Abstract
BackgroundThe oleaginous, carotenogenic yeast Rhodotorula toruloides has been increasingly explored as a platform organism for the production of terpenoids and fatty acid derivatives. Fatty alcohols, a fatty acid derivative widely used in the production of detergents and surfactants, can be produced microbially with the expression of a heterologous fatty acyl-CoA reductase. Due to its high lipid production, R. toruloides has high potential for fatty alcohol production, and in this study several metabolic engineering approaches were investigated to improve the titer of this product.ResultsFatty acyl-CoA reductase from Marinobacter aqueolei was co-expressed with SpCas9 in R. toruloides IFO0880 and a panel of gene overexpressions and Cas9-mediated gene deletions were explored to increase the fatty alcohol production. Two overexpression targets (ACL1 and ACC1, improving cytosolic acetyl-CoA and malonyl-CoA production, respectively) and two deletion targets (the acyltransferases DGA1 and LRO1) resulted in significant (1.8 to 4.4-fold) increases to the fatty alcohol titer in culture tubes. Combinatorial exploration of these modifications in bioreactor fermentation culminated in a 3.7 g/L fatty alcohol titer in the LRO1Δ mutant. As LRO1 deletion was not found to be beneficial for fatty alcohol production in other yeasts, a lipidomic comparison of the DGA1 and LRO1 knockout mutants was performed, finding that DGA1 is the primary acyltransferase responsible for triacylglyceride production in R. toruloides, while LRO1 disruption simultaneously improved fatty alcohol production, increased diacylglyceride and triacylglyceride production, and increased glucose consumption.ConclusionsThe fatty alcohol titer of fatty acyl-CoA reductase-expressing R. toruloides was significantly improved through the deletion of LRO1, or the deletion of DGA1 combined with overexpression of ACC1 and ACL1. Disruption of LRO1 surprisingly increased both lipid and fatty alcohol production, creating a possible avenue for future study of the lipid metabolism of this yeast.
Highlights
The oleaginous, carotenogenic yeast Rhodotorula toruloides has been increasingly explored as a platform organism for the production of terpenoids and fatty acid derivatives
Production of 8 g/L fatty alcohols has been reported from growth of R. toruloides on YP-sucrose media in a fed-batch fermentation using MaFAR [10], no metabolic engineering efforts towards production of fatty alcohols have been reported to date, likely due to the lack of advanced gene editing tools compared to E. coli and S. cerevisiae
While a recent study of constitutive promoters in R. toruloides identified various promoters stronger than pGAPDH such as the transcription elongation factor promoter and the adenine nucleotide transporter promoter in a GFP expression assay, when pANT1 and pGAPDH were compared for fatty alcohol production in another recent study in R. toruloides, pGAPDH-driven expression resulted in a four-fold greater titer of 50 mg/L [19, 22]. 1-hexadecanol and 1-octadecenol constituted approximately 25% each of the total fatty alcohols, while the remaining 50% were 1-octadecanol
Summary
The oleaginous, carotenogenic yeast Rhodotorula toruloides has been increasingly explored as a platform organism for the production of terpenoids and fatty acid derivatives. Fatty alcohols are natively produced in many organisms as a component of natural waxes by reduction of fatty acids or fatty acyl-CoA by carboxylic acid reductases (CARs) or fatty acyl-CoA reductases (FARs), respectively [1] These fatty alcohol-producing genes have been expressed in a variety of heterologous microbial hosts including Escherichia coli [7], Synechocystis species [8], Saccharomyces cerevisiae, Yarrowia lipolytica [9], and Rhodotorula toruloides [10], enabling fatty alcohol production by microbial fermentation. S. cerevisiae produced 6.0 g/L of fatty alcohols using FAR from Mus musculus after 9 distinct genome edits were combined including targets for deletion and overexpression, and replacing the negative regulator of the GAL1 promoter used for FAR expression with a positive regulator [14] Oleaginous yeasts such as Y. lipolytica and R. toruloides are promising candidate hosts for fatty alcohol bioproduction owing to their high levels of acylCoA production, which is natively used to produce lipids. With additional metabolic engineering work, the fatty alcohol titers produced in these yeast species can doubtlessly be further increased
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
