. Even though our knowledge of the spectrum of mobile elements and their occurrence in the various taxa is still limited, there is now sufficient information available on their molecular nature, their replication strategies, and their cellular function to outline some common and general features. This review will be focused on lower eukaryotes such as yeast, fungi, slime molds, and algae. Research on this group of organisms has contributed, although to a quite different degree, very much to our current knowledge on the nature of eukaryotic mobile elements. Furthermore, I will restrict this review to mobile sequences which interact physically in some way with the nuclear or extranuclear genomes. Thus, the reader is referred to the comprehensive treatise by (Berg and Howe 1989) which covers the entire field of “mobile DNA”. The presentation of material in this review is not only biased by my own research interests, but also by extreme variability in knowledge in the various systems investigated. Starting with an overview of transposons in lower eukaryotes and their possible relationship to each other and to other entities of cellular life, I will discuss the major strategies for expression of genetic information carried by transposons and for replication of the genome.

Function of the Genetic Material: Structure and Function of Elements Controlling Transcription in Lower Eukaryotes

The advances in gene technology with eukaryotic systems during the past 5 years has led to a considerable number of novel aspects on the function of DNA sequences which control transcription (promoters, terminators, regulators) of nuclear genes in lower eukaryotes. The mass of findings with regard to gene regulation has been obtained in baker’s yeast, Saccharomyces cerevisiae, and in a few other fungi and yeasts. From these studies it became very obvious that there are some basic differences in the mechanism of mRNA initiation and its regulation between prokaryotes and eukaryotes. This might not be surprising since prokaryotes lack an equivalent of the eukaryotic chromatin and a nucleus. However, in both systems it is a question of protein-nucleic acid interaction; that is to say the differences currently emerging can be largely viewed as variations of the same subject: how does a protein (RNA polymerase or a regulatory protein) recognize a specific site among thousands of other sites on the DNA.