Construction Of Synechocystis Sp.PCC6803::?-PpetE-petH And Strengthening Coenzyme NADPH Regeneration

Coenzyme NADPH has playing an important role in the intracellular redox system and production of other reducing substances.In the process of NADPH regeneration through photosynthesis,the ferredoxin: NADP⁺ oxioreductase?FNR?is the key enzyme which transfers the electron from the ferredoxin(Fd_red)to NADP⁺,to become reduced NADPH.So FNR is an important regulatory site for NADPH regeneration metabolic regulation.In this work,we hope to constructed an engineering cyanobacteria to overexpress FNR so that to strengthen the NADPH regeneration.Furthermore,its application in asymmetric reduction of prochiral carbonyl compounds to the corresponding chiral alcohol will be also investigated.First,the gene petH,which decodes the FNR,was cloned from Synechocystis sp.PCC6803 by PCR.It was inserted into plasmid pHB1524,which contains a promoter PpetE and terminator ? fragment,the recombinant plasmid pHB1524-petH was obtained.The ?-PpetE-petH fragment was obtained by PCR used pHB1524-petH as the template.For further,the ?-PpetE-petH fragment was integrated into the plasmid pKW1188 to obtain the recombinant plasmid pKW-?-PpetE-petH.The plasmid pKW1188 contains a part of homologous sequences of Synechocystis gene slr0168,which can be used to integrate ?-PpetE-petH into the Synechocystis sp.PCC6803 chromosome.The recombinant plasmid pKW-?-PpetE-petH has been successfully constructed which was determined with restriction enzyme digestion and gene sequencing.In order to stable overexpress the target gene,we used the homologous recombination technology to integrate FNR gene fragment ?-Ppet E-petH into Synechocystis sp.PCC6803 chromosome which can be induced with Cu²⁺.Through natural transformation,the recombinant plasmid pKW-?-PpetE-petH was transferred into Synechocystis sp.PCC6803.Under the guidance of homologous sequences of Synechocystis gene slr0168,the ?-PpetE-petH fragment was integrated into the Synechocystis chromosome by homologous recombination,then the engineering Synechocystis sp.PCC6803:: ?-PpetE-petH was obtained which can overexpress the FNR.With adjusting the concentration of Cu²⁺ in medium to regulation Synechocystis sp.PCC6803:: ?-PpetE-petH,the results show that the insertion of ?-PpetE-petH didn't influence the growth of Synechocystis sp.PCC6803.The FNR amount was increased in Synechocystis sp.PCC6803:: ?-PpetE-petH with the Cu²⁺ induction.These mean that engineering cyanobacteria was successfully constructed which can overexpress FNR.Furthermore,we studied the coenzyme regeneration efficiency in Synechocystis sp.PCC6803:: ?-PpetE-petH by overexpression of the FNR,and its application in catalytic asymmetric reduction.The results show that,under 320nmol/L Cu²⁺,intracellular FNR activity of Synechocystis sp.PCC6803:: ?-PpetE-petH was improved 40.74% compared to the wild type Synechocystis sp.PCC6803.And intracellular NADPH content of Synechocystis sp.PCC6803:: ?-PpetE-petH increased 80.75% compared to the wild type.Using the Synechocystis sp.PCC6803:: ?-PpetE-petH for the asymmetric reduction of acetophenone to S-phenyl ethanol,the yield improved 61.2% compared to the wild type Synechocystis sp.PCC6803,and the yield of catalytic reduction of ethyl acetoacetate to produce 3-hydroxybutyr improved 36.36%.This suggests that overexpression of FNR can promote NADPH coenzyme regeneration,and improve the efficiency of the catalytic asymmetric reduction.Compared to the vectors expression,we construct the engineering cyanobacteria use homologous recombination technology could be more long-term and stable to express the target gene.This work provides a theoretical and technical basis for the promoting coenzyme regeneration technical.