摘要:

AbstractIn this contribution we discuss the potential of methylotrophic yeasts as hosts for the high level production of valuable foreign proteins. Recent relevant achievements on the intracellular production or secretion of proteins are summarized. Special attention is paid to a specific advantage of the use of methylotrophic yeasts, namely the possibility of accumulating the foreign gene products inside peroxisomes. This approach may be of major advantage when the protein product is toxic for the host cell and, also, to protect these proteins these proteins from undesired side‐effects such as proteolysis or aggregatio

ISSN:0749-503X    

DOI:10.1002/yea.320111402

出版商:John Wiley&Sons, Ltd.    

年代:1995

数据来源: WILEY

摘要:

AbstractTwo genesPMT3andPMT4were identified by polymerase chain reaction of genomic DNA using primers derived from regions of high homology between the products of three genesPMT1, PMT2ofSaccharomyces cerevisiaeand part of aPMT1related sequence ofKluyveromyces lactis. Pmt1p and Pmt2p are mannosyltransferases involved in the transfer of a mannosyl residue from dolichyl phosphate‐D‐mannose (Dol‐P‐Man) to seryl and threonyl residues in proteins. The products encoded by thePMT3andPMT4genes have almost identical hydropathy profiles in comparison toPMT1andPMT2: a hydrophobic N‐ and C‐terminal third each with multiple potential transmembrane helices and a central hydrophillic part.The predicted Pmt3p contains 753 amino acids, four potential N‐glycosylation sites and it is significantly homologous to Pmt1p, Pmt2p and Pmt4p. Pmt4p contains 762 amino acids and two potential N‐glycosylation sites. Northern blot analysis showed a single mRNA transcript ofPMT3andPMT4of 2·8 kb. ThusPMT3andPMT4are two new members of thePMTgene family. Thepmt4null mutant thepmt3 pmt4double null mutant, but notpmt3null mutant, showed a small but significant shift of chitinase due to under glycosylation of the enzyme. The triple disruptionpmt2 pmt3 pmt4and the quadruple disruption result in a lethal phenotype. The EMBL data library Accession Number forPMT3is X83797 a

ISSN:0749-503X    

DOI:10.1002/yea.320111403

出版商:John Wiley&Sons, Ltd.    

年代:1995

数据来源: WILEY

摘要:

AbstractIn this investigation, a method for the accurate quantitative determination of net proton production or consumption in biological cultures has been devised. Cells are cultured under constant pH conditions. The specific rate of proton production or consumption by the culture (qH+, mmol h−1per g biomass) is proportional to the mmol of base or acid required to maintain constant pH per unit time, and this equivalence is independent of the buffering capacity of the culture medium.The above method has been applied to chemostat cultures ofCandida utilisgrowing on glucose or glycerol as carbon source, and different nitrogen sources. The results indicate that the nitrogen assimilation pathway alone determines the value of qH+, and a fixed stoichiometric relationship between nitrogen uptake rate qN (meq h−1per g biomass) and qH+has been found for each nitrogen source employed. Thus, qH+/qN values of +1, 0 and − 1 were found for ammonium ions, urea and nitrate respectively. Under oxidative metabolism, the contribution of carbon catabolism to the value of qH+was undetectable.Since qN may be related to growth and production of type 1 compounds in fermentation processes, the parameter qH+was incorporated into a model of growth and energy metabolism in chemostat culture (Castrillo and Ugalde,Yeast10, 185–197, 1994), resulting in adequate simulations of experimentally observed culture performance. Thus, it is suggested that qH+may be employed as a simple and effective control parameter for biotechnological processes involving biomass‐related

ISSN:0749-503X    

DOI:10.1002/yea.320111404

出版商:John Wiley&Sons, Ltd.    

年代:1995

数据来源: WILEY

摘要:

AbstractRepression or induction of the genes involved in arginine biosynthesis or catabolism, respectively, both require participation of the ArgRp/Mcm1p regulatory complex. Our previous work showed that those opposite effects were mediated by a similar arginine‐responsive element of 23 nucleotides (that we now call ARC, for ARginine Control) situated close to the start of transcription in the repressed promoters and far upstream of the TATA‐element in the induced promoters.To define more precisely the sequence and position requirements of the ARC element, we have now characterized by mutagenesis the promoter elements of the arginine‐repressibleARG1andARG8genes. We also identify a functional ARC in theCPA1promoter, thereby confirming, in agreement with our previous mRNA pulse‐labelling data, the participation of a transcriptional component in the arginine regulation of that gene otherwise submitted to a translational regulation.From the 12 ARC elements now characterized, we have derived a consensus sequence and show that such a synthetic element is able to mediate ArgRp/Mcm1p‐dependent arginine regulation.An important new finding illustrated byARG1andCPA1, is that contrary to what all the previous data suggested, repression can be mediated by ARC elements located far upstream of the TATA‐box. The new data suggest that the arginine repressor might inhibit transcription in an act

ISSN:0749-503X    

DOI:10.1002/yea.320111405

出版商:John Wiley&Sons, Ltd.    

年代:1995

数据来源: WILEY

摘要:

AbstractEscherichia coliβ‐lactamase was secreted into the culture medium ofSaccharomyces cerevisiaein biologically active form, when fused to the C‐terminus of the hsp150δ‐carrier. The hsp150δ‐carrier is an N‐terminal fragment of the yeast hsp150 protein, having a signal peptide and consisting mostly of a 19 amino acid peptide repeated 11 timesin tandem. Here we expressed the hsp150δ‐carrier fragment alone inS. cerevisiae. Apparently due to a positional effect of the gene insertion, large amounts of the hsp150δ‐carrier were synthesized. About half of thede novosynthesized carrier molecules were secreted into the culture medium, the rest remaining mostly in the pre‐Golgi compartment. The extensively O‐glycosylated carrier fragment was purified from the culture medium under non‐denaturing conditions. Circular dichroism spectroscopy showed that it had no regular secondary structure. Nuclear magnetic resonance spectroscopy showed that a non‐glycosylated synthetic peptide, the consensus sequence of the repetitive 19 amino acid peptide, also lacked secondary structure. The unstructured carrier polypeptide may facilitate proper folding and secretion of heterologo

ISSN:0749-503X    

DOI:10.1002/yea.320111406

出版商:John Wiley&Sons, Ltd.    

