Biomedical subjects

Jean-Marie Beckerich

Publications and source records attributed to Jean-Marie Beckerich.

8 recordsLinked to original sources

PubMed2005

Heterologous production of a laccase from the basidiomycete Pycnoporus cinnabarinus in the dimorphic yeast Yarrowia lipolytica.

Pycnoporus cinnabarinus lac1 gene was expressed in Yarrowia lipolytica. Different secretion signals and culture media were tested. Production was correlated to both culture growth rate and cell morphology (highest at low growth rate, without mycelium). Recombinant laccase was characterized (immunodetection, N-terminal sequencing) and purified. Production was estimated to 20 mgl(-1) in a bioreactor. Thus, complex metalloenzymes can be produced in Yarrowia, assuming some control of host physiology. Lac1p production was compared in Yarrowia, Pichia and Aspergillus: recombinant proteins were active, but host systems differed in transformation efficiency, production, and glycosylation. If not the best producer, Yarrowia offers very high transformation efficiencies, allowing the genetic engineering of laccases for industrial applications.

Basidiomycota↗

PubMed2005

Involvement of a branched-chain aminotransferase in production of volatile sulfur compounds in Yarrowia lipolytica.

The enzymatic degradation of L-methionine and the subsequent formation of volatile sulfur compounds (VSCs) are essential for the development of the typical flavor in cheese. In the yeast Yarrowia lipolytica, the degradation of L-methionine was accompanied by the formation of the transamination product 4-methylthio-2-oxobutyric acid. A branched-chain aminotransferase gene (YlBCA1) of Y. lipolytica was amplified, and the L-methionine-degrading activity and the aminotransferase activity were measured in a genetically modified strain and compared to those of the parental strain. Our work shows that L-methionine degradation via transamination is involved in formation of VSCs in Y. lipolytica.

Amino Acid Sequence↗

PubMed2004

Genome evolution in yeasts.

Identifying the mechanisms of eukaryotic genome evolution by comparative genomics is often complicated by the multiplicity of events that have taken place throughout the history of individual lineages, leaving only distorted and superimposed traces in the genome of each living organism. The hemiascomycete yeasts, with their compact genomes, similar lifestyle and distinct sexual and physiological properties, provide a unique opportunity to explore such mechanisms. We present here the complete, assembled genome sequences of four yeast species, selected to represent a broad evolutionary range within a single eukaryotic phylum, that after analysis proved to be molecularly as diverse as the entire phylum of chordates. A total of approximately 24,200 novel genes were identified, the translation products of which were classified together with Saccharomyces cerevisiae proteins into about 4,700 families, forming the basis for interspecific comparisons. Analysis of chromosome maps and genome redundancies reveal that the different yeast lineages have evolved through a marked interplay between several distinct molecular mechanisms, including tandem gene repeat formation, segmental duplication, a massive genome duplication and extensive gene loss.

Chromosomes, Fungal↗

PubMed2004

Heterologous protein expression and secretion in the non-conventional yeast Yarrowia lipolytica: a review.

The production of heterologous proteins is a research field of high interest, with both academic and commercial applications. Yeasts offer a number of advantages as host systems, and, among them, Yarrowia lipolytica appears as one of the most attractive. This non-conventional dimorphic yeast exhibits a remarkable regularity of performance in the efficient secretion of various heterologous proteins. This review presents the main characteristics of Y. lipolytica, and the genetic and molecular tools available in this yeast. A particular emphasis is given to newly developed tools such as efficient promoters, a non-homologous integration method, and an amplification system using defective selection markers. A table recapitulates the 42 heterologous proteins produced until now in Y. lipolytica. A few relevant examples are exposed in more detail, in order to illustrate some peculiar points of the Y. lipolytica physiology, and to offer a comparison with other production systems. This amount of data demonstrates the global reliability and versatility of Y. lipolytica as a host for heterologous production.

Gene Expression Regulation↗

PubMed2004

Identification and characterization of two alpha-1,6-mannosyltransferases, Anl1p and Och1p, in the yeast Yarrowia lipolytica.

In this study, the identification and characterization of the Yarrowia lipolytica homologues of Saccharomyces cerevisiae alpha-1,6-mannosyltransferases Anp1p and Och1p, designated YlAnl1p and YlOch1p, are described. In order to confirm the function of the Y. lipolytica proteins, including the previously isolated YlMnn9p, in the N-glycosylation pathway, a phenotypic analysis of the disrupted strains Delta Ylmnn9, Delta Ylanl1, Delta Yloch1, Delta Ylanl1 Delta Ylmnn9 and Delta Ylmnn9 Delta Yloch1 was performed. Disruption of the YlMNN9, YlANL1 and YlOCH1 genes caused an increased sensitivity to SDS, compatible with a glycosylation defect, and to Calcofluor White, characteristic of cell-wall defects. Moreover, Western-blot analysis of a heterologous glycosylated protein confirmed a direct role of YlMnn9p and YlAnl1p in the N-glycosylation process. These mutant strains, Delta Ylmnn9, Delta Ylanl1, Delta Yloch1, Delta Ylanl1 Delta Ylmnn9 and Delta Ylmnn9 Delta Yloch1 may thus be used to establish a model for the Y. lipolytica N-linked glycosylation pathway.

