Abstract
BackgroundYeasts 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.ResultsThe 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 MFα1/IL-6 fusion was performed in a faithful way.ConclusionrDNA 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.
Highlights
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
Design and optimization of the CoMedTM vector system Since vector systems of different yeast species are based on different basic vectors it is very difficult to exchange single cassettes between the yeast systems
A selection of rDNA elements derived from the A. adeninivorans or H. polymorpha clusters has been assessed for suitability as targeting sequences
Summary
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. Production procedures had to be developed that employ platforms which meet both the demand for efficient mass production and criteria of safety and authenticity of the produced compounds [1,2]. In this respect, yeasts offer considerable advantages over alternative microbial and eukaryotic cellular systems in providing low-cost screening and production systems for authentically processed and modified compounds. Yeasts offer considerable advantages over alternative microbial and eukaryotic cellular systems in providing low-cost screening and production systems for authentically processed and modified compounds These organisms meet safety prerequisites in that they do not harbour pyrogens, pathogens or viral inclusions. Examples for the application of such vectors are provided in the following overview
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