Effects of Gene Orientation and Use of MultiplePromoters on the Expression of XYL1 and XYL2in Saccharomyces cerevisiae

摘要

Orientation of adjacent genes has been reported to affect their expression ineukaryotic systems, and metabolic engineering also often makes repeated use of a fewpromoters to obtain high expression. To improve transcriptional control in heterologousexpression, we examined how these factors affect gene expression and enzymatic activity inSaccharomyces cerevisiae. We assembled D-xylose reductase (XYL1) and D-xylitoldehydrogenase (XYL2) in four ways. Each pair of genes was placed in two differenttandem (1→2→ or ←1←2), convergent (1→ ←2), and divergent (←1 2→ ) orientations inautonomous plasmids. The TEF1 promoter was used to drive XYLI and the TDH3 promoterto drive XYL2 in each of the constructs. The effects of gene orientation on growth,transcription, and enzyme activity were analyzed. The transcription level as measured byquantitative PCR (q-PCR) correlated with enzyme activities, but our data did not show asignificant effect of gene orientation. To test the possible dilution of promoter strength dueto multiple use of the same promoter, we examined the level of expression of XYLI drivenby either the TEF1 or TDH3 promoter when carried on a single copy plasmid. We then co-expressed XYL2 from either a single or multicopy plasmid, which was also driven by thesame promoter. XYL2 transcript and enzyme expression increased with plasmid copynumber, while the expression of XYL1 was constant regardless of the number of other TEF1or TDH3 promoters present in the cell. According to our data, there is no significant effectof gene orientation or multiple promoter use on gene transcription and translation whengenes are expressed from plasmids; however, other factors could affect expression ofadjacent genes in chromosomes.