Increasing lipid yield in Yarrowia lipolytica through phosphoketolase and phosphotransacetylase expression in a phosphofructokinase deletion strain

Abstract

Background Lipids are important precursors in the biofuel and oleochemical industries. Yarrowia lipolytica is among the most extensively studied oleaginous microorganisms and has been a focus of metabolic engineering to improve lipid production. Yield improvement, through rewiring of the central carbon metabolism of Y. lipolytica from glucose to the lipid precursor acetyl-CoA, is a key strategy for achieving commercial success in this organism. Results Building on YB-392, a Y. lipolytica isolate known for stable non-hyphal growth and low citrate production with demonstrated potential for high lipid accumulation, we assembled a heterologous pathway that redirects carbon flux from glucose through the pentose phosphate pathway (PPP) to acetyl-CoA. We used phosphofructokinase (Pfk) deletion to block glycolysis and expressed two non-native enzymes, phosphoketolase (Xpk) and phosphotransacetylase (Pta), to convert PPP-produced xylulose-5-P to acetyl-CoA. Introduction of the pathway in a pfk deletion strain that is unable to grow and accumulate lipid from glucose in defined media ensured maximal redirection of carbon flux through Xpk/Pta. Expression of Xpk and Pta restored growth and lipid production from glucose. In 1-L bioreactors, the engineered strains recorded improved lipid yield and cell-specific productivity by up to 19 and 78%, respectively. Conclusions Yields and cell-specific productivities are important bioprocess parameters for large-scale lipid fermentations. Improving these parameters by engineering the Xpk/Pta pathway is an important step towards developing Y. lipolytica as an industrially preferred microbial biocatalyst for lipid production.

Impact of central carbon metabolism bypasses on the production of beta-carotene inYarrowia lipolytica

“…In contrast, the introduction of phosphoketolase bypass, which has been shown to be largely beneficial for enhancing the production of metabolites downstream of acetyl-CoA in various studies (15–18, 20, 21), led to decreased production, yield, and content of beta-carotene in our study. In three studies that have utilized the same set of genes for the phosphoketolase bypass as we did, the authors have observed positive impact on the production of acetate (17) and lipids (15), and no impact on the production of alpha-humulene (19).…”

Lipid Readjustment in Yarrowia lipolytica Odd-Chain Fatty Acids Producing Strains

“…Over the past several years, great progress has been made to improve lipid and lipid-derivatives production by Y. lipolytica [ 48 , 49 , 50 ]. Different strategies have been used for increasing lipid accumulation and recovery from Y. lipolytica [ 51 , 52 ].…”

Advances in the optimization of central carbon metabolism in metabolic engineering

“…The knockout of phosphofructokinase (PFK) in Yarrowia lipolytica blocked the metabolic flux of G6P in glycolysis and caused the redox imbalance with excess NADPH production. Then the introduction of the PHK pathway resulted in the accumulation of acetyl-CoA and correction of the redox imbalance by providing a route towards the NADPH-oxidizing lipid synthesis pathway, and a 19% increase in total lipid production was derived in the host strain [ 26 ]. In the TCA cycle, citrate can be catalyzed by ATP:citrate lyase (ACL) to produce acetyl-CoA [ 27 ].…”