title: Application of a wide-range yeast vector (CoMed) system to recombinant protein production in dimorphic Arxula adeninivorans, methylotrophic Hansenula polymorpha and other yeasts.
abstract: BACKGROUND
  Yeasts provide attractive expression platforms in combining ease of genetic manipulation and fermentation of a microbial organism with the capability to secrete and to modify proteins according to a general eukaryotic scheme. However, early restriction to a single yeast platform can result in costly and time-consuming failures. It is therefore advisable to assess several selected systems in parallel for the capability to produce a particular protein in desired amounts and quality. A suitable vector must contain a targeting sequence, a promoter element and a selection marker that function in all selected organisms. These criteria are fulfilled by a wide-range integrative yeast expression vector (CoMed) system based on A. adeninivorans- and H. polymorpha-derived elements that can be introduced in a modular way.
  
  
  RESULTS
  The vector system and a selection of modular elements for vector design are presented. Individual single vector constructs were used to transform a range of yeast species. Various successful examples are described. A vector with a combination of an rDNA sequence for genomic targeting, the E. coli-derived hph gene for selection and the A. adeninivorans-derived TEF1 promoter for expression control of a GFP (green fluorescent protein) gene was employed in a first example to transform eight different species including Hansenula polymorpha, Arxula adeninivorans and others. In a second example, a vector for the secretion of IL-6 was constructed, now using an A. adeninivorans-derived LEU2 gene for selection of recombinants in a range of auxotrophic hosts. In this example, differences in precursor processing were observed: only in A. adeninivorans processing of a MFalpha1/IL-6 fusion was performed in a faithful way.
  
  
  CONCLUSIONS
  rDNA targeting provides a tool to co-integrate up to 3 different expression plasmids by a single transformation step. Thus, a versatile system is at hand that allows a comparative assessment of newly introduced metabolic pathways in several organisms or a comparative co-expression of bottleneck genes in cases where production or secretion of a certain product is impaired.
authors.affiliation: Institut für Pflanzengenetik und Kulturpflanzenforschung, Corrensstr. 3, 06466 Gatersleben, Germany. steinbor@ipk-gatersleben.de
authors.forename: Gerhard
authors.initials: G
authors.lastname: Steinborn
authors.forename: Erik
authors.initials: E
authors.lastname: Böer
authors.forename: Anja
authors.initials: A
authors.lastname: Scholz
authors.forename: Kristina
authors.initials: K
authors.lastname: Tag
authors.forename: Gotthard
authors.initials: G
authors.lastname: Kunze
authors.forename: Gerd
authors.initials: G
authors.lastname: Gellissen