Effects Of Anti-sense RNA Mediated Inhibition Of The Yeast Squlene Synthase Expression On The Engineered Amopha-4,11-diene Biosynthesis

Artemisinin is the preferred drug for treating malaria.Because of the limited natural resource,one of the potential methods is firstly to produce the intermediate artemisinic acid by the metabolic pathway engineering,and then to obtain artemisinin by chemical semi-synthesis with artemisinic acid as the substrate.To get the engineered yeast with high-yield of artemisinic acid,we carried out the research on the metabolic pathway engineering of amorpha-4,11-diene in the engineered yeast,and some of the results are presented here:1.Construction of the engineered Saccharomyces cerevisiae that produces amorpha-4,11-diene.According to homologous recombination mechanism,the yeast expression vector was constructed,which harbors the ADS gene ofArtemisia annua and the twoλDNA fragments of yeast.After the ADS gene was introduced into the yeast host, a new biosynthetic pathway of amorpha-4,11-diene was established.The twoλDNA fragments were ligated to the up-and down-streams of the ADS gene,respectively,so that the ADS gene might be inserted in theλDNA sites on the yeast genomic DNA. There are about 200λDNA copies in the yeast genome,and the multi-copies integration of the ADS gene on the yeast genome might greatly improve the amount of ADS in yeast, leading to the increase of the product amorpha-4,11-diene.The procedure is as follows: firstly,the yeast ADH1-promoter and terminator were cloned into the vector,respectively. Then the ADS gene,the auxotrophic marker URA3 and theλDNA sequences were subcloned into the vector step by step.This recombinant plasmid was transformed into the URA-deficient yeast W303A and the engineered yeast was screened on the SD-ura" medium at 28℃.After 3-4 days cultivation,the positive clones were characterized by PCR.The fermentation products of these positive engineered yeasts were detected by GC-MS and the amorpha-4,11-diene yield in one of these strains reached up to 117.0 mg·L^-1.2.Comparison of the AD yield between the episomic plasmid-transformed and the integrative plasmid-transformed yeasts.Comparison of the AD yields between the two kinds of the engineered yeasts was performed as follows:three strains were chosen from each of the engineered yeasts.Their fermentation products were extracted and then detected by GC-MS.The results indicated that the AD yield of the integrative plasmid-transformed yeast was much higher than that of the episomic plasmid-transformed yeast(P＜0.01).3.Construction of the antisense RNA expression vectors of the yeast squalene synthase.Normally,the DNA fragments with 0.5kb-3.0kb in length,which harbor the translation start coding site,or with the 3'-untranslated regions(UTR) are used for the construction of the antisense RNA expression vectors.In this paper,the four SQS fragments,namely SQSI(5'-UTR),SQS7(Y-UTR),SQS3^*4(5'-UTR plus 5'-coding region) and SQS5^*6(3'-coding region plus 3'-UTR),were used to construct the SQS antisense RNA expression vectors for the SQS antisense RNA transcription in the host yeast after transformation and cultivation.All of the DNA fragments were reversely inserted at the downstream of the galactose-induced promoter GAL10-CYC1 in the vector pYeDP60,leading to the four recombinant vectors:pYeD/SQS1,pYeD/SQS7, pYeD/SQS3^*4,and pYeD/SQS5^*6.4.Effects of the SQS anti-sense RNA expression on the AD biosynthesis of the engineered yeast.The four SQS antisense RNA expression plasmids mentioned above were transformed into the AD producing strain.Products of the transformants were quantified by GC-MS.The results indicated that both the squalene and AD yields were declined and the further analysis is ongoing.Besides,the SARS-CoV recombinant protein work was also carried out.5.Expression,purification,characterization and antibody preparation of SARS-CoV putative unknown proteins.The SARS-CoV putative unknown proteins X4,X5 and ORF 10 were expressed by the E.coli recombinants,respectively.ORF 10 and X5 proteins were purified on a Ni⁺ affinity chromatography.With the low concentration (60mM·L^-1) of imidazole washing elution,two protein bands(22 KD and 44 KD) of the ORF10 transformant were observed by SDS-PAGE.But when the imidazole concentration increased(120raM·L^-1),only one protein band(22 KD) was obtained. Both the 22KD and 44 KD protein bands on SDS-PAGE gel were cut off for the LC-ESI-MS/MS analysis.It showed that the detected sequences of the 22KD band match 38.3%of the ORF 10 protein sequence with 100%identity,while those of the 44KD band match 45.1%of the ORF 10 protein sequence with 100%identity.This result implied that the ORF 10 protein can be expressed in E.coli in the dimmer form.The recombinant X5 protein was expressed in the form of inclusion body.The inclusion body was dissolved in high concentration of urea.The supernatant was purified by Ni⁺ affinity chromatography,and then the denatured protein was refolded in a series of gradient solutions of urea.The purified protein was obtained with the purity of＞95% and the yield of 93.3mg·L^-1(five times of the non-inclusion protein ORF10). Polyclonal antibody of this protein was obtained,and Western blot assay indicated that the antibody could specifically bind to the X5 protein produced by the recombinant strain..