Development and Application of Advanced Synthetic Biology Tools for Engineering Chemical Production in Yarrowia lipolytica

Renewable chemical production via microbial fermentation is a growing and critical industry. To overcome limitations in the productivity of commonly used hosts, nonconventional microbes that have uniquely advantageous metabolisms and phenotypes are needed. One such organism is the yeast Yarrowia lipolytica, which has a high native capacity to synthesize lipids and grow on a range of substrates. A significant bottleneck in engineering Y. lipolytica is a lack of synthetic biology tools for multiplexed genome editing and rapid strain development. To overcome these limitations, we have developed CRISPR-Cas9 based tools for (i) targeted gene disruption, (ii) expression cassette integration into predefined genomic loci, (iii) repression of native genes using CRISPR interference, (iv) activation of cryptic genes using CRISPR activation, and (v) genome-wide knockout screening. Straightforward adaptation of CRISPR-Cas9 strategies used in other eukaryotes had limited success, resulting in low gene disruption rates in Y. lipolytica. To improve this, we designed novel synthetic RNA polymerase III promoters for sgRNA expression, the best of which achieved gene disruption rates >90% and genome integration rates >50%. CRISPR-mediated markerless genome integration was used to make a strain of Y. lipolytica capable of producing over 21 mg/g DCW of the valuable carotenoid lycopene. We also adapted the CRISPR-Cas9 system for gene repression and significantly increased homologous recombination by transiently repressing genes involved in nonhomologous end-joining. The system was further adapted for gene activation and used to express natively silent β-glucosidase genes to enable growth on cellobiose. To accelerate strain engineering, we designed and constructed a library of plasmids expressing ~48,000 sgRNAs that target all protein coding sequences in the genome with 6-fold coverage. By transforming this pooled library into Y. lipolytica strains expressing Cas9 and quantifying sgRNA enrichment or depletion after outgrowth, genes important for growth under different conditions can be identified. By performing screening experiments in different strain backgrounds, determinants of CRISPR-Cas9 function can be characterized and industrially relevant phenotypes can be selected for