Efficient microbial cell factories require the assembly of biosynthetic and metabolic pathways within a microbial host. The genetic information for these pathways needs to be integrated into the genome of the microbe. Often, genomic integration is limited by the availability of so-called neutral sites whose modifications do not impact cell fitness. The study “Expanding the neutral sites for integrated gene expression in Saccharomyces cerevisiae” in FEMS Microbiology Letters characterized a multitude of neutral sites in the S. cerevisiae genome to be used for genomic-editing studies. Yongjin Zhou explains for the #FEMSmicroBlog how these can expand the metabolic capacity of S. cerevisiae. #FascinatingMicrobes
Genomic integration sites in Saccharomyces cerevisiae
To construct stable microbial cell factories, target genes are usually integrated into the genome of a microbial host. Yet, these genes need to be integrated at sites where the target genes do not compromise cellular fitness or interfere with the metabolism.
Integrated synthetic genes should not compromise cellular fitness or interfere with the metabolism of the microbial host.
Modifying these locations does not affect cell viability or cause any significant phenotypic changes. Often, interval sequences between coding gene sequences are potential neutral sites for gene integration. Knowledge of such neutral sites in microbial hosts is limited. Hence, identifying additional neutral sites in microbes is of utmost importance.
The baker’s yeast Saccharomyces cerevisiae is an ideal host for the bioproduction of natural products, biofuels and chemicals. Improving the performance of yeast cells always involves extensive metabolic rewiring, which usually requires a large number of neutral sites to integrate long biosynthetic pathways.
The study “Expanding the neutral sites for integrated gene expression in Saccharomyces cerevisiae” in FEMS Microbiology Letters aimed to identify additional neutral sites on the yeast genome to facilitate gene expression studies in yeast cells. The work avoided disrupting telomeres and mitotic sites as well as encoding genes, and yet, managed to identify dozens of candidate sites on the 16 chromosomes.
Evaluating genomic neutral sites
To achieve this, the study used an approach based on the fluorescence intensity of green fluorescent protein (GFP), which has always been a popular method to characterize gene expression. As a marker for characterizing neutral sites, the signal from GFP activity gives conclusions about the gene expression levels at the integrated neutral site.
Interestingly, the intensity of the GFP signal varied among the different neutral sites. These results suggest that the chromosome structure affects gene expression at the transcriptional level by interfering with the binding of transcriptional enzymes or factors.
The study also evaluated the neutral sites by integrating the gene MaFAR1 which encodes a fatty-acyl-CoA reductase. This enzyme catalyzes the biosynthesis of certain fatty alcohols. Expressing this gene from different integration sites and measuring the expression levels can give an idea about the suitability of the integration site.
These results imply that integrating target genes at different neutral sites can be a feasible strategy to regulate gene expression and biosynthetic pathways. Taken together, this study identified a plethora of neutral sites for the genomic integration of genes in the genome of S. cerevisiae. Each one of these sites can be used for metabolic engineering procedures in S. cerevisiae. This strategy can further help identify genome-neutral sites in other yeasts to facilitate the metabolic engineering of yeast cell factories.
- Read the Editor’s Choice article “Expanding the neutral sites for integrated gene expression in Saccharomyces cerevisiae” by Kong et al. (2022).
Yongjin Zhou is a Chair Professor at the Dalian Institute of Chemical Physics (DICP), Chinese Academy of Sciences. He received his PhD in Biochemical Engineering from the Dalian Institute of Chemical Physics in 2012. After finishing a Postdoc research project at the Chalmers University of Technology, he started the Microbial Synthetic Biology lab at DICP in 2017. His research mainly focuses on synthetic microbiology for cell factory construction, yeast genetics and metabolic engineering.
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The section #FascinatingMicrobes for the #FEMSmicroBlog explains the science behind a paper and highlights the significance and broader context of a recent finding. One of the main goals is to share the fascinating spectrum of microbes across all fields of microbiology.
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