Screening And Characterizing FNR Mutant With NCD Preference

The ferredoxin-NADP reductase(FNR)is an enzyme performing function at the end of the photosynthesis system of photoautotrophic organisms,it regenerates NADPH using electrons generated by photosynthesis and provides reducing power for biosynthesis.However,the natural coenzyme NADPH participates in many intracellular metabolic pathways,and it can not transmit reducing power pathway selective way.This limits the efficiency and controllability of reducing power utilization.The artificial coenzyme nicotinamide cytosine dinucleotide(NCD)can transmit reducing power as NAD(P),and it is not recognized by natural metabolic pathways.NCD can reduce the complexity of the metabolic systems and selectively transmit the reducing power to the target pathway.This study combined analyzing structure of Synechocystis sp.PCC 6803 derived FNR and designing high-throughput screening method,and mutated the FNR through protein directed evolution strategy to make FNR obtain the ability of generating NCDH.Regenerating NCDH by the reducing power generated by photosynthesis would realize pathway selective transmission of the reducing power.We also constructed an efficient NCD transport strategy to overcome the inability of intracellular synthesis of NCD and low transport efficiency of NCD.The main achievements are summarized as follows:A high-throughput activity detection method based on coenzyme cycle color development and a high-throughput screening strategy for plate color development based on HpaB-FNR fusion protein were constructed.10 active sites that may affect the coenzyme preference of FNR were identified through simulation and analysis of FNR structure.The single-site saturation mutation library,two-site saturation mutation library and random mutation library were constructed.This study screened 4 single-site saturation mutation libraries,9 double-site saturation mutation libraries and one random mutation library.Each single-site library was screened for 200 transformants.Each double-site library was screened for 2000 transformants.Random mutation library was screened for 4000 transformants.Wild-type FNR has no detectable NCD activity.The NAD activity of wild-type FNR is 340 U/mg and NADP activity reaches 344679 U/mg.The mutant FNR 1158R/K311T/R340A/R349D had an activity of 80 U/mg with NCD,642 U/mg with NAD,and 3419 U/mg with NADP.The NCD activity of the mutant FNR K311T/R340A/R349D is 58 U/mg.The NAD and NADP activity are 561 U/mg and 164 U/mg,respectively.The mechanism for the enhancement of FNR coenzyme specificity was explored by discussing protein structure simulation and analysis,K31 1.R340.R349 are located in the NADP binding domain and binding to the 3'-phosphate of NADP.The binding domain space becomes smaller.Basic amino acids become acidic or neutral amino acids,which is not conducive to NADP binding,and the affinity for NAD and NCD is increased.1158 is also located in the NADP binding domain.The I1588 mutation makes the space of NADP binding domain open become smaller,which improves the NAD/NADP specificity of the enzyme.A strategy for efficiently transporting NCD across cell membranes was constructed,by improving the stability of exogenous coenzymes and selecting highly active coenzyme transporters.The degradation of extracellular coenzyme was limited by exploring the Escherichia coli BW25113 strain with pyrophosphate hydrolase(UshA)encoding gene knocked(E.coli AushA)as the host.The stabilities of NAD and NCD were improved by 8 times and 11 times respectively.Protochlamydia amoebophila UWE25 derived transporter NTT4 and Arabidopsis thaliana mitochondria derived transporter AtNDT2 transported NAD and NCD.The activity of NTT4 is higher than AtNDT2.The transporting efficiency of NAD and NCD by NTT4 is 3 times and 18 times of AtNDT2,respectively.Transporting NAD analogs such as NCD and NAD by E.coli ?ushA expressing NTT4 had no effect on the growth of the strain,suggested that NCD has little cell toxicity.This study screened FNR mutants with NAD or NCD preference and investigated the relation between the structure and function of the mutant FNR.These studies deepened our understanding on the function and structure of FNR,and provided the basis for efficient and selective transmission of the reducing power generated by photosynthesis.The construction of the efficient NCD transporting strategy would promote the intracellular study and application of NCD and other NAD analogs.