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.
Highlights
Genome-scale engineering is indispensable in understanding and engineering microorganisms, but the current tools are mainly limited to bacterial systems
Cloning from complementary DNA (cDNA) libraries, both overexpression and knockdown mutations can be created on a genome scale, which is proved effective in studying various traits including protein and chemical production, substrate utilization and inhibitor tolerance
In E. coli, recombineering enables insertion of synthetic DNA cassettes upstream of 495% genes to achieve both up- and downregulation with antibiotic selection
Summary
Genome-scale engineering is indispensable in understanding and engineering microorganisms, but the current tools are mainly limited to bacterial systems. Multiplex genome-scale modifications will result in enormous diversity, which requires robotic automation to facilitate creation and screening of genomic libraries, but very few examples have been reported to automate such efforts[6], and there is a general lack of standardized procedures of microbial genome-scale engineering To address these limitations, we sought to devise an automated system to integrate genome-wide screening and multiplex optimization. Our system integrated three major designs for automated yeast engineering: (1) To achieve genome-wide coverage, a normalized full-length-enriched complementary DNA (cDNA) library was used to construct genetic modulation parts encoding both overexpression and knockdown mutations. On the basis of these designs, multiplex genome-wide mutations can be accumulated in a scalable manner using a standardized workflow, enabling automated genome-scale engineering in S. cerevisiae Using this system, we performed genotype–phenotype mapping of both gain- and reduction-of-function mutations for 492% yeast genes and successfully created multiplex diversity on a genome scale in a fully automated manner. A variety of industrially relevant traits were successfully improved in S. cerevisiae
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