| Nootkatone is a high-value sesquiterpene with a unique pleasant aroma and is extremely popular in the food and cosmetics industries.Nootkatone is mainly extracted from the grapefruit peel,but its content is extremely low,resulting in a low yield and unable to meet the demand of increasing market.nootkatone was chemically synthesized successfully in lab.However,the low yield rate and using of carcinogens,heavy metals,flammables and strong oxides makes chemically synthesized,nootkatone is high cost and is not recognized by the market.Therefore,synthetic biology approach might be a more effective solution for the production of nootkatone.Like the other sesquiterpenes,the nootkatone biosynthetic precursor is farnesyl diphosphate(FPP),which is widespread exist in plants.There are two enzymatic reactions involved in converting FPP into nootkatone:1)valencene synthase(VS)catalyzes the cyclization of FPP to valencene;2)valencene was converted to nootkatone by valencene oxidase(VO).The identification of nootkatone biosynthesis gene provides essential elements for the production of nootkatone by synthetic biology technology.At present,the reconstruction of nootkatone synthesis pathway in Pichia pastoris has been successful.It is also feasible to reconstruct the nootkatone biosynthetic pathway in plants using the similar synthetic biology strategy.Medicinal plant Artemisia annua is rich in sesquiterpene compounds,such as artemisinin,farnesene,caryophyllene as well as the precursor of sesquiterpenes,FPP,making it an ideal chassis plant for the production of nootkatone.Cultivating nootkatone-producing Artemisia annua can also can improve the comprehensive economic value of Artemisia annua.In this study,two synthetic biology strategy were designed to reconstitute the nootkatone biosynthetic pathway in Artemisia annua:The first one is to reconstruct the nootkatone biosynthetic pathway in cytoplasm by introducing VS and VO into the cytoplasm of Artemisia annua;The second is to reconstruct the nootkatone biosynthetic pathway in plastid by introducing farnesyl diphosphate synthase(FPS),VS and VO into the plastids of Artemisia annua through addition plastid transit peptides.The following results have been obtained:1.Construction of plant expression vectors.The 35S promoter in p BI121 and the OCS terminator in the plant expression vector p EG104 were cloned and inserted into plant binary expression vector p1305.1 in Hind III/Pst I sites and Bam HI/Eco RI sites respectively,generated plasmid p ZZG100,which was used to insert related biosynthetic genes.According to the previously reported VO gene coding sequence(Gen Bank Accession:JX518290.1)and VS gene coding sequence(Gen Bank Accession:JX040471.1)in Alaska cork heartwood,the VO and VS were obtained by PCR amplification.Constructing of plant expression vector which was used to co-express VO and VS in cytoplasm:Firstly,Insert the VS gene fragment into plasmid p ZZG100 in the Sal1 and Bam H1 sites,p ZZG100-VS;then VO fragment was inserted into plasmid p ZZG100-VS in Sac1 and Kpn1 sites,generated p ZZG100-VS+VO,is used to reconstruct the biosynthetic pathway of nootkatone in the cytoplasm of Artemisia annua.Construction of plant expression vectors co-expressing FPS,VO and VS in plastids with the help of plastid transit peptide(TP).The homologous recombination tp gene fragment at the restriction site Sac1 of plant expression vector p ZZG100-VS+VO was named p ZZG100-VS+tp VO;homologous recombination tp FPS fusion gene fragment at the recombination plasmid vector p ZZG100-VS+tp VO restriction site Sal1 was named p ZZG100-tp FPSVS+tp VO,and the recombinant plant expression vector is used to reconstruct the synthetic pathway of nootkatone in the plastids of Artemisia annua.2.Obtaining and molecular detection of transgenic Artemisia annua.Obtaining and molecular detection of transgenic Artemisia annua reconstructing the nootkatone biosynthetic pathway through introducing VS and VO into the cytoplasm of Artemisia annua by the plant-expression vector p ZZG100-VS+VO:Transgenic Artemisia annua was obtained by leaf disc infestation mediated by Agrobacterium EHA105.After obtaining antibiotics resistance positive Artemisia annua plants,genomic DNA of Artemisia annua was exacted and used as templets of PCR amplification to detect the target genes VO,VS and Hygr.Five transgenic lines,including C1,C2,C3,C8 and C13 showed VO,VS and Hygr-positive;After then the total RNA of those lines and wild type Artemisia annua grown in the same condition were exacted,and reversed into c DNA to analyze the expression levels of VO and VS in plants by q PCR technology.The q PCR assay showed that VO and VS were expressed in both of transgenic Artemisia annua lines C1,C2,C3,C8 and C13,but not in wild-type Artemisia annua.Obtaining and molecular detection of transgenic Artemisia