Effects of Gene Orientation and Use of Multiple Promoters on the Expression of XYL1 and XYL2 in Saccharomyces cerevisiae

Abstract

Orientation of adjacent genes has been reported to affect their expression in eukaryotic systems, and metabolic engineering also often makes repeated use of a few promoters to obtain high expression. To improve transcriptional control in heterologous expression, we examined how these factors affect gene expression and enzymatic activity in Saccharomyces cerevisiae. We assembled D-xylose reductase (XYL1) and D-xylitol dehydrogenase (XYL2) in four ways. Each pair of genes was placed in two different tandem (1-->2--> or <--1<--2), convergent (1--><--2), and divergent (<--1 2-->) orientations in autonomous plasmids. The TEF1 promoter was used to drive XYL1 and the TDH3 promoter to 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 by quantitative PCR (q-PCR) correlated with enzyme activities, but our data did not show a significant effect of gene orientation. To test the possible dilution of promoter strength due to multiple use of the same promoter, we examined the level of expression of XYL1 driven by 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 the same promoter. XYL2 transcript and enzyme expression increased with plasmid copy number, while the expression of XYL1 was constant regardless of the number of other TEF1 or TDH3 promoters present in the cell. According to our data, there is no significant effect of gene orientation or multiple promoter use on gene transcription and translation when genes are expressed from plasmids; however, other factors could affect expression of adjacent genes in chromosomes.

Transcription Interference and ORF Nature Strongly Affect Promoter Strength in a Reconstituted Metabolic Pathway

“…These effects were abolished when the two transcriptional processes were not simultaneous, confirming the strong TI in the case of simultaneous transcription. Contrarily to previous studies (Bae et al, 2008; Lee et al, 2014), our results provide a clear indication that relative positioning of three contiguous transcription units and their orientations probably induce TI on a multicopy plasmid. Large TI effects demonstrated in our design may be related to terminators efficiency, and are probably accentuated by the absence of extra-space between our three transcription units.…”

“…TI is defined as the suppressive influence of one transcriptional process, directly and in cis, on a second transcriptional process (Shearwin et al, 2005). This phenomenon was well-studied with native S. cerevisiae genes (Prescott and Proudfoot, 2002; Wang et al, 2014) but only two studies addressed the case of TI in heterologous expression, demonstrating transcription inhibition effects only in the case of divergent genes (Bae et al, 2008; Lee et al, 2014). It is known that the genome of S. cerevisiae is highly compact (an ORF every 2 kb in average) and less than 6% of the convergent adjacent genes show a pattern of co-expression.…”

Suppression of Expression Between Adjacent Genes Within Heterologous Modules in Yeast

“…Coexpression can be explained by bidirectional transcription attributable to various factors, including common cis -regulatory elements (in MAL6T-MAL6S ) (Bell et al 1995) or chromatin organization (in UGA3-GLT1 ) (Ishida et al 2006). In contrast, some studies reported no dependence on gene orientation (Bae et al 2008) and others reported anticorrelated gene expression between divergent genes (Lin et al 2007). Multiple factors may account for the differences in how divergent promoters behave, including the structure and properties of promoters ( i.e.…”