Functional Investigation Of The Nmt1 Promoter Of Fission Yeast Schizosaccharomyces Pombe

Yeasts are widely used in the production of recombinant proteins of medical or industrial interest.For each individual product,the most suitable expression system has to be identified and optimized,both on the genetic and fermentative level,by taking into account the properties of the product,the organism and the expression cassette.Several species were engineered to have further advantages,such as humanized glycosylation pathways or lack of proteases.Additionally,with a large variety of vectors,promoters and selection markers to choose from,combined with the accumulated knowledge on industrial-scale fermentation techniques and the current advances in the post-genomic technology,it is possible to design more cost-effective expression systems in order to meet the increasing demand for recombinant proteins and glycoproteins.The nmt1 promoter that is repressible by addition of thiamine into a medium has been most widely used for expressing heterologous genes.Although its expression is known to be strong,it is not fully known how much its transcriptional activity varies among different p H conditions.For this purpose,we cultivated recombinant fission yeast strains under different buffer conditions ranging from p H 2 to p H 10(in the absence of thiamine)and monitored the expression levels of three test genes as well as that of the endogenous nmt1 gene by q PCR.A strong dependence of the relative m RNA expression levels of nmt1 genes on the p H conditions was observed,with highest values being found under acidic conditions,but surprisingly,different expression constructs did not show a uniform pattern.In contrast,expression levels for genes present in single copies on a chromosome showed a broader range of maxima.These results suggest that in a typical over night batch culture,which upon exhaustion of the buffer capacity of the media display a p H gradient,the reproducibility of recombinant protein expression experiments might be compromised when using constructs that harbor the nmt1 promotor.Inducible/repressible promoters are useful for the maintenance of toxic genes or timely expression.For ectopic expression of cloned genes in the fission yeast S.pombe,the thiamine-regulatable nmt1 promoter has been widely used,since the transcriptional activity of this promoter can be controlled by thiamine.However,this property sometimes limits a certain type of research,since the expression inevitably requires cells to be cultivated under the conditions that induce promoter activation.The nmt1 promoter,for example,is not suitable for overexpression of genes in YES medium(containing thiamine),which is commonly used for yeast cell culture.We have made stepwise deletions of the nmt1 promoter and quantitated the effects of the deletions by assaying the expression of the cytochrome P450 CYP2C9 cloned downstream.To allow constitutive expression of heterologous genes,we cloned a set of nmt1 promoter deletion constructs(-800,-600,-400,-200)of p REP1-CYP2C9.Construction of a series of vectors comprising these promoters and their introduction into the fission yeast cells demonstrated that the activity was different among these promoters but was not affected by cultured media commonly used in fission yeast.Therefore,a promoter with appropriate strength would be selectable from these promoters,depending on the genes to be expressed.