annua reconstructing the nootkatone biosynthetic pathway through introducing VS,VO and FPS into the plastid of Artemisia annua by the plant-expression vector p ZZG100-tp FPSVS+tp VO:Transgenic Artemisia annua was obtained by leaf disc infestation mediated by Agrobacterium EHA105.After obtaining antibiotics resistance positive Artemisia annua plants,genomic DNA of Artemisia annua was exacted and used as templets of PCR amplification to detect the target genes VO,VS,FPS and Hygr.Five transgenic lines,including P5,P6,P9,P10and P11 showed VO,VS,tp FPS and Hygr-positive;After then the total RNA of those lines and wild type Artemisia annua grown in the same condition were exacted,and reversed into c DNA to analyze the expression levels of VO and VS in plants by q PCR technology.The q PCR assay showed that VO and VS were expressed in both of transgenic Artemisia annua lines P5,P6,P9,P10 and P11,but not in wild-type Artemisia annua.3.Determination of nootkatone in transgenic Artemisia annua.GC-MS was used to detect the content of nootkatone in transgenic Artemisia annua in which nootkatone biosynthesis pathway was reconstructed in cytoplasm.Different levels of nootkatone were detected in the transgenic Artemisia annua lines,but no in wild type.The contents of nootkatone in different transgenic lines were listed below:C1 line was 7.65×10-3 mg·g-1(FW),line C2:5.01×10-3 mg·g-1(FW),line C3:8.51×10-3 mg·g-1(FW),line C8:3.64×10-3 mg·g-1(FW),line C13:0.88×10-3 mg·g-1(FW).The same method was used to detect the content of nootkatone in transgenic Artemisia annua in which nootkatone biosynthesis pathway was reconstructed in plastid.Different levels of nootkatone were detected in the transgenic Artemisia annua lines,but no in wild type.The contents of nootkatone in different transgenic lines were listed below:P5 line was 21.45×10-3 mg·g-1(FW),line P6:47.80×10-3 mg·g-1(FW),line P9:12.92×10-3 mg·g-1(FW),line P10:12.11×10-3 mg·g-1(FW),line P11:19.50×10-3 mg·g-1(FW).These results indicated that the engineering design of rebuilding nootkatone biosynthesis in plastids is more effective than that in cytoplasm.Interestingly,it was found that the CYP enzyme VO works even in plastids.4.Determination of artemisinin and dihydroartemisinic acid in transgenicArtemisia annua.HPLC was used to detect the content of artemisinin and dihydroartemisinic acid in transgenic Artemisia annua in which nootkatone biosynthesis pathway was reconstructed in cytoplasm.The artemisinin content of wild-type Artemisia annua in the control group was 14.05 mg·g-1(DW),the artemisinin content of transgenic Artemisia annua line C1,C2,C3,C8 and C13 was 12.22 mg·g-1(DW),15.23 mg·g-1(DW),10.91 mg·g-1(DW),13.66 mg·g-1(DW)and 12.56 mg·g-1(DW),respectively.The dihydroartemisinic acid content of wild-type Artemisia annua in the control group was 1.60 mg·g-1(DW),and the dihydroartemisinic acid content of transgenic Artemisia annua line C1,C2,C3,C8 and C13 was 1.14 mg·g-1(DW),1.50 mg·g-1(DW),0.83 mg·g-1(DW),1.20 mg·g-1(DW)and1.67 mg·g-1(DW),respectively.No significant change of artemisinin and dihydroartemisinic acid production between transgenic Artemisia annua and wild-type Artemisia annua was observed according to statistical analysis.The same method was used to detect the content of artemisinin and dihydroartemisinic acid in transgenic Artemisia annua in which nootkatone biosynthesis pathway was reconstructed in plastid.The artemisinin content of wild-type Artemisia annua in the control group was 14.05 mg·g-1(DW),the artemisinin content of transgenic Artemisia annua line P5,P6,P9,P10 and P11 was 13.33 mg·g-1(DW),14.73 mg·g-1(DW),11.77 mg·g-1(DW),16.24 mg·g-1(DW)and13.27 mg·g-1(DW),respectively.The dihydroartemisinic acid content of wild-type Artemisia annua in the control group was1.60 mg·g-1(DW),and the dihydroartemisinic acid content of transgenic Artemisia annua line P5,P6,P9,P10 and P11 was 0.99 mg·g-1(DW),1.55 mg·g-1(DW),1.17 mg·g-1(DW),1.01 mg·g-1(DW)and 1.19 mg·g-1(DW),respectively.No significant change of artemisinin and dihydroartemisinic acid production between transgenic Artemisia annua and wild-type Artemisia annua was observed according to statistical analysis.To sum up,this study successfully reconstructed the nootkatone biosynthesis pathway in the cytoplasm and plastids of Artemisia annua through two synthetic biology strategy.For the first time,a new method for producing nootkatone has been developed by synthesizing nootkatone in a plant that does not produce it.And the production of nootkatone in plastid engineering is significantly higher than that in cytoplasmic engineering;Meanwhile,it did not influence the production of artemisinin and dihydroartemisinic acid.This nootkatone producing Artemisia annua is a value-added material with huge market potential. |
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