Study On The Ultra-centrifugal Screening And Mechanism Analysis Of High Lipid Producing Trichosporon Cutaneum Mutant

Oleaginous microorganism can convert fermentable sugars derived from lignocellulose into microbial lipid,which is a reliable method to ensure the sustainable supply of biodiesel lipid raw materials.The oleaginous yeast Trichosporon cutaneum is the most suitable strain for lignocellulose system oil fermentation.In this paper,a mutant strain T.cutaneum MS 28 with low intracellular density was successfully obtained in a lignocellulose culture system by high-throughput ultra-centrifugation screening method.The mutant strain T.cutaneum MP11 with higher lipid content was screened more quickly in synthetic medium,which confirmed the feasibility and universality of ultracentrifugal screening.The mutant strains can transform hexose and pentose at the high solid content of 30%wheat straw simultaneous saccharification and co-fermentation,but the parental T.cutaneum ACCC 20271 can only use part of glucose to produce 6 g/L lipid.T.cutaneum MS 28 and T.cutaneum MP 11 accumulated high lipid yields of 32.8 g/L and 34.4 g/L,respectively.The mutant strain has excellent lipid production capacity.Microscopic observations show that ultra-centrifugal screening of two mutants has obvious differences in cell budding growth and morphology.The parenal T.cutaneum ACCC 20271 had a single apical bud at the top,and the cell morphology was a uniform single cell ellipse.Both mutants had multilateral buds.Their diverse cell morphology and larger cell size provide a broader space for lipid accumulation.Yeast cell morphology may be related to the integrity of the cell wall.The cell wall can adjust the synthesis and decomposition of mannan,chitin,and glucan to make the structure change dynamically to adapt to environmental changes.Transmission electron microscopy observations showed that the mutant strains had larger cell volume and more active metabolism.At the same time,the cell wall of the mutant strain was thinner and the intracellular lipid content was higher.The results of qRT-PCR indicated that genes related to cell wall synthesis and degradation were differentially expressed.The ratio of cell wall unit surface area chitin to monosaccharide polymer in the parental strain T.cutaneum ACCC 20271,mutant strains T.cutaneum MS 28 and T.cutaneum MP 11 was 0.05,0.09 and 0.28,respectively.The results show that the ratio of high chitin to monosaccharide polymer is conducive to changes in cell morphology and accumulation of lipid.The content of acetyl-CoA and NADPH in the two mutants was higher than the parental T.cutaneum ACCC 20271.T.cutaneum MS 28 and T.cutaneum MP 11 have a stronger ability to supply key precursors for lipid synthesis.The results showed that some genes related to acetyl-CoA,NADPH and fatty acid synthesis were up-regulated,and the transcription level of two mutants were significantly different from T.cutaneum ACCC 20271.Therefore,the supply of acetyl-CoA and NADPH has become a key factor for improving the ability of fat synthesis.In this article,the ultra-centrifugal screening method can obtain oleaginous yeast with excellent substrate conversion ability,which is of popularization value.First,the potential of T.cutaneum to produce lipids using lignocellulose was evaluated.Then,the budding propagation and cell wall structure of T.cutaneum mutants were analyzed.Finally,the effect of centrifugal screening mechanism on cell morphology and lipid anabolism is further understood.