Metabolic engineering strategies for increasing lipid production in oleaginous yeast

Abstract

Although petroleum and other fossil fuels have traditionally been used to fulfill our energy needs, rising concerns over energy security and the climate-changing effects of our continual greenhouse gas emissions have led to great interest in developing a domestic source of renewable fuel with low net carbon emissions. Biodiesel is an attractive option for replacing petroleum-based fuels used in the transportation sector due to its compatibility with existing infrastructure. Single cell oils from heterotrophic oleaginous microorganisms as a source of bio diesel allow for high productivity from a wide array of potential feedstocks, including agroindustrial and municipal waste streams. The goal of this work is to use the tools of rational metabolic engineering to improve lipid production in the non-conventional oleaginous yeast Yarrowia lipoytica on two representative carbon sources, glucose and acetate. Previous work in this area achieved considerable success with the simultaneous overexpression of the native acetyl-CoA carboxylase (ACC 1) and diacylglycerol acyltransferase (DGA2) genes; the resulting strain was used as a benchmark to evaluate our own efforts. We began with the compilation of a set of 44 genes and evaluated the effects of the individual overexpression of each gene on the ability of the resulting strain to produce lipids in fermentations of glucose and acetate. The genes tested here represent many different functions potentially important to lipid production, including the Kennedy pathway, fatty acid synthesis, central carbon metabolism, NADPH generation, regulation, and metabolite transport. Our results demonstrate that a diverse subset of gene overexpressions led to significant improvements in lipid production on at least one substrate. The largest improvements unsurprisingly came from overexpressing genes directly related to triacylglycerol synthesis, such as diacylglycerol acyltransferase DGAI, which on glucose increased the lipid titer, content and yield by 236%. 165%, and 246%, respectively, over our wild-type control strain, and the acylglycerolphosphate acyltransferase SLC1 gene, which increased titer/content/yield on glucose by 86%/73%/87% and on acetate by 99%/91%/151%. Significant improvements were also detected from genes that more indirectly effect lipogenesis, such as glycerol-3-phosphate dehydrogenase GPD (which produces head groups for triacylglycerol molecules) and the 6-phosphogluconolactoase SOL3 (catalyzing the middle step of the NADPH-producing oxidative pentose phosphate pathway). We next chose the aforementioned SLCl, GPD, and SOL3 genes for use in continued rational engineering of our benchmark strain due to the significance of their effects and the lack of redundancy in their likely mechanism of improving lipogenesis when overexpressed along with ACC I and DGA2. The results of this investigation indicate that the strain overexpressing ACC 1, DGA2,'and SLC 1 may be superior to our benchmark strain, increasing lipid content and yield by 24% and 20%, respectively, with a statistically equivalent titer on acetate. This strain produces the highest reported overall lipid yield of an oleaginous yeast on acetate, at 0.207 g lipids/g acetate.