Identification Of Glyceraldehyde-3-phosphate Dehydrogenase Pseudogene GapC In Escherichia Coli MG1655 And Preliminary Analysis Of Its Physiological Function

Pseudogenes are genetic sequences that are similar to those of functional genes in genome but cannot be translated into functional proteins or encode abnormal proteins due to some genetic defect.Pseudogenes are present in almost all forms of life and have been considered non-functional "junk DNA" or genetic fossils.Researchers have studied pseudogenes in different species and they discovered that pseudogenes are able to perform different functions in a variety of forms.Glyceraldehyde-3-phosphate dehydrogenase(GAPDH)catalyzed reaction sits at the intersection of multiple metabolic pathways,including glycolysis pathway,gluconeogenesis pathway,pentose phosphate pathway and glycerol metabolism pathway.The pseudogenes of GAPDH are distributed in a variety of organisms,but their specific physiological functions and the role of pseudogenization in the evolution of species remain to be revealed.The research object of this thesis was the laboratory type strain Escherichia coli MG 1655.There are three GAPDH homologous protein coding genes,gapA,gapB and gapC in E.coli MG 1655,among which gapC is a pseudogene(named gapCP).Firstly,the physiological functions of gapA,gapB and gapC were investigated.Mutant strains of gapA and gapB were constructed and it was confirm that GapA encoded by gapA in E.coli MG1655 performs the major GAPDH function and played a key role in central metabolism.Then,a mutant strain E.coli MG 1655(ΔgapA ALE)that restored normal growth and GAPDH activity was obtained by adaptive laboratory evolution(ALE)of E.coli MG 1655(ΔgapA)in minimum medium with 20 mM glycerol as the sole carbon source.Further analysis revealed that the mutation of the pseudogene gapCP to the real gene gapCE was the key to the restoration of growth of E.coli MG1655(ΔgapA ALE),and the pseudogene gapCP has the biological function as "adaptive repertoire" of E.coli MG1655.GAPDH homologs GapA and GapB in E.coli MG1655,GapCE encoded by gapCE in the adaptive laboratory evolution strain E.coli MG1655(ΔgapA ALE)and GapCR encoded by the real gene gapCR in the environmental strain E.coli ZK126 were expressed and purified.The cofactor preferences and kinetics of the four homologs were investigated.It was revealed that GapA,GapCE and GapCR possessed high GAPDH activity with NAD+as cofactor,and GapB possessed high erythrose-4-phosphate dehydrogenase(E4PDH)activity with NAD+as cofactor.Among the four GAPDH homologous proteins,GapA exhibited the highest GAPDH activity,GapCE and GapCR had approximate GAPDH activity and substrate affinity,while GapB only had low GAPDH activity.Further bioinformatics analysis of the genes encoding GAPDH homologous proteins including gapA,gapB and gapC in the sequenced E.coli genomes in the PATRIC database was performed.It was confirmed that gapC showed a significant pseudogenization trend compared to gapA and gapB in E.coli,accounting for about 25.68%of the sequenced E.coli.The pseudogenization of gapCP mainly occurred at bases 115,419 and 750 of the DNA sequence,resulting in early termination of translation and inability to translate normal functional proteins.In addition,the percentage of gapC pseudogenes in laboratory-derived E.coli was higher than that in natural environment and other sources of E.coli.A preliminary analysis of the physiological significance of gapC pseudogenization in E.coli MG1655 was conducted.Compared with strain E.coli MG1655(gapCR),in which both gapA and gapC are functional genes,gapC pseudogenization caused the reduced motility and increased biofilm formation of E.coli MG1655 in rich nutrient media.In summary,this thesis found that the pseudogene gapCP in E.coli MG 1655 can be repaired by mutation to the real gene gapCE after gapA deletion,which can restore GAPDH activity and normal growth of E.coli MG 1655(ΔgapA).A large number of E.coli have gapC in the form of pseudogene,and the pseudogenization of gapC resulted in reduced motility but increased biofilm production in E.coli MG1655 in rich nutrient media.The results of the thesis demonstrate the important role of pseudogenes in enhancing the metabolic flexibility and adaptability of microorganisms,which can provide useful reference for studying the physiological functions of gapC pseudogenes in other species and the causes of pseudogenization of some GAPDH-encoded genes.