Screening Of Phaffia Rhodozyma Strain And The Mechanism Of Increasing Its Astaxanthin Production Under TiO₂ Stress

Natural astaxanthin is found to be the strongest antioxidant at present and the only fourth generation antioxidant,its antioxidant activity is ten times or even more than a hundred times that of lycopene,β-carotene,lipoic acid,lutein and vitamin E.It has many biological functions such as preventing or treating diabetes,cardiovascular diseases,anti-tumor and improving immunity,and has broad market prospects and economic value in food,health care products,pharmaceuticals,cosmetics and feed industries.However,there is a lack of high-yield astaxanthin strains with independent intellectual property rights in China,and the metabolic regulation mechanism of astaxanthin is not clear,which limits the application of astaxanthin.As a microbial candidate for natural astaxanthin production,Phaffia rhodozyma has the advantages of not requiring light to accumulate astaxanthin,being able to metabolize a variety of sugars and growing rapidly.In this study,a high astaxanthin producing strain PR106 was screened,and its whole genome sequencing,resequencing and genetic evolution were analyzed.Then,the stress conditions were screened,and TiO₂ was determined as the best stress condition to promote the astaxanthin synthesis of PR106.The mechanism of TiO₂ improving the astaxanthin production of PR106 was determined by high-throughput omics technology and metabolic flux analysis.It provided theoretical guidance for the development and utilization of astaxanthin in P.rhodozyma,and provided new ideas for developing genetic engineering methods to improve astaxanthin production of P.rhodozyma.The main contents and results of this study are as follows:1.The strain with high astaxanthin yield was screened from the orchard soil,then,was isolated and identified,and the whole genome of the screened strain was sequenced by the second generation high-throughput sequencing technology.The variation and genetic evolution of the strain were studied by resequencing and phylogenetic analysis.The results showed that the astaxanthin content of the screened strain was 1.69 mg/g dry cell weight,the 26S r DNA D1/D2sequence of the strain was sequenced,after comparing with the sequence in NCBI database,it was found that the sequence of the selected strain was 99.9%similar to that of P.rhodozyma,which was named as P.rhodozyma PR106.The genome size of PR106 was 16.18 Mb with a GC content of 47%,compared with the genome of P.rhodozyma CBS7918,the sequencing depth and coverage were 125.31×and 99.41%,respectively.The phylogenetic tree showed that the genetic evolution relationship between PR106 and CBS7918 was the closest.The divergence times of PR106 with CBS7918 and Xden1 were 1.25 and 1.34 MYA,respectively.72 expansion gene families and 1128 contraction gene families were generated in PR106.2.Taking astaxanthin yield of P.rhodozyma PR106 as an important index,the stress conditions for improving astaxanthin yield were screened,including different reactive oxygen species generators,metal ions,ethanol and initial p H value.After confirming that TiO₂ was the stress condition to significantly improve the astaxanthin yield of PR106,the basic parameters of PR106 were determined,and the mechanism for TiO₂ increased the astaxanthin yield of PR106were preliminarily discussed.The results showed that 500 mg/L TiO₂ was the best stress condition to improve the astaxanthin content of PR106,the astaxanthin content was 4.03 mg/g,which was twice that of the control group.TiO₂ stress did not change the biomass of PR106,however,it promoted the cell division in the later-log phase and stable phase of yeast（although the biomass did not change significantly,but it increased）and changed the cell morphology.TiO₂ could significantly increase the concentrations of H₂O₂、O₂^-and·OH in yeast medium.Before PR106grew to the mid-log phase,TiO₂ stress caused yeast cell apoptosis（consistent with the decreased of biomass）,and with the extension of culture time,yeast showed strong adaptability.TiO₂ stress induced oxidative stress in yeast cells,resulting in the increase of ROS level in cells,and CAT activity increased significantly in the mid-log phase of yeast.3.TiO₂ in the medium first contacted the cell wall and cell membrane of P.rhodozyma PR106.The effect of reactive oxygen species produced by TiO₂ on the cell structure of PR106 was explored by measuring the integrity and permeability of cell wall,and the integrity and fluidity of cell membrane.Then,the changes of ergosterol and fatty acid contents of cell membrane components were determined,and their relationship with the increase of astaxanthin content was analyzed.The results showed that TiO₂ stress did not destroy the integrity of PR106 cell wall and cell membrane,but decreased the permeability of cell wall and increased the fluidity of cell membrane to resist the adverse growth environment stress,furthermore,decreased ergosterol content and increased the content of oleate,linoleate andα-linolenate in cell membrane.And compared with the control,TiO₂ combined with oleate increased the astaxanthin content in PR106by 2.75 times,and the astaxanthin content reached 5.5 mg/g dry cell weight.Under TiO₂ stress,the content of astaxanthin ester formed by fatty acids and astaxanthin in PR106 increased significantly,and the maximum content of astaxanthin ester was 2.21 times that of the control.Astaxanthin was negatively correlated with palmitic and stearic,the correlation was weak,and was positively correlated with oleate,linoleate andα-linolenate,the correlation