Engineering oleaginous yeast Rhodotorula toruloides for production of alkanes and alkenes

Abstract

Due to limited reserves and excessive carbon emission of fossil fuels, there has been an increasing interest in developing advanced biofuels with high energy density such as alkanes and alkenes. Here we report the design and construction of three heterologous biosynthetic pathways of alkanes and alkenes in oleaginous yeast Rhodotorula toruloides, including the AAR/ADO, UndA/UndB and FAP pathways. The performance of various enzymes from different organisms was evaluated within R. toruloides for each pathway. Various metabolic engineering strategies were used to enhance the production of alkanes and alkenes across all three pathways, including enzyme screening, byproduct elimination, and precursor supply enhancement. Notably, the FAP pathway demonstrated significantly superior performance compared to the AAR/ADO and UndA/UndB pathway. As a result, 1.73 g/L alkanes and alkenes were produced from glucose, and 0.94 g/L alkanes and alkenes were produced from lignocellulosic hydrolysates, representing the highest alkanes and alkenes titers reported in yeast. This work establishes R. toruloides as a promising host for hydrocarbons production from glucose and CO₂-neutral feedstocks and paves the way for further strain and process optimization towards industrial production of alkanes and alkenes.

Keywords: Alkanes; Alkenes; CRIPSR/Cas9; Fatty acid-derived compounds; Metabolic engineering; Rhodotorula toruloides.