Model-driven engineering of Cutaneotrichosporon oleaginosus ATCC 20509 for improved microbial oil production

Consumption of plant-based oils, especially palm oil, is increasing at an alarming rate. This boosted demand for palm oil has drastic effects on the ecosystem as its production is not sustainable. C. oleaginosus is an oleaginous yeast with great potential as a source for microbial-based oil production which is a sustainable alternative to palm oil. However, microbial processes are not yet economically feasible to replace palm oil, unto a large extent due to limited lipid accumulation in the microbe, which limits titers and productivity. Therefore, obtaining enhanced lipid accumulation is essential to render this process commercially viable. Herein we deployed a systematic, iterative Design-Build-Test-Learn (DBTL) approach to establish C. oleaginosus as an efficient fatty acid production platform. In the design step, we identified genes and medium supplements that improved lipid content. To this end, we compared its transcriptional landscape in conditions with high and low amounts of lipid production. A metabolic map was reconstructed and integrated with the expression data. Finally, the genome-scale metabolic model of C. oleaginosus was used to explore metabolism under maximal growth and maximal production conditions. The combination of these four analyses led to the selection of four overexpression targets (ATP-citrate lyase (ACL1), acetyl-CoA carboxylase (ACC), threonine synthase (TS), and hydroxymethylglutaryl-CoA synthase (HMGS)) and five media supplements (biotin, thiamine, threonine, serine, and aspartate). We established an electroporation-based co-transformation method to implement selected genetic interventions. These findings were experimentally validated in the build and test steps of the DBTL approach by adding supplements into the medium and overexpressing the identified genes. Characterization of ACL, ACC, and TS at various C/N ratios, and the addition of medium supplements provided up to 56% (w/w) lipid content, and a 2.5-fold increase in total lipid in the glycerol and urea-based defined medium. In the learn step, quadratic models identified the optimum C/N ratios shifted towards around C/N240. These results firmly confirm C. oleaginous as a sustainable alternative to replace palm as an oil source. HighlightsO_LITranscriptional profile and metabolic model analyzed, predicting genetic targets and medium supplements. C_LIO_LIGenetic targets and medium supplements for improved oil production. C_LIO_LIThe genetic toolbox for C. oleaginosus was expanded (co-transformation method, promoters, genes, and terminators). C_LIO_LIExperimental validations showed that biotin, and threonine increased lipid content. C_LIO_LIOverexpression of ACL1, ACC, and TS in C. oleaginosus provided higher oil content. C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=76 SRC="FIGDIR/small/585731v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@e3b6org.highwire.dtl.DTLVardef@65d016org.highwire.dtl.DTLVardef@40952eorg.highwire.dtl.DTLVardef@22724_HPS_FORMAT_FIGEXP M_FIG C_FIG