Harnessing Yarrowia lipolytica lipogenesis to create a platform for lipid and biofuel production

Abstract

Economic feasibility of biosynthetic fuel and chemical production hinges upon harnessing metabolism to achieve high titre and yield. Here we report a thorough genotypic and phenotypic optimization of an oleaginous organism to create a strain with significant lipogenesis capability. Specifically, we rewire Yarrowia lipolytica's native metabolism for superior de novo lipogenesis by coupling combinatorial multiplexing of lipogenesis targets with phenotypic induction. We further complete direct conversion of lipid content into biodiesel. Tri-level metabolic control results in saturated cells containing upwards of 90% lipid content and titres exceeding 25 g l À 1 lipids, which represents a 60-fold improvement over parental strain and conditions. Through this rewiring effort, we advance fundamental understanding of lipogenesis, demonstrate non-canonical environmental and intracellular stimuli and uncouple lipogenesis from nitrogen starvation. The high titres and carbon-source independent nature of this lipogenesis in Y. lipolytica highlight the potential of this organism as a platform for efficient oleochemical production.

Integrated bioprocess for conversion of gaseous substrates to liquids

“…One promising direction has been the production of microbial oil from carbohydrate feedstocks. This oil can be readily converted to biodiesel and recently there has been significant progress in the engineering of oleaginous microbes for the production of lipids from sugars (2)(3)(4)(5). A major problem with this approach has been the relatively high sugar feedstock cost.…”

Synthetic Biology and Metabolic Engineering Approaches and Its Impact on Non-Conventional Yeast and Biofuel Production

“…Disruption of the genes involved in the NHEJ pathway Ku70 (Näätsaari et al, 2012;Verbeke et al, 2013) or Ku80 (Kooistra et al, 2004;Saraya et al, 2012) enhanced the efficiency of homologous recombination. The frequency of homologous recombination in S. cerevisiae is high achieved with 40 bp, while in the case of non-conventional yeast 500-3,000 bp of flanking sequence is required (Blazeck et al, 2014;Horwitz et al, 2015). Construction of expression cassettes for such long adaptor sequences requires a large number of PCR reactions, cloning steps, etc.…”

“…Yarrowia lipolytica is a well-studied oleaginous organism and extensively used for industrial biofuel production, and it has been served as a model organism for biofuel research, especially for fatty acid-derived fuels (Beopoulos et al, 2009;Tai and Stephanopoulos, 2013;Blazeck et al, 2014;Zhou et al, 2016). Several metabolic engineering tools are available for Y. lipolytica (Juretzek et al, 2001;Madzak, 2015).…”

“…Further, they proved that lipid production from starch can be enhanced by both metabolic engineering and culture condition optimization. Blazeck et al (2014) rewired Y. lipolytica native metabolism for increased lipid titers, by coupling combinatorial multiplexing of lipogenesis targets with phenotypic induction. The tri-level metabolic control results in accumulation of 90% lipid content and 60-fold improvement over wild-type strain.…”

Microbes paired for biological gas-to-liquids (Bio-GTL) process

“…Similarly, the oleaginous yeast, Yarrowia lipolytica, has evolved the ability to use available carbon feedstocks to synthesize (with high yield) and store large amounts of lipids when growth is limited by inorganic nutrient starvation. Strains of Y. lipolytica have been engineered to store up to 90% of dry weight as lipid (3). The approach of combining evolved specialists is a sharp contrast to the more frequently observed synthetic biology strategy of combining desired traits into a single organism [e.g., a consolidated bioprocessing microbe (16)].…”