Development of a reporter system for the yeast Schwanniomyces occidentalis: influence of DNA composition and codon usage

Abstract

In this paper we report on searching for suitable reporters to monitor gene expression and protein secretion in the amylolytic yeast Schwanniomyces occidentalis. Several potential reporter and marker genes, formerly shown to be functional in other yeasts, were cloned downstream from the homologous invertase gene (INV ) promoter and their activity was followed in conditions of repression and derepression of the INV promoter. However, neither β-glucuronidase nor β-lactamase nor phleomycin resistance-conferring gene, all originating from E. coli, were expressed in S. occidentalis cells to such a level to allow for monitoring of their activity. All the reporter genes tested have a higher percentage of GC (47-62%) in their DNA compared to the DNA composition of S. occidentalis genes that are more ATrich (36% GC). The codon usage of all the reporter genes also varies from that of 16 so far sequenced S. occidentalis genes. This suggests that an appropriate composition of DNA and a codon usage similar to S. occidentalis genes might be very important parameters for an efficient expression of a heterologous gene in Schwanniomyces occidentalis. Indeed, two genes originating from Staphylococcus aureus, with an AT-content in their DNA similar to that of S. occidentalis, were functionally expressed in S. occidentalis cells. Both a phleomycin resistance-conferring gene and a chloramphenicol acetyltransferase-encoding gene thus represent suitable reporters of gene expression and protein secretion in S. occidentalis. Additionally, we show in this work that the transcription-regulating region and the signal peptide sequence of the S. occidentalis invertase gene were efficient to direct gene expression and subsequent protein secretion in Saccharomyces cerevisiae.

New Insights into the Fructosyltransferase Activity ofSchwanniomyces occidentalisβ-Fructofuranosidase, Emerging from Nonconventional Codon Usage and Directed Mutation

“…It has been proposed that the codon usage bias is generally correlated with the overall genome GϩC content (8) and that the codons favored are due to the tDNA gene copy number (20). CUG is a rare leucine codon in S. cerevisiae (GϩC, 40%), and it is also a rare codon for serine in S. occidentalis (GϩC, 36%) (13). The decoding of the leucine CUG codon as serine has been reported previously in the cytoplasm of some Candida species (15) and in P. farinosa (33).…”

“…However, our study of the Ffase sequence provides the first direct evidence for CUG decoding as serine in S. occidentalis. In this context, it has not been possible to express the ␤-glucuronidase (gusA) or ␤-lactamase (bla) gene or the phleomycin resistance-conferring gene (Tn5ble) from E. coli in S. occidentalis (13). Thus, an appropriate DNA composition and codon usage bias might be important parameters for efficient expression of heterologous genes in this host.…”

Yarrowia lipolytica Mutants Devoid of Pyruvate Carboxylase Activity Show an Unusual Growth Phenotype

“…We hypothesize that the difference in codon usage between the two yeasts (19) may explain the lack of functional enzyme. The genome from Y. lipolytica is richer in GϩC than that of S. cerevisiae and differences in this content have been reported in some cases to be responsible for lack of functionality of heterologous proteins (30).…”

Codon optimization enables the Zeocin resistance marker’s use in the ascomycete yeast <i>Debaryomyces occidentalis</i>

“…The phleomycin resistance-conferring gene is a suitable marker because of its DNA composition and codon usage, which influence expression. The DNA of D. occidentalis genes is very ATrich (36% GC content), and a high percentage of GC-rich (47-62%) genes are not expressed (Janatova et al, 2003). The Staphylococcus aureus bleomycin (Sa ble) gene having an AT-rich composition conferred to the phleomycin resistance could, therefore, be expressed in D. occidentalis (Janatova et al, 2003).…”