with linoleate was the strongest.4.The mechanism of TiO₂ stress promoting astaxanthin synthesis in P.rhodozyma PR106was explored by high-throughput transcriptomics and proteomics techniques,and the map of gene transcription and protein translation of PR106 under TiO₂ stress were established.The transcriptomic and proteomic results were verified by real-time fluorescence quantitative PCR and parallel reaction monitoring（PRM）techniques respectively,and the content and enzyme activity of proteins were determined.The results showed that a total of 6 differentially expressed genes were found by transcriptomics,these differentially expressed genes were annotated as uncharacterized protein,transcription factor MEIS1 and related HOX domain protein,Mfs monosaccharide transporter,uncharacterized protein,acid phosphatase and retrovirus related gag-pol poly protein,the differential expression multiples were 3.47,4.05,2.79,2.42,0.48 and 0.45times,respectively.In the GO enrichment pathway,up-regulated genes were responsible for the cell response to stimulation and the activity of DNA binding transcription factors,down-regulated genes were mainly related to 3-phytase activity,acid phosphatase activity and hydrolase activity.Differentially expressed genes were enriched in three KEGG pathways:thiamine metabolism,riboflavin metabolism and yeast meiosis.There was no significant difference in the relative expression of phytoene dehydrogenase gene crt I and astaxanthin synthase gene ast in PR106under TiO₂ stress by real-time fluorescence quantitative PCR analysis,which was consistent with the results of transcriptomics.Proteomics results found that a total of 3193 proteins could be quantified,and there were 61 proteins with differential expression multiples greater than 1.2 times,of which 5 proteins were up-regulated and 56 proteins were down-regulated.The differential expression of proteins related to redox balance,ribosomal translation and ion transmembrane transport in PR106 under TiO₂ stress,and were mainly enriched in the RNA degradation pathway.PRM analysis showed that the 5 differentially expressed proteins（bud-site selection protein（BUD22）,ubiquitin-40s ribosomal protein s31 fusion protein,cell cycle control protein,C-4methyl sterol oxidase and glutaredoxin）related to astaxanthin synthesis in PR106 after TiO₂ stress changed by 0.8,0.39,1.2,1.12 and 0.63 times,respectively,and the results of PRM validation showed a similar trend with the results of proteomics.When the yeast grew to the stable stage,the content of reduced glutathione in the non-enzymatic antioxidant system was significantly increased by TiO₂ stress,the content of oxidized glutathione was significantly decreased,and there was no significant difference in the content of total glutathione.5.By analyzing the changes of protein post-translational modification（PTM）,and measuring the enzyme activity and metabolite content related to astaxanthin synthesis in P.rhodozyma PR106 cells,the close relationship between the related metabolic regulatory network and astaxanthin synthesis after TiO₂ stress on PR106 was explored.The results showed that PTMs such as acetylation,succinylation,phosphorylation,2-hydroxyisobutylation,ubiquitination and lactation in PR106 were not directly related to astaxanthin synthesis under TiO₂ stress.After TiO₂stress,PR106 could rapidly utilize glucose to synthesize primary metabolites for cells growth,and the lactic acid content in PR106 at the early-log phase and mid-log phase was significantly reduced,but the addition of lactic acid did not affect the astaxanthin synthesis of PR106.In the later-log phase of yeast,TiO₂ stress significantly increased the ethanol content,on the contrary,in the stable phase,the ethanol content significantly decreased.TiO₂ stress significantly increased the enzyme activities of pyruvate dehydrogenase,citrate synthase,2-ketoglutarate dehydrogenase,malate dehydrogenase and mitochondrial respiratory chain complex I-V,as well as acetyl Co A content in PR106.The increase of carbon flux provided sufficient substrate for astaxanthin synthesis,and the enhancement of aerobic respiration promoted ATP synthesis and provided energy for astaxanthin synthesis.Based on the above results,a P.rhodozyma strain PR106 with high astaxanthin production was screened and obtained,and the astaxanthin content reached 1.69 mg/g dry cell weight.PR106had great variation in the process of evolution,and had the closest genetic evolutionary relationship to P.rhodozyma CBS7918.500 mg/L TiO₂ was the best stress condition to significantly increase the astaxanthin content of PR106,and the astaxanthin content was increased by 2 times to 4.03 mg/g dry cell weight.The increase of astaxanthin yield in PR106 promoted by TiO₂ was not due to the changes of gene transcription level,protein translation level and the regulation of protein post-translational modification in the astaxanthin synthesis pathway.It was because the oxidative stress of PR106 caused by TiO₂ stress promoted astaxanthin accumulation to maintain redox balance,increased astaxanthin precursor substances and weakened the synthesis of other carotenoids in the branch pathway of astaxanthin synthesis,increased astaxanthin esters content to relieve the feedback inhibition of astaxanthin,increased carbon flux to provide sufficient substrate for astaxanthin synthesis,promoted aerobic respiration of yeast to increase ATP synthesis and provided energy for astaxanthin synthesis.Moreover,TiO₂ combined with oleate stress PR106 increased the astaxanthin content by 2.75 times,and the astaxanthin content reached 5.5mg/g dry cell weight.