(129d) A Tool for Rapid Integration of Long Metabolic Pathways in Yarrowia Lipolytica

Plant natural products (PNPs) represent a large and vital class of medicinal compounds regularly used to treat a variety of ailments. PNPs can be extremely complex molecules, with great regio- and stereo-selectivity required for their synthesis, prohibiting economical conventional chemical synthesis. Additionally, the plants that natively produce these compounds generally grow slowly and produce a very small amount of the product of interest, making extraction from plant matter very expensive and inefficient. Heterologous biosynthesis of PNPs is the most promising avenue for a reliable and cost-effective method of production. However, the metabolic pathways that facilitate production of PNPs are generally as long as the products are complex. The targeted integration of these pathways using CRISPR-based toolkits is achievable, as has been demonstrated in S. cerevisiae and P. pastoris, however, the genes must be integrated one or two at a time, severely limiting the speed at which a strain or library of strains can be generated. Additionally, the use of a non-conventional, oleaginous yeast such as Y. lipolytica may be superior compared to S. cerevisiae due to the enhanced flux towards precursors and the formation of intracellular lipid droplets. Both attributes can be leveraged to increase titers by increasing flux to the product of interest and sequester potentially toxic non-polar products and intermediates. One class of medicinally relevant PNPs, monoterpene indole alkaloids (MIAs), includes the anti-malarial quinine, the anti-addiction ibogaine and the anti-cancer vincristine and vinblastine as well as many others. All MIAs are derived from a single compound, strictosidine, itself fairly complex. The heterologous biosynthesis of strictosidine requires the stable overexpression of 20 genes, with more genes necessary for production of any downstream product. The rapid generation of strain libraries capable of producing MIAs requires the integration of these pathways in as few integration events as possible. Serine integrases have been used to integrate extremely large DNA fragments (>50 kb) in mammalian cells and have demonstrated activity in a variety of other organisms such as S. cerevisiae and E. coli, but their use has not been demonstrated in Y. lipolytica. The serine integrase under investigation here (ΦC31) recognizes several different attachment sites, allowing for the design of a marker recycling system that is not predicated on other enzymes or plasmid curing, further enhancing the throughput. Additionally, a three fluorescent protein system for identification and quantification of recombination outcome has been developed. This will aid in optimization of the recombination reaction and can be used to sort recombination positive cells if efficiencies are low with extremely large DNA integrations. We expect to optimize the recombination procedure and to generate a strain capable of strictosidine production by a single integration event. The development of this integration system is expected to enable easy and efficient integration of long metabolic pathways in Y. lipolytica, allowing for a much higher throughput Design-Build-Test-Learn cycle. It may be adapted in the future to a variety of different organisms.