COMPARISON OF EXPRESSION SYSTEMS IN THE YEASTS SACCHAROMYCES-CEREVISIAE, HANSENULA-POLYMORPHA, KLYVEROMYCES-LACTIS, SCHIZOSACCHAROMYCES-POMBE AND YARROWIA-LIPOLYTICA - CLONING OF 2 NOVEL PROMOTERS FROM YARROWIA-LIPOLYTICA

We have compared expression systems based on autonomously replicating vectors in the yeasts Saccharomyces cerevisiae, Schizosaccharomyces po mbe, Kluyveromyces lactis, Hansenula polymorpha and Yarrowia lipolytic a in order to identify a more suitable host organism for use in the ex pression cloning method (Dalboge and Heldt-Hansen, 1994) in which S. c erevisiae has traditionally been used. The capacity of the expression systems to secrete active forms of six fungal genes encoding the enzym es galactanase, lipase, polygalacturonase, xylanase and two cellulases was examined, as well as glycosylation pattern, plasmid stability and transformation frequency. All of the examined alternative hosts were able to secrete more active enzyme than S. cerevisiae but the relative expression capacity of the individual hosts varied significantly in a gene-dependent manner. One of the most attractive of the alternative host organisms, Y. lipolytica, yielded an increase which ranged from 4 .5 times to more than two orders of magnitude. As the initially employ ed Y. lipolytica XPR2 promoter is unfit in the context of expression c loning, two novel promoter sequences for highly expressed genes presen t in only one copy on the genome were isolated. Based on sequence homo logy, the genes were identified as TEF, encoding translation elongatio n factor-lu and RPS7, encoding ribosomal protein S7. Using the heterol ogous cellulase II (celII) and xylanase I(xyl/I) as reporter genes, th e effect of the new promoters was measured in qualitative and quantita tive assays. Based on the present tests of the new promoters, Y. lipol ytica appears as a highly attractive alternative to S cerevisiae as a host organism for expression cloning. GenBank Accession Numbers: TEF g ene promoter sequence: AF054508; RPS7 gene promoter sequence: AF054509 . (C) 1998 John Wiley & Sons, Ltd.