Mutation Of Carotenoid Production Pathway In Rhodosporidium Toruloides And The Metabolic Analysis Therewith

Carotenoids are essential for protecting cells from free radical damage, synthesizing vitamin A, maintaining normal life of cell metabolism, deferring the senescence of organism, preventing and treating cancer, and improving immune function and so on. Rhodosporidium toruloidesis an excellent carotenoid and lipid producing strain. R. toruloidesis decomposes glucose into pyruvate through the glycolytic pathway. Under aerobic conditions, pyruvate is further oxidized and decomposed to generate acetyl CoA. Lipid and carotenoids achieve their biosynthesis through the competition for acetyl-CoA precursor. However, the carotenoid biosynthetic pathway has not been clearly clarified yet. Thus, it is difficult to increase the yield of carotenoids through the genetic engineering. Screening of carotenoids synthesis deficient strains using mutagenesis techniques and the metabolic analysis therewith will provide new insight into the clarification of the key nodes in the biosynthetic pathway of carotenoids in R. toruloidesis.In this work, we began from R. toruloidesis wild-type strain, conducted the mutagenesis by utilizing atmospheric temperature plasma (ARTP). We screened out four carotenoid synthesis deficient strains, named RT W5, RT W5-1, RT W5-1B and RT W5-1B1, respectively. Subsequently, R. toruloidesis wild-type the deficient strains were nitrogen limited cultured, and then the cell dry weight, lipd content, carotenoid content, organic acids content and the pH of the supernatant were measured, respectively. Meanwhile, gas chromatography (GC) and high performance liquid chromatography (HPLC) were used to analyze fatty acids and organic acids. The results showed that, compared with the wild-type strain, the cell dry weight, lipid content and the content of carotenoid of RT W5 reduced 39.27%,34.36% and 53.92%, respectively. Moreover, the pH value of the culture supernatant was increased slightly. Analysis results of organic acids showed that, compared with the wild-type strain, contents of glyoxylic acid, lactic acid and succinic acid of RT W5 increased 68.75%,232.65% and 13%, respectively. Malic acid could not be observed. However, tartaric acid, oxaloacetate, ketoglutarate and citrate were not detected both in wild-type R. toruloidesis and carotenoid-deficient strains. Fatty acids in wild-type R. toruloidesis and RT W5 were measured using gas chromatography. By contrast, we found that the relative content of palmitic acid was very low, and even invisible. The contents of stearic acid and oleic acid reduced 16.98% and 60%, respectively, in RT W5, compared with wild-type strain.Our work obtained carotenoid synthesis deficient R. toruloides strains using ARTP technology. And the analysis results of cell dry weight, lipd content, carotenoid content, organic acids content and fatty acid would be valuable for the clarification of the biosynthetic pathway of carotenoids in R. toruloidesis.