Amino Acid Sequence↗

PubMed2002

Sbh1p, a subunit of the Sec61 translocon, interacts with the chaperone calnexin in the yeast Yarrowia lipolytica.

The core component of the translocation apparatus, Sec61p or alpha, was previously cloned in Yarrowia lipolytica. Using anti-Sec61p antibodies, we showed that most of the translocation sites are devoted to co-translational translocation in this yeast, which is similar to the situation in mammalian cells but in contrast to the situation in Saccharomyces cerevisiae, where post-translational translocation is predominant. In order to characterize further the minimal translocation apparatus in Y. lipolytica, the beta Sec61 complex subunit, Sbh1p, was cloned by functional complementation of a Deltasbh1, Deltasbh2 S. cerevisiae mutant. The secretion of the reporter protein is not impaired in the Y. lipolytica sbh1 inactivated strain. We screened the Y. lipolytica two-hybrid library to look for partners of this translocon component. The ER-membrane chaperone protein, calnexin, was identified as an interacting protein. By a co-immunoprecipitation approach, we confirmed this association in Yarrowia and then showed that the S. cerevisiae Sbh2p protein was a functional homologue of YlSbh1p. The interaction of Sbh1p with calnexin was shown to occur between the lumenal domain of both proteins. These results suggest that the beta subunit of the Sec61 translocon may relay folding of nascent proteins to their translocation.

Amino Acid Sequence↗

PubMed2002

Secretion of active anti-Ras single-chain Fv antibody by the yeasts Yarrowia lipolytica and Kluyveromyces lactis.

Yarrowia lipolytica and Kluyveromyces lactis secretion vectors were constructed and assessed for the expression of heterologous proteins. An anti-Ras single-chain antibody fragment (scFv) coding sequence was fused in-frame to different pre- or prepro-regions, or downstream from a reporter secretory gene (Arxula adeninivorans glucoamylase), separated by a Kex2 protease (Kex2p)-like processing sequence. Both organisms are able to secrete soluble scFv, with yields depending on the nature of the expression cassette, up to levels ranging from 10 to 20 mg l(-1). N-terminal sequence analysis of the purified scFv showed that fusions are correctly processed to the mature scFv by a signal peptidase or a Kex2p-type endoprotease present in Y. lipolytica and K. lactis. The scFv protein also retains the capacity to bind to a glutathioneS-transferase (GST)-Harvey-Ras(Val12) fusion, indicating that the antibody is functional. These results indicate that the yeasts Y. lipolytica and K. lactis have potential for industrial production of soluble and active scFv.

Fungal Proteins↗

PubMed2002

Nucleotide exchange factor for the yeast Hsp70 molecular chaperone Ssa1p.

We report on the identification of Fes1p (yBR101cp) as a cytosolic homologue of Sls1p, an endoplasmic reticulum (ER) protein previously shown to act as a nucleotide exchange factor for yeast BiP (M. Kabani, J.-M. Beckerich, and C. Gaillardin, Mol. Cell. Biol. 20:6923-6934, 2000). We found that Fes1p associates preferentially to the ADP-bound form of the cytosolic Hsp70 molecular chaperone Ssa1p and promotes nucleotide release. Fes1p activity was shown to be compartment and species specific since Sls1p and Escherichia coli GrpE could not substitute for Fes1p. Surprisingly, whereas Sls1p stimulated the ATPase activity of BiP in cooperation with luminal J proteins, Fes1p was shown to inhibit the Ydj1p-mediated activation of Ssa1p ATPase activity in steady-state and single-turnover assays. Disruption of FES1 in several wild-type backgrounds conferred a strong thermosensitive phenotype but partially rescued ydj1-151 thermosensitivity. The Delta fes1 strain was proficient for posttranslational protein translocation, as well as for the ER-associated degradation of two substrates. However, the Delta fes1 mutant showed increased cycloheximide sensitivity and a general translational defect, suggesting that Fes1p acts during protein translation, a process in which Ssa1p and Ydj1p are known to be involved. In support of this hypothesis, Fes1p was found to be associated with ribosomes.

Adenosine Triphosphatases↗