Ricerc@Sapienza

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 efect. 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-specifc 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 purifed A1, A2
and A3 isoforms displaying Km, Vmax and Sildenafl 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 defciencies and interferences with the endogenous cAMP/cGMP signal transduction
pathways.
Conclusions: To our knowledge, this is the frst 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 specifc PDE inhibitors for therapeutic applications in many pathologies.