| Nelumbo nucifera Gaertn is a kind of medicine food homologous crop,containing betulinic acid,oleanolic acid,lupeol and other triterpenoids.It is detected that there is betulinic acid in Nelumbo nucifera Gaertn and the content is high in it.Betulinic acid itself and its derivatives have an obvious inhibitory effect on many human cancer diseases,and have preferable for drug preparation.Relying on chemical synthesis and plant extraction,there are some problems of high energy consumption and low yield.Microbial metabolic engineering provides a new way for the production of betulinic acid.In this study,a high-throughput sequencing platform(DNBSEQ)was used to analyze the transcriptomics of six different growths periods of Nelumbo nucifera Gaertn of the "Elian No.5 " variety.Screening of betulinic acid synthesis-related P450 s cytochrome oxidase gene(CYP),P450 s cytochrome reductase gene(CPR)and lupeol synthase gene(LUP),prokaryotic and eukaryotic expression,and detection of enzyme activity.The enzyme genes with corresponding functions were screened by adding substrates to the fermentation products and then fermented by Saccharomyces cerevisiae to produce betulinic acid.The yield of betulinic acid was increased by specific gene optimization,chassis strain modification and fermentation condition optimization.The main findings of this study are as follows:(1)83,475 Unigenes were obtained from transcriptome sequencing,and the length was mainly concentrated in 1000 bp-2600 bp.66 705 Unigenes were annotated in the GO database.The number of annotated genes in the KEGG metabolic pathway reached 11212,and there were 251 Unigenes related to terpenoid biosynthesis.According to the results of transcriptome data analysis and homologous sequence alignment,the genes of biosynthesis-related enzymes of betulinic acid were excavated,including CYP candidate genes LOC104593846 and LOC104593852;CPR candidate genes LOC104595501 and LOC104599186;LUP candidate genes LOC104589206,LOC104589208,LOC104588379,and LOC104593853.(2)Total DNA was extracted from Nelumbo nucifera Gaertn S1 period,and two CYP gene sequences,Lr CYP-93846 A and Lr CYP-93852B;two CPR gene sequences,Lr CPR-95501 G and Lr CPR-99186H;four LUP gene sequences,Lr LUP-89206 C,Lr LUP-89208 D,Lr LUP-93853 F,and Lr LUP-88379 E were amplified.The prokaryotic and eukaryotic expression vectors were constructed respectively,and the results of prokaryotic expression showed that Lr CYP-93846 A,Lr CYP-93852 B protein was about 55 k Da;Lr LUP-89206 C,Lr LUP-89208 D,Lr LUP-93853 F,Lr LUP-88379 E,Lr LUP-93853 F,and Lr LUP-88379 E protein was about 55 k Da.Lr LUP-88379 E protein was about 85 k Da;eukaryotic expression results showed that Lr LUP-93853 F could catalyze 2,3 oxidosqualene into lupinol.The combination of Lr CYP-93846 A,Lr CYP-93852 B and Lr CPR-99186 H could catalyze lupeol to produce betulinic acid,and the other enzyme genes had no corresponding enzymatic activity.The candidate genes with enzymatic activity were combined to construct a strain for de novo synthesis of Saccharomyces cerevisiae.The results showed the yield of betulinic acid was low,and the combination of Lr CYP-93846 A and Lr CPR-99186 F was only 0.036 mg/L.(3)The candidate genes with enzymatic activity were selected for codon adaptation,and the Lj CPR P450 reductase gene was introduced,and different combinations were transferred into Saccharomyces cerevisiae strains to produce betulinic acid.The results of HPLC showed that the content of betulinic acid produced by the combination of Lr CYP-93846 A,Lj CPR and Lr LUP-93853 F was the highest,reaching 1.02 mg/L.The key ratelimiting enzyme HMG1 and the important enzyme gene ERG20 were overexpressed in the Saccharomyces cerevisiae genome,and the high-yield squalene strain BY4742 was introduced for fermentation production.The test results showed that the yield of betulinic acid after the transformation,and the yield of betulinic acid in BY4742 reached the maximum of 4.01 mg/L in the combination of Lr CYP-93846 A,Lj CPR,and Lr LUP-93853 F.The optimization of the fermentation condition showed that the initial p H of the medium was 6,the initial OD value was 1,the galactose concentration was 6%,and the time was 60 h.The content of betulinic acid in BY4742 could reach 10.65 mg/L.The results of the substrate extensiveness study on the Nelumbo nucifera Gaertn CYP gene showed that the Lr CYP-93846 A gene could catalyze α-amyrin and β-amyrin to produce ursolic acid and oleanolic acid respectively,respectively,the Lr CYP-93852 B gene could catalyze β-amyrin to produce oleanolic acid.In summary,this study analyzed the synthesis pathway of betulinic acid in Nelumbo nucifera Gaertn and identified two CYP450 enzyme genes,two CPR enzyme genes,and four LUP enzyme genes that might be involved in betulinic acid biosynthesis based on transcriptome analysis.Through functional validation analysis,Lr LUP-93853,Lr CYP-93846 A,Lr CYP-93852 B,and Lr CPR-99186 H were involved in betulinic acid,which laid the foundation for the de novo synthesis of betulinic acid by Saccharomyces cerevisiae. |
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