Heterologous Efficient Biosynthesis Of Phycoerythrin In Escherichia Coli

Phycobiliprotein is an important light catching pigment protein for some algae.There are mainly two sources of phycobiliprotein,including natural extraction and heterologous biosynthesis.This study is based on the principle of metabolic engineering.Firstly,the double promoter plasmid pRSFDuet was used to make phycobiliprotein subunit gene cpcB and split synthase gene cpcS to form polycistron and insert it into the first expression box,alga red bilirubin biosynthesis gene Ho1 and pebS to form polycistron and insert it into the second expression box.After plasmid transformation of Escherichia coli,an expression strain was obtained to optimize some fermentation conditions such as inducer concentration,induction temperature and induction time.The recombinant phycoerythrin was separated and purified by affinity chromatography,and the spectroscopic and antioxidant activity of the recombinant protein was analyzed.An highly efficient biosynthesis of phycoerythrin in Escherichia coli strains?STEC?were obtained.The optimal induction condition with lactose as an inducer were:2 g/L lactose,inducting 28 h at 25^oC,and 211.6 mg/L of phycoerythroprotein expression.When IPTG was used as inducer,the best induction conditions were:0.4 mmol/L IPTG,inducting 28 h at 25^oC,and 188.7mg/L of phycoerythroprotein expression.The maximum absorption peak of phycoerythroprotein was 555 nm,the maximum fluorescence emission peak was 565nm,the Chromophore binding rate was 92%,OD₅₅₅/OD₂₈₀80 was 8.The efficient biosynthesis of phycoerythroprotein in Escherichia coli was successfully achieved,and the recombinant phycoerythroprotein exhibited hydroxyl radical scavenging activity.Secondly,the antioxidant activity of phycoerythroprotein is positively correlated with the binding rate of chromophore,through different lyase catalytic chromophore to combine different apoprotein.The combination of color groups at different sites produces recombinant proteins with different spectral properties.Stokes shift of recombinant proteins is increased by energy resonance transfer.Firstly,selecting four different sources of cpcT.The required genes?ho1?pcyA?cpcB?cpcT?of the phycoerythroprotein were synthesized to constructed in the double promoter plasmid pRSFDuet.The plasmid was transformed into the Escherichia coli to obtain the expression strain,and the recombinant phycohemoglobin was isolated and purified by affinity chromatography.The spectroscopic properties of recombinant proteins were analyzed to obtain Nostoc PCC 7120 source cpcT catalyzed PEBspecific binding of PEB to the Cys-153 of CpcB had a characteristic absorption peak at 535nm.Secondly,the Bp-1 source cpcS was constructed in the carrier pBAD?His-A?to obtain the pBAD?His-A?-cpcS,and combined with the above plasmid pRSFDuet to co-transform the Escherichia coli into the expression strain.The CpcS catalyzed the lyase with PEB combined at Cys-84 sites,to separate and purify the recombinant phycohemoglobin,analysis the spectroscopic properties of recombination protein,and obtained the recombination phycohemoglobin with a combination of the double chromophore.Finally,the activity of cleft synthase directly affects the chromophore binding rate,and then affects the physiological properties of the phycoerythroprotein.This study aims to lay the foundation for the discovery of new type of lyase and provide a broader approach to the synthesis of recombinant phycoerythroprotein in vitro.The genomic library of polychlorella?Synechococcus sp.?CC9311 was used as the screening object,and Sau3A I was used to carry out incomplete digestion conditions on the CC9311 genome DNA of polychlorella,try to connect,transform,and induce expression with the carrier G110200 and G112939.To screen the colony with fluorescence characteristics by flow cytometry.To determine the extraction method of polychlorella CC9311 genomic DNA,that is,plant genome extraction kit.1 mg polychlorella CC9311 algae can extract genomic DNA about 1.8?g.The optimum condition that Sau3A I was used to carry out incomplete digestion conditions on the CC9311 genome DNA of polychlorella was 1 U/?g DNA,37^oC,10min.The colony with fluorescence characteristics were selected by flow cytometry.In this study,the effective biosynthesis of recombinant phycosalin in Escherichia coli was realized,and the linear relationship between the chromophore binding rate and the antioxidant activity was determined.The recombinant phycoerythroprotein with a combination of the double chromophore was obtained by co-conversion,and the experimental process for the discovery of a new type of lyase was established.It provided a new idea for heterologous biosynthesis of phycobiliprotein and laid a foundation for its wide application.