Qiongqiong Chen

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.

Terpenoids have tremendous biological activities and are widely employed in food, healthcare and pharmaceutical industries. Using synthetic biology to product terpenoids from microbial cell factories presents a promising alternative route compared to conventional methods such as chemical synthesis or phytoextraction. The red yeast Rhodotorula mucilaginosa has been widely studied due to its natural production capacity of carotenoid and lipids, indicating a strong endogenous isoprene pathway with readily available metabolic intermediates. This study constructed several engineered strains of R. mucilaginosa with the aim of producing different terpenoids. Monoterpene α-terpineol was produced by expressing the α-terpineol synthase from Vitis vinifera. The titer of α-terpineol was further enhanced to 0.39 mg/L by overexpressing the endogenous rate-limiting gene of the MVA pathway. Overexpression of α-farnesene synthase from Malus domestica, in combination with MVA pathway rate-limiting gene resulted in significant increase in α-farnesene production, reaching a titer of 822 mg/L. The carotenoid degradation product β-ionone was produced at a titer of 0.87 mg/L by expressing the β-ionone synthase from Petunia hybrida. This study demonstrates the potential of R. mucilaginosa as a platform host for the direct biosynthesis of various terpenoids and provides insights for further development of such platforms.