Effects of gene orientation and use of multiple promoters on the expression of XYL1 and XYL2 in Saccharomyces cerevisiae

Applied Biochemistry and Biotechnology
Ju Yun BaeThomas W Jeffries

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 sing...Continue Reading

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Citations

Dec 18, 2013·Proceedings of the National Academy of Sciences of the United States of America·Eric M YoungHal S Alper
Dec 17, 2015·Applied Microbiology and Biotechnology·Sun-Mi LeeHal S Alper
May 8, 2010·Metabolic Engineering·Nikhil U Nair, Huimin Zhao
Mar 15, 2015·Frontiers in Bioengineering and Biotechnology·Marie CarquetGilles Truan
Nov 28, 2013·G3 : Genes - Genomes - Genetics·Tae J LeeHarold D Kim
Feb 5, 2015·The Journal of Biological Chemistry·Veneta GerganovaGeorgi Muskhelishvili

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