Comparative Selection Of Promoters And Construction Of Ethanol-producing Vectors In The Euryhaline Cyanobacterium Synechococcus Sp.PCC7002

With the increase in fossil fuel consumption and the large amount of CO2 emissions,serious energy crises and environmental problems have arisen.As the largest and most important primary producer of biomass in the ocean,cyanobacteria have been receiving much attention for their development and utilization.The euryhaline cyanobacterium Synechococcus sp.PCC7002 has the characteristics of high temperature tolerance,high salt tolerance,and clear genetic background,which was regarded as a new generation of "cell factory".In recent years,a large number of metabolic engineering studies have been carried out in Synechococcus sp.PCC7002,which has made the development and utilization of various molecular components more mature.However,there are limited research on the expression intensity of promoters in Synechococcus sp.PCC7002 and the effects of promoter strength on metabolic pathways.In this study,different promoters were used to drive expression of green fluorescent protein(GFP)in the Synechococcus sp.PCC7002,and the strength of the green fluorescent protein was examined to determine the strength of the promoter.After obtaining the strength of different promoters,promoters were used to drive the expression of pyruvate decarboxylase(pdc)gene and alcohol dehydrogenase(adh)gene in the ethanol biosynthesis pathway,respectively.And the effect of promoter on ethanol synthesis was studied by detecting the yield of ethanol.(1)GFP was used as a reporter protein to study the expression intensity of eight constitutive promoters involved in photosynthesis and CO2 fixation.The results showed that the 7002PcpcB and 6803PpsaAB related to photosynthesis were strong promoters;6803PpsbDC,6803PcpcB,7002PrbcL and 7002PpsbDC were weak promoters;while 7002PpsaAB and 6803PrbcL were weaker promoters.At the same time,the inducible promoters induced by salt or ions were also studied.The results showed that the expression of the inducible promoter 7002PggpS was the strongest when the NaCl concentration was 5%(W/V),and the induction promoter 7002smtB-PsmtA was significantly induced when the concentration of Zn2+ was 50 ?mol.(2)According to the strength of the promoters,12 vectors were constructed,and finally 7 ethanol mutants were obtained.Among ethanol-producing mutants,2 strains employed inducible-constitutive promoter combinations,and 5 strains employed constitutive promoter combination mutants.