Acyl-Carrier-Protein-Engineering Rewires the Fatty Acid Profile in the Oleaginous Yeast Rhodotorula toruloides

Abstract

The oleaginous yeast Rhodotorula toruloides has emerged as a host for the production of lipids mainly consisting of long-chain fatty acid triacylglycerides as alternative feedstock for oleochemicals and biofuels. The R. toruloides fatty acid synthase (RtFAS) complex contains two acyl carrier protein (ACP) domains within its Fas2 subunit; it remains unclear whether inactivation of one ACP domain can lead to any biological effects, especially for fatty acid biosynthesis. Here, we constructed R. toruloides strains in which either one of the ACP domains was inactivated, and found that there was little difference in terms of cell growth and overall lipid production, suggesting these ACPs are functionally redundant. However, lipids from those strains contained lower contents of palmitic acid (C16:0). When one ACP was replaced with a truncated Escherichia coli thioesterase (‘TesA), known for the premature termination of the chain elongation process of fatty acid synthesis, these strains produced lipids with substantially higher contents of medium-chain fatty acids, corresponding to 6.3-fold and 4.2-fold increases in the relative abundance of C12:0 and C14:0, respectively, compared with the wild-type strain. This work shows that engineering the ACP domain of RtFAS can be explored as a new strategy for modulating fatty acid compositional profiles in oleaginous yeasts.