Abstract
BackgroundIntegration of heterogeneous genes is widely applied in synthetic biology and metabolic engineering. However, knowledge about the effect of integrative position on gene expression remains limited.ResultsWe established a genome-wide landscape of position effect on gene expression in Saccharomyces cerevisiae. The expression cassette of red fluorescence protein (RFP) gene was constructed and inserted at 1044 loci, which were scattered uniformly in the yeast genome. Due to the different integrative loci on the genome, the maximum relative intensity of RFP is more than 13-fold over the minimum. Plots of the number of strains to RFP relative intensity showed normal distribution, indicating significant position effect on gene expression in yeast. Furthermore, changing the promoters or reporter genes, as well as carbon sources, revealed little consequences on reporter gene expression, indicating chromosomal location is the major determinant of reporter gene expression.ConclusionsWe have examined the position effects to integration genes expression in large number loci around whole genome in S. cerevisiae. The results could guide the design of integration loci for exogenous genes and pathways to maximize their expression in metabolic engineering.
Highlights
Integration of heterogeneous genes is widely applied in synthetic biology and metabolic engineering
The position effect refers to that the heterologous genes inserted at different loci of chromosomes presented various expression levels, which have been reported in Escherichia coli [17, 18], Salmonella typhimurium [19, 20], Bacillus subtilis [21], Lactobacillus lactis [22], Saccharomyces cerevisiae [22,23,24,25,26], Drosophila melanogaster [27], mouse cells [28], and human cells [29]
Expression of red fluorescence protein (RFP) at different chromosome loci In this study, RFP gene with URA3 promoter was integrated into deletion strains from the Yeast Knock-Out library by replacing the kanMX gene (Fig. 1a)
Summary
Integration of heterogeneous genes is widely applied in synthetic biology and metabolic engineering. Knowledge about the effect of integrative position on gene expression remains limited. The effect of genomic location will open another door to regulate gene expression. In consideration of stability and efficiency of gene expression, chromosomal integration is preferred to episomal plasmid [7,8,9]. The structure of chromosome affects and regulates the cellular processes, including gene expression [10,11,12], DNA replication [13, 14] and transformation efficiency [15, 16]. The position effect refers to that the heterologous genes inserted at different loci of chromosomes presented various expression levels, which have been reported in Escherichia coli [17, 18], Salmonella typhimurium [19, 20], Bacillus subtilis [21], Lactobacillus lactis [22], Saccharomyces cerevisiae [22,23,24,25,26], Drosophila melanogaster [27], mouse cells [28], and human cells [29]
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