Directed Evolution Of Near-infrared Fluorescent Proteins Derived From Cyanobacteria Phycobiliprotein And Its Application In The Zebrafish Bleeding Detection

Cyanobacteria are a group of prokaryotes capable of photosynthesis.They can capture light energy through phycobilisome and transmit it to photosystems I and II.The phycobiliprotein that make up phycobilisome bind linear tetrapyrrole pigments and thus has the ability to absorb red or far-red light,and are capable of emitting intense fluorescence.Therefore,phycobiliprotein are suitable materials for the evolution of near-infrared fluorescent proteins.Core-membrane linker(Apc E),acts as the terminal energy emitter of the phycobilisome and play a connector between the phycobilisome and the endocyst membrane.Due to the poor water-solubility of Apc E,the deletion mutant Apc E(35)obtained through lacking loop domain of Apc E has good water-solubility.However,the solubility of Apc E-14,which can bind biliverdin(BV)and evolved from Apc E(35)(11)is still low.Thus,in this study,site-directed PCR was performed on the basis of Apc E-14,and it was found that the mutant Apc E-14(F76E)had good water solubility.It has a molecular weight of only 12.5 k Da and has a far-red fluorescence emission spectrum(～667 nm).Therefore,we named it mini Apc E and used mini Apc E as a template to evolve fluorescent proteins that can be used for mammalian cell imaging via error-prone PCR.Ultimately,we obtained mutants that co-expressed with heme oxygenase(HO1)and were able to have weak fluorescence in mammalian cells.Our lab has evolved a series of Bei Dou fluorescent proteins(BDFPs)from Apc F2 coming from Chroococcidiopsis thermalis sp.PCC7203.Among them,BDFP1.2 and 1.6 have a far-red(～670 nm)fluorescence spectrum,while BDFP1.1has a near-infrared(～710 nm)fluorescence spectrum.In this study,BDFP1.6 was used as the template,and a mutant with fluorescence spectrum similar to BDFP1.1 but6.25 times brighter than BDFP1.1 in mammalian cells was obtained by site-directed PCR,and named BDFP1.7.By simulating and analyzing the structure of BDFP1.7,we used BDFP1.7 as a template and selected residues of BDFP1.7 that interacted with BV chromophore for site-directed PCR,and finally obtained a mutant that was 3.24 times brighter than BDFP1.7 and named BDFP1.8.Moreover,the brightness of BDFP1.8 was 2.38 times brighter than that of i RFP720.Furthermore,our results showed that the effective brightness(brightness in mammalian cells)of BDFPs does not correlate with the molecular brightness(product of fluorescence quantum yield and molar extinction coefficient).The effective brightness mainly depends on the speed of integration of BDFPs with BV chromophore and the affinity between them.The stability of BDFPs,IFP2.0 and i RFP720 was determined.The results showed that the photostability,thermal stability and acid stability of BDFPs were significantly stronger than those of IFP2.0 and i RFP720.In addition,BDFP1.6 and BDFP1.8 can be used to two-color imaging of mammalian cells.In these studies,we also developed a biosensor that can detect bleeding in animals using E.coli.as a vector and the far-red fluorescent signal as an output signal.The biosensor was constructed by deletion of the hemf gene in E.coli.strain BL21(DE3)and expression of HO1,Chu A,and BDFP1.6 proteins.The fluorescence of BDFP1.6,which outputs a fluorescent signal,is located at 667 nm in the far-red light spectral region.Therefore,we named this biosensor as FRLBD(Far-Red Light for Bleeding Detector).The reaction of FRLBD with blood or heme was measured in vitro.Results showed that the FRLBD is efficient and specific for the detection of heme or blood.To verify whether FRLBD can be used to detecting the bleeding in vivo.Zebrafish was used as a model animal and induced bleeding in zebrafish with high concentration of aspirin.Finally,convolutional neural network(CNN)was used to identify the fluorescent features in the images.Results showed that FRLBD can be used to detecting the intestinal bleeding in zebrafish.And the CNN model predicted the zebrafish bleeding with an accuracy of 91%.Moreover,the intestinal microbial composition of zebrafish with intestinal bleeding was compared with that of normal zebrafish.And it was found that the content of harmful microorganisms Aeromonas was greatly increased and the content of beneficial microorganisms Deefgea and Chitinibacter was decreased in the intestinal bleeding zebrafish.