Combining metabolic engineering and fermentation optimization to achieve cost-effective oil production by Cutaneotrichosporon oleaginosus

Microbial oils, produced by oleaginous microorganisms, offer a sustainable alternative to plant-derived oils. Among these microorganisms, Cutaneotrichosporon oleaginosus attracts attention as a promising microbial cell factory for sustainable oil production due to its capacity to accumulate lipids with a similar composition to palm oil. Although C. oleaginosus can reach higher lipid contents than other oleaginous yeasts, suboptimal lipid yields and productivity limit economic feasibility. Enhanced productivity is necessary to have a feasible microbial oil production process via C. oleaginosus. In this study, we followed a combinatorial approach for strain design and bioprocess development to improve the lipid content and lipid yield. Initially, we deployed a full factorial design with genetic factors (ATP-citrate lyase (ACL), acetyl-CoA carboxylase (ACC), threonine synthase (TS)) and carbon-to-nitrogen ratio (C/N) in the medium. The C/N ratio appeared to have the most impact on oil accumulation. Combined with genetic modifications, lipid content and lipid yield increased by 1.6-fold. In a two-stage fermentation approach at a 2L scale, the triple transformant overexpressing ACL, ACC, and TS outperformed the wild-type by achieving a lipid content of 75.4% (w/w) with lipid productivity of 0.40 g L-1 h-1 and around 0.30 g lipids/g glycerol. In all, we established a cultivation strategy and strain that reached almost the theoretical maximum yield, and highest lipid content reported for a medium containing glycerol as a carbon source. These results strengthen the basis of using C. oleaginous as a platform for microbial oil production, thereby facilitating the development of processes substituting palm oil with a sustainable alternative. HighlightsO_LIFull factorial design allowed selection of the best performer transformant. C_LIO_LIOverexpression of ACL1, ACC, and TS in C. oleaginosus provided higher lipid yield. C_LIO_LITwo-stage fermentation enhanced the lipid content of wild-type and transformant. C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=62 SRC="FIGDIR/small/624102v1_ufig1.gif" ALT="Figure 1"> View larger version (17K): org.highwire.dtl.DTLVardef@1371a7aorg.highwire.dtl.DTLVardef@ab5ae1org.highwire.dtl.DTLVardef@1db84caorg.highwire.dtl.DTLVardef@eb19e9_HPS_FORMAT_FIGEXP M_FIG C_FIG