Molecular Analysis Of Lipid Biosynthesis Mechanism And Genetic Manipulation In Ultra-centrifugal Screened Trichosporon Cutaneum Mutants

The accumulated lipid in microorganism is a significant source of biodiesel and bio-jet fuel.Lipid fermentation of oleaginous yeast using lignocellulosic material provide an important path for lipid production.Trichosporon cutaneum is a typical oleaginous yeast which adapt lignocellulosic system excellently.Long-term adaptation of T.cutaneum ACCC 20271 in lignocellulosic hydrolysate was conducted and upper cells after centrifugation were inoculated in fresh hydrolysate to obtain lightest mutants.The lipid content of those upper cells were highest and the lipid productive capacity of them was enhanced through continuous screening until Trichosporon cutaneum MS28 was screened.The lipid accumulation of T.cutaneum MS28 was increased more than 2.4 folds than that of T.cutaneum ACCC 20271 and T.cutaneum MS28 was further screened in fermentation broth with reduced viscosity and density.Trichosporon cutaneum WL97 was finally screened and the lipid yield of it was much improved than parental T.cutaneum.The lipid content of ultra-centifugal screened T.cutaneum mutants were increased to a large extent and assimilation of all the five monosaccharides derived from lignocellulose biomass were also enhanced.Moreover,cell wall became thinner and the cell volume were enlarged in mutants.Therefore,qRT-PCR were conducted to explore probable reasons of all these changes.Among the fatty acid synthesis related genes,several genes responsible for Acetyl-CoA and NADPH production were extremely significantly up-regulated in mutants.Content of Acetyl-CoA and NADPH in parental T.cutaneum and mutants were consistent with the qRT-PCR result.The genes in glycolysis were significantly up-regulated partly when each monosaccharide was used as sole sugar source.The enhanced assimilation capacity of all fermentable sugars derived from lignocellulose improved the utilization of hydralysate and provided more carbon source during lipid production in mutants.In addition,genes encoding the enzymes in synthesis and degradation of glucan and chitin were significantly up-regulated in mutants.The determination of mannan,glucan and chitin content in the cell walls on the weight basis per cell surface area suggested the declined mannan,glucan and chitin content in the mutant cells.The significant variation of cell morphology was probably led by the lessened glucan and chitin content and the enlarged cell volume provided more protein and nucleic acid for lipid biosynthesis and created the prerequisite for the final extraordinary lipid accumulation in the mutant cells.Genetic operating system was first contracted in T.cutaneum mutant.Hygromycin resistance gene HYR was set as target gene and selectable marker in expression cassette to construct single-copy integrated plasmid pUC19-△ECH1::HYR.The plasmid was transformed into T.cutaneum MS28 though electrotransformation to obtain engineered T.cutaneum MS28 AECHl::HYR.The green fluoresence protein gene GFP was then expressed in T.cutaneum MS28 △ECH1::HYR to examine the general applicability of the newly built system.However,engineered strains lost their target genes during continuous culture and all optimization methods failed to overcome this issue,which revealed the difficulty of the homologous recombination in T.cutaneum.To sum up,molecular analysis of lipid biosynthesis mechanism was conducted in ultra-centrifugal screened T.cutaneum mutants to lay foundation for further metabolic engineering and utilization of T.cutaneum.Exogenous gene was first expressed in T.cutaneum and the developed genetic operating system can become effective tools in molecular engineering process.