Discovery And Application Of The Constitutive Promoter In Rhodosporidium Tor Uloides

Rhodosporidium toruloides,a non-model yeast that can produce high yields of oils and fats,is able to accumulate more than 70%of its own cell dry weight when cultured under nitrogen limitation,and can introduce different modifying enzymes or metabolic pathways to synthesize various types of fatty acid fuels and chemicals.Meanwhile,this yeast has a wide range of carbon and nitrogen sources available,and it is naturally tolerant to toxic substances such as acetic acid in cellulose hydrolysates,which is regarded as an oil producing strain with a wide prospect of industrial applications.Although several strains of this yeast have been previously genome sequenced and annotated for gene function,tools for their genetic manipulation are still relatively lacking,such as the lack of available auxotrophic and resistance screening markers,promoters and terminators,and thus metabolic engineering and synthetic biology studies in Rhodosporidium toruloides remain very difficult.These severely limit the development and application of this yeast.Promoters are indispensable regulatory elements for gene expression and can significantly influence the initiation,duration,transcriptional efficiency and expression levels of endogenous or exogenous gene transcription;Meanwhile,promoters are also the most effective tools for regulating metabolic pathways,and mining promoters of different strengths can help optimize the expression levels of different genes,thereby optimizing the target metabolic pathways and increasing the efficiency of synthesizing the products of interest.Therefore,to establish a promoter library suitable for use in Rhodosporidium toruloides with different expression strengths,we mined its transcriptome data.First,we identified genes with different expression intensities by transcriptome analysis of Rhodosporidium toruloides in four culture media,two growth periods,and cloned the corresponding promoter sequences to control the expression of the marker gene green fluorescent protein(EGFP).Next,the intensity of each promoter under different conditions was measured by the change of fluorescence intensity.In comparison to the promoters already reported for this yeast,we mined and identified 9 highly expressed promoters,12 promoters with moderate expression intensity,and 10 promoters with better expression.Subsequently,we further applied the identified promoters of different strengths to optimize the synthetic pathways of oleic acid and linoleic acid,achieving differential expression of different genes of linoleic acid synthetic pathway and increasing the production of linoleic acid and its precursor oleic acid by 48.03%and 95.29%,respectively,illustrating the effectiveness of using different strengths promoters to achieve differential expression in optimizing metabolic pathways.This work enriches the promoter library of Rhodosporidium toruloides and has important implications for the optimization of metabolic pathways and synthetic biology studies in Rhodosporidium toruloides.