Strong hybrid promoters and integrative expression/secretion vectors for quasi-constitutive expression of heterologous proteins in the yeast Yarrowia lipolytica.
The industrial yeast Yarrowia lipolytica secretes high amounts of an alkaline extracellular protease encoded by the XPR2 gene. The industrial use of the XPR2 promoter was however hindered by its complex regulation. We designed hybrid promoters, based on tandem copies of the XPR2 promoter UAS1 region. In contrast to native XPR2 promoter, these hybrid promoters were not repressed by the preferred carbon and nitrogen sources, nor by acidic conditions, and they did not require the presence of peptones in the culture medium. They exhibited a strong quasi-constitutive activity, similar when carried on either integrative or replicative plasmids. We used these hybrid promoters to direct the production of bovine prochymosin, using XPR2 secretion signals. The production of active chymosin was several fold higher than with previously available Y. lipolytica promoters (up to 160 mg/l). Integrative vectors based on the hybrid promoters, allowing the easy insertion of a heterologous gene and its expression or expression/secretion in Y. lipolytica, were designed. We also designed new Y. lipolytica recipient strains with good secreting abilities, able to grow on sucrose, and devoid of extracellular proteases. These new tools will add to the interest of Y. lipolytica as a host for heterologous protein production.
Madzak
C
C
Laboratoire de Génétique Moléculaire et Cellulaire, INRA (UR216)-CNRS (URA1925), Centre de Biotechnologies Agro-Industrielles, Institut National Agronomique Paris-Grignon, Thiverval-Grignon, France. madzak@platon.grignon.inra.fr
Tréton
B
B
Blanchin-Roland
S
S
D000818:Animals; D001483:Base Sequence; D001709:Biotechnology; D002417:Cattle; D012085:Chymosin / Q000096:biosynthesis / Q000235:genetics; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D004792:Enzyme Precursors / Q000096:biosynthesis / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics*; D004718:Saccharomycetales / Q000235:genetics* / Q000378:metabolism; D016385:TATA Box / Q000235:genetics
D017931:DNA Primers; D004792:Enzyme Precursors; D011994:Recombinant Proteins; C032443:prorennin; D012085:Chymosin