Isolation And Identification Of Pigmented Serratia Marcescens And Construction Of Plasmid Containing Prodigiosin Synthetase

During the growth of serratia marcescens,it could produce a secondary metabolite prodigiosin.Prodigiosin has a variety of biological activities that can resist malaria,anti-tumor,and immunosuppression.Wild-type serratiamarcescens produced a little of prodigiosin,which cannot supply the demand for industrial production.Only optimizaing the culture condition,serratiamarcescens could not greatly increase the production of prodigiosin.With the development of biotechnology,it is a way to increase the production of prodigiosin through modifying the prodigiosin gene of serratia marcescens by genetic engineering.In this paper,a red pigment-producing strain was isolated and purified from lake water by streak plate method.Through strain's 16S rDNA identification with primers27F and 1492R amplification,the results of sequence were compared with the NCBI gene bank to confirm the strain's species.Then,the pigment in the bacterial cells was extracted by acidic methanol,and the crude extract was separated and purified by column chromatography to obtain purified red pigment.The red pigment was analysis by visible spectrophotometer,FTIR and LC-MS.The paper also studied prodigiosin genes and plasmids construction.Based on the homologous sequences of the strains in the NCBI,we designed primers for the prodigiosin synthase genes with the restriction site,upstream and downstream of non-coding sequence.The prodigiosin synthase genes–pigF and pigC was amplified and ligated with pBM20-T vector.In addition,upstream of non-coding sequences were amplified.The cloned non-coding sequences were detected by overlap PCR.After,the recombined genes were ligated with pBM16A-T and transformed into serratia marcescens.The expression of the recombinant gene was analyzed.The paper obtained the following results:1.Through streaked and separated from the lake water,we obtained a bacterium with producing red pigment.The 16S rDNA of strain was cloned using primers 27F and 1492R.The sequencing results were contrast to the sequence of NCBI database.The results showed that the strains had 98%similarity with serratia marcescens.The crude extract was obtained by spin-drying with methanol and purified using column chromatography to obtain a purified red pigment.A thin layer analysis of the purified pigments determined that the best developing solvent was petroleum ether:ethyl acetate=4:1.After visible spectrophotometer scanning at wavelengths of 400-700 nm,maximum absorption peak of pigment was 535 nm.FTIR analysis showed that have-CH₃,C=C,-NH absorption peaks.HPLC-MS analysis shows m/z peaks were at 324.5m/z and 325.5 m/z,which is consistent with the characteristic absorption peak,molecular weight,and infrared spectrum of the erythromycin standard product.It was confirmed that we obtained serratiamarcescensstrain with producing prodiogisin.2.With the designed primers,the synthesis enzyme genes pigF and pigC were amplified about 1200 bps and 2700 bps respectively.The pigF and pigC gene amplification products were recombined with the pBM20-T vector and transformed into E.coli DH5?.The strains recombined were sequenced.Results were compared with the sequence of NCBI database.It was showed that pigF and pigC genes had the highest homology with serratia marcescens FS14.With the primers coa-1,non-coding region genes were amplified about and 600 bps.The part of coa-1 genes with pigC and pigF genes was amplified to coa-pic and coa-pif genes.Then,recombined genes were recombined with pBM16A-T vector and transformed into E.coli DH5?.The transformants recombined successfully were isolated to sequencing.The sequence results were compared with the NCBI database.It was showed that pigC and pigF with Coa-1 genes was recombined successful.coa-pic and coa-pif genes were recombined with pBM16A-T to be pBM16A-coa-pic and pBM16A-coa-pif plasmids?3.Then plasmids were transferred into serratia marcescens for selection of recombinants that S.m-pif and S.m-pic.It was result that recombinants expressied protein which analyzed by SDS-PAGE electrophoresis.By comparing the change of pigment absorbance in bacterial growth,it was found that the S.m-pif strain increased the prodigiosin yield by 6.8%compared with the wild strain and the S.m-pic strain increased the prodigiosin yield by 17.1%.