年代:1995

数据来源: WILEY

摘要:

AbstractIt has been claimed that the low‐affinity component of glucose transport inSaccharomyces cerevisiaeis due to passive diffusion of the sugar across the plasma membrane. We have investigated this possibility. For this purpose we have measured the permeability coefficient of hexoses in this organism. We have found that this coefficient is at least two to three orders of magnitude lower than required to account for the low‐affinity component of glucose transport, and have concluded that this component is not due to passive diffus

ISSN:0749-503X    

DOI:10.1002/yea.320111407

出版商:John Wiley&Sons, Ltd.    

年代:1995

数据来源: WILEY

摘要:

AbstractYeast strains which form velum on the surface of Sherry wine during the aging process have been isolated and characterized. According to their metabolic and molecular features most of the yeasts that were isolated belong to different races ofSaccharomyces cerevisiae (beticus, cheresiensis, montuliensisandrouxii). Due to the conditions under which these yeasts were isolated, all strains have in common the capacity to develop a film as an adaptive mechanism which allows them to grow and survive in 15·5% vol. ethanol. All strains were prototrophs for amino acids and most vitamins but they gave different responses to the killer factor. However, whereas their physiological features were similar, they showed a great heterogeneity with regards to the nuclear and mitochondrial genome (mtDNA): DNA content per cell was quite variable (1·3 to 2n), electrophoretic karyotypes of nuclear genomes indicated a main pattern with some variations, and polymorphism shown by the mtDNA was very high. Under extreme conditions such as Sherry wine with 15·5% vol. ethanol, no fermentable sugar and an exclusively oxidative metabolism, cells hardly grow and the maintenance of a live population depends on survival and respiration, which in turn depend on the mtDNA. At the same time these environmental conditions are mutagenic for the mtDNA, causing an increase in variation. Thus, the polymorphism observed might reflect the enormous variability induced by the ethanol followed by the selection of those mtDNA sequences which make the mitochondria metabolically active under these conditio

ISSN:0749-503X    

DOI:10.1002/yea.320111408

出版商:John Wiley&Sons, Ltd.    

年代:1995

数据来源: WILEY

摘要:

AbstractWe present DNA sequence data from a 35 364 bp region on the left arm of chromosome VII ofSaccharomyces cerevisiae. This region contains 19 open reading frames (ORFs). ORF G1821 corresponds to theRAD54gene involved in repair and recombination (Emeryet al., 1991). G1810 is identical to theACE1gene sequenced by Szczypka and Thiele (1989), required for copper‐inducible transcription of theCUP1gene. The first 693 bp on the minus strand represent part of the 3′ non‐coding region from the P‐type ATPase genePMR1, previously sequenced by Rudolphet al.(1989), which is identical to theSSC1gene (Smithet al., 1988). G1845 corresponds to theRCK1protein kinase gene fromS. cerevisiae(Dahlkvist and Sunnerhagen, 1994). G1861 is almost identical to the α‐mannosidase geneAMS1reported by Yoshihisa and Anraku (1989) and G1864 has 100% identity with the yeastCAL1gene (Ohyaet al., 1989)/CDC43gene (Johnsonet al., 1990) which is involved in control of cell polarity. This region also contains a gene specifying a Leu‐tRNA precursor and a remnant of a tau element. ORF G1880 shows some similarity to theS. cerevisiae SNF2, STH1andNPS1genes and to the humanERCC1gene. A 93 bp region shows similarity to yeast EST sequenced by Burnset al.(1994). None of the remaining ORFs has similarity to any sequence within the databases screened. The sequence described in this paper has been deposited in the EMBL Data Library under the Accession N

ISSN:0749-503X    

DOI:10.1002/yea.320111409

出版商:John Wiley&Sons, Ltd.    

年代:1995

数据来源: WILEY

摘要:

AbstractThe DNA sequence of a 26 677 bp fragment from the right arm of chromosome VII fromSaccharomyces cerevisiaereveals 18 open reading frames (ORFs) longer than 300 bp. Eight ORFs correspond to previously characterized genes. G6620 is the 3′ end of theMOL1gene coding for a polypeptide similar to stress‐inducible proteins fromFusarium; G6630 is theNAT2gene which encodes a methionine N‐acetyltransferase; G6635 is theRPL30Bgene coding for the ribosomal protein L30; G6658 isRSR1encoding a ras‐related protein; G6667 isCYS4, the gene for cystathionine β‐synthase; G6670 is identical to ORF2 located close toCYS4; G6673 isPEM1/CHO2encoding a phosphatidylethanolamine methyltransferase; G7001 is theNSR1gene coding for a nuclear signal recognition protein. G6664 shares significant homology with the ORF YKR076w from chromosome XI. The other nine ORFs show no significant homology to any protein sequence presently available in the public data bases. The sequence has been deposited in the EMBL data library under Accession Num

ISSN:0749-503X    

DOI:10.1002/yea.320111410

出版商:John Wiley&Sons, Ltd.    

年代:1995

数据来源: WILEY

ISSN:0749-503X    

DOI:10.1002/yea.320111412

出版商:John Wiley&Sons, Ltd.    

年代:1995

数据来源: WILEY