Abstract
As biotechnological applications of synthetic biology tools including multiplex genome engineering are expanding rapidly, the construction of strategically designed yeast cell factories becomes increasingly possible. This is largely due to recent advancements in genome editing methods like CRISPR/Cas tech and high-throughput omics tools. The model organism, baker’s yeast (Saccharomyces cerevisiae) is an important synthetic biology chassis for high-value metabolite production. Multiplex genome engineering approaches can expedite the construction and fine tuning of effective heterologous pathways in yeast cell factories. Numerous multiplex genome editing techniques have emerged to capitalize on this recently. This review focuses on recent advancements in such tools, such as delta integration and rDNA cluster integration coupled with CRISPR-Cas tools to greatly enhance multi-integration efficiency. Examples of pre-placed gate systems which are an innovative alternative approach for multi-copy gene integration were also reviewed. In addition to multiple integration studies, multiplexing of alternative genome editing methods are also discussed. Finally, multiplex genome editing studies involving non-conventional yeasts and the importance of automation for efficient cell factory design and construction are considered. Coupling the CRISPR/Cas system with traditional yeast multiplex genome integration or donor DNA delivery methods expedites strain development through increased efficiency and accuracy. Novel approaches such as pre-placing synthetic sequences in the genome along with improved bioinformatics tools and automation technologies have the potential to further streamline the strain development process. In addition, the techniques discussed to engineer S. cerevisiae, can be adapted for use in other industrially important yeast species for cell factory development.
Highlights
Saccharomyces cerevisiae (Baker’s yeast) has been exploited by humans for millennia for the production of fermented foods and beverages
GRAPHICAL ABSTRACT | Overview of key approaches used for multiplex genome engineering for yeast. (A) Delta sequences and ribosomal DNA (rDNA) cluster found in the yeast genome can be targeted for multi-copy integration. (B) For donor DNA delivery, multiple linearized plasmids or multiple oligonucleotides can be used to target multi-loci. (C) Multiple sgRNAs can be used to target specific regions in the yeast genome
This review considers recent studies in which traditional genome editing methods such as delta integration, rDNA clusters and plasmid-based integration were coupled with CRISPR/Cas technology for enhanced efficiency
Summary
Saccharomyces cerevisiae (Baker’s yeast) has been exploited by humans for millennia for the production of fermented foods and beverages. The species has gained substantial interest as a model platform for the renewable production of valuable chemicals in recent times (Nowrouzi et al, 2020; Walls et al, 2020). Such chemicals have extensive applications across the agricultural, Yeast Multiplex Genome Engineering Methods. Its genetic and physiological features are well characterized (Hong and Nielsen, 2012; Buchholz and Collins, 2013; Li and Borodina, 2014) and relevant tools and knock-out libraries are widely available for straightforward strain manipulation (Jensen and Keasling, 2014)
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