(597e) Synthetic Biology Tools Development and Metabolic Engineering of Yarrowia Lipolytica for Producing Lipid-Based Chemicals

The industrially relevant yeast Yarrowia lipolytica with import applications has attracted growing attention due to its high capability of accumulating lipid. To facilitate genetic manipulation of Y. lipolytica, a comprehensive synthetic biology toolbox has been developed. The molecular tools including natural and engineered promoters, which can be used for fine-tuning target genes expression, and a toolkit for sequential gene knockout in Y. lipolytica with high frequency of homologous recombination were characterized and developed. Construction of pathways involving serval steps is required for metabolic engineering of microorganisms for biofuel and chemicals production, and usually multiple-gene expression in eukaryotes such as yeast is labor-intensive and time-consuming. To overcome this obstacle, we first created a set of modular expression vectors similar with BioBricks-compatible plasmids by combination of replication origins, auxotrophic marker genes, characterized promoters and terminators. We further introduced a novel methodology that could generate the individual active enzymes from the polycistronic mRNA of a gene cluster under a single promoter in Y. lipolytica by using viral 2A peptide mediated cleavage. As proof of the concept, the entire carotenoid biosynthesis pathway was assembled and engineered into the heterologous host, Y. lipolytica, and a mutant of the gene encoding ERG20 from Y. lipolytica was identified as a more efficient enzyme for producing lycopene. After establishment of the genetic manipulation platform, lipid metabolism in cell factories of Y. lipolytica was tailored for successfully overproducing oleochemical including free fatty acid, fatty alcohol and wax esters with wide applications as ingredient of detergents, surfactants, and personal care products. Metabolic engineering strategies for enhancing production of lipid precursors (“push”), overexpressing genes for tailored products formation (“pull”) and deleting genes for β-oxidation (“block”) were employed. More significantly, guided with a genome-scale metabolic network model of Y. lipolytica, fatty alcohol production was remarkably improved and the by-product, citric acid accumulation was eliminated by boosting cellular energy generation. These combined efforts eventually resulted in the development of synthetic biology tools for genetic manipulation of this important strain Y. lipolytica and paved the way for production of tailor-made products through interception and modification of yeast lipid metabolism.