Automated multiplex genome-scale engineering in yeast

Abstract

Genome-scale engineering is indispensable in understanding and engineering microorganisms, but the current tools are mainly limited to bacterial systems. Here we report an automated platform for multiplex genome-scale engineering in Saccharomyces cerevisiae, an important eukaryotic model and widely used microbial cell factory. Standardized genetic parts encoding overexpression and knockdown mutations of >90% yeast genes are created in a single step from a full-length cDNA library. With the aid of CRISPR-Cas, these genetic parts are iteratively integrated into the repetitive genomic sequences in a modular manner using robotic automation. This system allows functional mapping and multiplex optimization on a genome scale for diverse phenotypes including cellulase expression, isobutanol production, glycerol utilization and acetic acid tolerance, and may greatly accelerate future genome-scale engineering endeavours in yeast.

Unraveling the genetic basis of fast l‐arabinose consumption on top of recombinant xylose‐fermenting Saccharomyces cerevisiae

“…Finally, 57.07 g/L ethanol was produced in YPD80X40A20 medium, which is the highest rate among the published records ( Figure 5, Table 1, and Supporting Information Table S5). If a highly efficient, multiple copy integration method is implemented (Si et al, 2017), then we should obtain a fast L-arabinose-fermenting strain more quickly and easily. Here, we report our discovery of the phenomenon of amplification of AUC genes after adaptive evolution and confirmed its decisive role in arabinose utilization.…”

Engineering biological systems using automated biofoundries

“…In most cases, these DNA molecules need to be introduced into cells to be expressed and tested. There is already a collection of well-defined protocols for transforming, transfecting, or transducing model organisms, while some of these operations has been automated (Ben Yehezkel et al, 2011; Si et al, 2017), some, however still remains challenges to be automated in high throughput. First, some protocols require strict experimental conditions, such as low temperature, precise timing, as well as quick but gentle pipetting.…”

“…This DNA assembly workflow was also applied to pathway engineering in different organisms for various products. In addition to bottom-up assembly of synthetic DNA constructs, a genome engineering workflow for Saccharomyces cerevisiae was also implemented on the iBioFAB (Si et al, 2017). According to the above-mentioned roadmap, two bottlenecks were identified as lack of standardized and modular methods to introduce genetic modulations, and low genome modification efficiency during each engineering cycle.…”

The renaissance of yeasts as microbial factories in the modern age of biomanufacturing

“…To date, generating these deletion collections was a laborious process limiting their applications to a handful of strains, but several methods have been developed for genome‐scale engineering in a variety of species (e.g., Si et al, ). CRISPR‐Cas9 gene editing has been combined with genetic barcoding to enable efficient, traceable, markerless, and genome‐scale editing (Bao et al, ; Guo et al, ; Roy et al, ).…”