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Background  Phosphodiesterases (PDE) are a superfamily of enzymes that hydrolyse cyclic nucleotides (cAMP/cGMP), signal molecules in transduction pathways regulating crucial aspects of cell life. PDEs regulate the intensity and duration of the cyclic nucleotides signal modulating the downstream biological effect. Due to this critical role associated with the extensive distribution and multiplicity of isozymes, the 11 mammalian families (PDE1 to PDE11) constitute key therapeutic targets. PDE5, one of these cGMP-specific hydrolysing families, is the molecular target of several well known drugs used to treat erectile dysfunction and pulmonary hypertension.  Kluyveromyces lactis , one of the few yeasts capable of utilizing lactose, is an attractive host alternative to  Saccharomyces cerevisiae  for heterologous protein production. Here we established  K. lactis  as a powerful host for the quantitative production of the murine PDE5 isoforms.    Results  Using the promoter of the highly expressed  KlADH3  gene, multicopy plasmids were engineered to produce the native and recombinant  Mus musculus  PDE5 in  K. lactis . Yeast cells produced large amounts of the purified A1, A2 and A3 isoforms displaying K m , V max  and Sildenafil inhibition values similar to those of the native murine enzymes. PDE5 whose yield was nearly 1 mg/g wet weight biomass for all three isozymes (30 mg/L culture), is well tolerated by  K. lactis  cells without major growth deficiencies and interferences with the endogenous cAMP/cGMP signal transduction pathways.    Conclusions  To our knowledge, this is the first time that the entire PDE5 isozymes family containing both regulatory and catalytic domains has been produced at high levels in a heterologous eukaryotic organism.  K. lactis  has been shown to be a very promising host platform for large scale production of mammalian PDEs for biochemical and structural studies and for the development of new specific PDE inhibitors for therapeutic applications in many pathologies.

Use of the KlADH3 promoter for the quantitative production of the murine PDE5A isoforms in the yeast Kluyveromyces lactis