| With the excessive consumption of fossil energy,the search for renewable energy is becoming more and more important.The use of microorganisms to convert biomass to biodiesel is one of the ways to solve the energy shortage.Microbial oil is a special oil produced by yeast,algae,mold and other microorganisms accumulated in the body through metabolism under certain growth conditions.Its main composition is triglyceride(TAG),which is similar to vegetable oil such as palm oil and rapeseed oil in composition,mainly C16 and C18.The use of microorganisms to produce oil has the following advantages: short cycle of oil production by microorganisms,not affected by season and climate,simple nutrition,easy industrial large-scale culture and so on.However,at present,the yield of microbial oil is not high,and the breeding of high-yield strains has become the bottleneck of microbial oil industrialization.In order to improve the ability of microbial oil accumulation,excavate the key enzymes in the way of oil synthesis,and regulate the fermentation process.In this study,using Trichosporon cutaneum(Trichosporon cutaneum)as the starting strain,a high oil-producing strain was successfully screened from the mutant library by atmospheric room temperature plasma mutagenesis instrument(ARTP)and combined with sesamol resistance screening.At the same time,a method for the determination of fatty acid composition of the oil produced by gas chromatography was established,which can analyze its composition quickly and accurately.The transcriptome analysis of the oil-producing strain was carried out,and two genes related to oil synthesis were excavated compared with the original strain.The fermentation medium and fermentation process were optimized by response surface method and fermentor.The corresponding experimental results are as follows:(1)Detection of fatty acid composition by gas chromatography: the produced oil was quickly esterified,and then the composition was detected by gas chromatography.A gas chromatographic method for simultaneous determination of palmitic acid,stearic acid,oleic acid,linoleic acid and eicosenoic acid in Trichosporon cutaneum was established.Five kinds of fattyacid methyl esters were well separated in 14 min by using DB-FFAP capillary column(30mx0.25 mm x 0.25um).Each component shows a good linear relationship in the concentration range of 156?2000mg/L,the linear range is more than 0.99,the recovery is between 92% and 96%,and the relative deviation is less than 2%,which can meet the detection of fatty acid composition of yeast oil.(2)Atmospheric Room Temperature Plasma mutagenesis breeding of Trichosporon cutaneum: the effect of atmospheric room temperature plasma(ARTP)mutagenesis time on the lethality of Trichosporon cutaneum(Trichosporoncutaneum)was studied,the inhibition of different concentrations of sesamol on the growth of Trichosporon cutaneum was investigated,and a rapid and efficient method for screening high oil yield strains was established.The results showed that after 40 s mutagenesis of ARTP,the strains were cultured in sesamol medium with 0.15mg/m L concentration at 28 ? for 2 days,and then inoculated into 48-well liquid culture medium.96-well plate Nile red fluorescence staining was used for high-throughput screening,and a high-yield oil-producing strain Trichosporoncutaneum A1 was selected.Compared with the original strain,the biomass,oil yield and oil yield of this strain increased by 41.6%,120.3% and58.3%,respectively.(3)Optimizing the composition of fermentation medium by response surface method: the fermentation medium for oil production was studied by using Trichosporon cutaneum(Trichosporoncutaneum)as the original strain.Taking oil yield as an index,the effects of carbon source,nitrogen source and exogenous factors in fermentation medium on oil yield were studied by single factor experiment,and then the medium was optimized by response surface experiment.The results showed that the best medium formula was glucose97.6g/L,corn pulp dry powder 4.4g/L and sodium acetate 0.09 g max L.under these conditions,the oil yield of Trichosporon cutaneum reached 14.4g max L,which was 114.9% higher than that before optimization.(4)Scale-up culture in fermentor: the conditions and technology of oil production by Trichosporon cutaneum in fermentor were studied,mainlyfocusing on the initial fermentation glucose concentration,initial corn pulp dry powder concentration and rotational speed.The results showed that the fermentor was more suitable for cell growth and oil accumulation,and the utilization amount of glucose increased from 90g/L to 120 g / L,and more nitrogen sources were needed for protein synthesis,growth and development,so the addition of corn pulp dry powder was increased from 4.5g/L to 7.5g / L,under these conditions,the cell growth was greatly improved.At the same time,it was found that the rotational speed had an obvious effect on the dissolved oxygen.At low rotational speed,the dissolved oxygen was at zero level for a long time,which was not conducive to the growth and reproduction of bacteria.At higher rotational speed,the dissolved oxygen is sufficient,the cell growth is good,the oil yield is high,and the fermentation time becomes shorter,which is more beneficial to the production.Under the optimal conditions,the cell biomass reached 41.5g / L,the oil yield reached 25 g / L,and the fermentation was completed in 110 h.The fermentation kinetics were studied to obtain the fitting curve of biomass,product and substrate.(5)Transcriptome sequencing and gene analysis of key enzymes: in order to further study the changes of gene expression and metabolic pathway of high-yielding strain A1,transcriptome sequencing was used to ferment the original strain WT and high-producing strain A1 to the maximum oil accumulation,and then transcriptome sequencing analysis was carried out.The results showed that 1185 genes were differentially expressed between A1 and WT,of which 767 genes were up-regulated and 418 genes were down-regulated.In the classification of differential gene GO enrichment,cell process,metabolic process,catalytic activity,cell part and other related genes showed enrichment.In the KEEG metabolic pathway,differential genes are mainly involved in yeast cell cycle,gene replication,mutual transformation of pentose and glucuronic acid,glycolysis / gluconeogenesis and so on.It was found that three enzymes related to fatty acid accumulation were overexpressed,including citrate synthase,glycerol phosphodiester phosphodiesterase and glyceraldehyde 3-phosphate dehydrogenase.Theseresults can provide a theoretical basis for the later gene modification and metabolic pathway regulation of Trichosporon cutaneum. |
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