Engineering Yarrowia lipolytica for high lipid production

Among potential value-added fuels and chemicals, fatty acid-based chemicals are important due to their wide use in industrial processes and in daily life. Fatty acids produced from microbial systems could provide a sustainable supply to replace the current costly and unsustainable process using plant oil or animal fat. The oleaginous yeast Yarrowia lipolytica naturally possesses moderate lipid production capacity and grows on different kinds of biomass and organic waste. However, fatty acid production from native, un-engineered strains is not economically viable. Therefore, this work develops strategies inspired from synthetic biology and metabolic engineering to expand the engineering potential of Y. lipolytica — helping to establish this organism as a premier platform for industrial-level, high lipid production as well as providing a platform for uncovering novel understanding of lipogenesis. \nTo do so, first, novel synthetic promoters and high expression plasmid were necessary to achieve the ability to tune gene expression levels inside the cell. We developed a hybrid promoter engineering strategy to create a promoter library exhibiting a range of more than 400-fold in terms of mRNA levels as well as engineered plasmids with regulated centromeric function to achieve a 2.7 fold expression range. Next, a rational and evolutionary metabolic engineering approach was coupled with genomic and transcriptomic studies to both engineer and understand underlying lipogenesis in this organism. Through the engineering efforts, we successfully increased the lipid production titer to over 40 g/L in bioreactor as well as identified novel lipogenic enhancers and mechanisms. In addition, we identified and characterized a mutant mga2 protein with superior lipogenesis enhancing capacity, which can regulate fatty acid desaturation and carbon flux inside the cells. Collectively, these studies have facilitated the utilization of Y. lipolytica as an industrially relevant microbial lipid production platform and supplied novel understanding of its lipogenesis process. The methods and concepts developed here can also be adapted to other oleaginous microbes and serve as a template for enabling value-added chemical production in other nonconventional organism.