Cloning Of Chromophore Lyase Gene CpcS, CpcT, CpcU From Arthrospira Platensis FACHB314and The Study Of The Founction On The Expression Of A Fluorescent Phycocyanin In Heterologous Host

Arthrospira platensis is a well-known economically important Cyanobacteria onfood and pharmacy industry while its phycocyanin is especially useful which not onlyhas biological activity on anti-oxidation, anti-tumor, anti-inflammatory and so on, butalso contains the fluorescence property to be used as fluorescent tags and possiblyapplied to the photodynamic therapy of cancers. In recent years, the research ofexpressing optically active phycocyanin has made many achievements throughgenetic engineering, providing the foundation for application of optical phycocyaninand establishment new photosynthesis system in transgenic plants.An important step in phycobiliprotein biosynthesis is the covalent coupling of thechromophores to the apoproteins, while the properly connection requires specificchromophore lyases catalysis. The biological synthesis of optically active phycocaningenerally requires three components-apo-phycocyanin (cpcA, cpcB), thechromophore synthetase (hox1, pcyA) and the chromophore lyase to participatetogether. The research of chromophore lyases for phycocyanin α subunit has beenrelatively completed in our laboratory, but there are no studies about chromophorelyases of phycocyanin β subunit from Arthrospira platensis. So this thesis made aseries of related studies about chromophore lyases on phycocyanin β subunit.In this thesis, chromophores lyases cpcS, cpcT and cpcU were first cloned fromArthrospira platensis FACHB314, and some bioinformatics analyses were made ofthese lyases. The gene cpcS contains594base pairs and encodes a197amino acidprotein. The molecular weight of CpcS is22.1kDa. It belongs to CpeS superfamilyand contains5conserved domains, which are THHL, ENH, LRERGYAEI, YETMand ERF. Homologous sequence alignment showed the similarity with other cyanobacteria was72.52%. The gene cpcT contains597base pairs and encodes a198amino acid protein. The molecular weight of CpcT is22.8kDa. It belongs to CpeTsuperfamily and contains7conserved domains, including FSN, NPP, AHI, RPL,EQAY, PYR and FKG respectively. Homologous sequence alignment showed thesimilarity with other cyanobacteria was71.14%. The gene cpcU contains525basepairs and encodes a174amino acid protein. The molecular weight of CpcU is22.8kDa. It belongs to CpeU superfamily and contains7conserved domains,including EFF，SAGKWFS，GKS，EER，PNLR，ASF and SEIR. Homologoussequence alignment showed the similarity with other cyanobacteria was73.22%.In order to research the function of chromophore lyases CpcS, CpcT and CpcU,three recombinant expression vectors: pACYCDuet-cpcB-cpcS (containing cpcBcoding for β subunit of phycocyanin and cpcS)，pACYCDuet-cpcB-cpcT (containingcpcB and cpcS) and pACYCDuet-cpcB-cpcU (containing cpcB and cpcS) wereconstructed. These three vectors and pACYCDuet-cpcB (only containing cpcB) weretransformed into E. coli BL21respectively with pET-hox1-pcyA(containing hox1andpcyA coding for chromophore synthetase) to construct four recombinant strains-B, BS,BT and BU. SDS-PAGE and western blotting were used to detect the expression ofphycocyanin and fluorescence emission spectra of the recombinant expression strainswas measured using a fluorescence spectrophotometer (HITACHI F-4500).The resultshowed that phycobiliprotein were expressed in all the recombinant strains, and theunit fluorescence intensity of the strains containing chromophore lyases were higherthan that of strains lack of chromophore lyases, indicating that the chromophorelyases CpcS, CpcT and CpcU had certain roles for the biosynthesis ofphycobiliprotein with optical activity.In order to further study of the acting site of the lyases-CpcS, CpcT and CpcU onβ subunit of phycobiliprotein, Cys at the sites of82and153of the β subunit had beenmutated to Ala, and mutant strains B(C82A)S，B(C153A)S，B(C82A)T，B(C153A)T，B(C82A)U and B(C153A)U were constructed. The acting site can be analyzed by thecomparison of fluorescence intensity between mutant strains and strains withoutmutation. The results showed that the unit fluorescence intensity of strains B(C82A)T and BT were kept consistent, while the unit fluorescence intensity of mutant strainB(C153A)T was lower, and therefore it can be deduced that the acting site ofchromophore lyase CpcT was the Cys153of β subunit. Compared with BS, the unitfluorescence intensity of mutant strain B(C153A)S had no change, while thefluorescence intensity of B(C82A)T was decreased, which indicated the acting site ofCpcS was the Cys82of β subunit. The unit fluorescence intensity of strainsB(C153A)U and BU were kept consistent, while that was lower in mutant strainB(C82A)U, which indicated the acting site of chromophore lyase CpcU was theCys82of β subunit.This research first cloned and studied the function of chromophore lyases CpcS,CpcT and CpcU from Arthrospira platensis FACHB314, which provided anexperimental foundation for assembling the phycocyanin β-subunit and synthesizingoptically active phycocyanin in a heterologous host.