Development Of Kluyveromyces Cicerisporus Expression System

In order to develop a yeast expression system of Kluyveromyces cicerisporus CBS4857, we first cloned the KcURA3 gene and KcHIS3 gene by the complementation of the ura3 and his3 mutation in Saccharomyces cerevisiae W303-1B with a genomic library of K. cicerisporus CBS4857 we constcucted. The KcURA3 gene encoded a 267 amino acid protein with an 87% amino acids identity to that of S. cerevisiae. The KcHIS3 gene encoded a 230 amino acid protein with an 70% amino acids identity to that of S. cerevisiae.An ura3 mutant strain from K. cicerisporus CBS4857, named as Y179U was obtained by selection on the 5-FOA plate. The sequence analysis of this mutated gene revealed that it contained a point mutation at nucleotide position +278. Several vectors including the integrating type and autoreplicating type were constructed bearing the KcURA3 gene as selection marker. The autoreplicating plasmids can transform Y179U strain with a high efficiency by the optimal LiAc method and electroporation. So we successfully constructed a transformation system of K. cicerisporus CBS4857. Then a new plasmid pUKD-S was constructed by modifying the plasmid pUKD, and it showed a more higher stability and transform efficiency than the original plasmid pUKD.An his3 strain was constructed by disruption the KcHIS3 gene of Y179U through homologous recombination and named as Y179H. The strain can be tranformed with a autoreplicating vector pHK1. Thus we constructed a new host-vector system of K. cicerisporus CBS4857 with the KcHIS3 gene as a selection marker.With the promoter and terminator of the INU gene we constructed several expression vectors of the HBsAg S-preS1 fusion gene. These expression vectors were transformed to Y179U strain and the tranformants showed that the HBsAgS-preS1 fusion gene had been expressed by detecting the fermentation products with ELISA.This result was also confirmed by the Western-blot test. The expression vectors pUKD-S-PLT showed a highest expression level with a 3000 titer among all the expression vectors. With the promoter, terminator of the INU gene andα-factor we constructed expression vector of the interferon α2b gene. The fermentation product of transformants show that interferon α2b had been secreted by Y179U throgh SDS-PAGE which also had been confirmed by Western-blot. After optimizing the fermentation condition the expression level of interferon α2b was estimated as 62.5mg/L. Thus we developed a new yeast expression system of K. cicerisporus CBS4857 and successfully expressed heterologous gene in the K. cicerisporus CBS4857 for the first time.