Kayayyaki

Fahimtar metabolism na mai masaukin ƙananan ƙwayoyin cuta yana da mahimmanci don haɓakawa da haɓaka tsarin tsarin biocatalytic gaba ɗaya tantanin halitta, kamar yadda yake faɗar ingantaccen samarwa.Wannan gaskiya ne musamman ga redox biocatalysis inda ake amfani da sel masu aiki na rayuwa saboda cofactor / cosubstrate mai haɓaka ƙarfin haɓakawa a cikin mai watsa shiri.An yi amfani da Recombinant Escherichia coli don haɓakar proline-4-hydroxylase (P4H), dioxygenase yana haɓaka hydroxylation na L-proline kyauta zuwa trans-4-hydroxy-L-proline tare da a-ketoglutarate (a-KG) azaman cosubstrate.A cikin wannan biocatalyst gabaɗaya tantanin halitta, tsakiyar carbon metabolism yana ba da cosubstrate a-KG da ake buƙata, haɗa P4H biocatalytic aikin kai tsaye zuwa metabolism na carbon da aiki na rayuwa.Ta hanyar yin amfani da kayan aikin gwaji da na lissafin lissafi, irin su injiniya na rayuwa da (13) C-metabolic flux analysis ((13)C-MFA), mun bincika kuma mun kwatanta da ƙididdiga na ilimin lissafin jiki, na rayuwa, da amsawar bioenergetic na biocatalyst gabaɗayan tantanin halitta. zuwa canjin yanayin halitta da aka yi niyya da gano yuwuwar ƙullawar rayuwa don ƙarin aikin injiniya mai ma'ana. An gina nau'in lalata-rashin E. coli ta hanyar share kwayar halittar putA da ke ɓoye proline dehydrogenase.Dukan-kwayoyin halitta biotransformations tare da wannan mutant iri ya kai ba kawai ga adadi proline hydroxylation amma kuma zuwa ninki na takamaiman trans-4-L-hydroxyproline (hyp) adadin samuwar, idan aka kwatanta da na daji nau'in.Nazarin juzu'in iskar carbon ta hanyar sinadari na tsakiya na nau'in mutant ya nuna cewa karuwar buƙatar a-KG na ayyukan P4H bai inganta haɓakar a-KG ba, yana nuna ingantaccen tsarin sake zagayowar TCA a ƙarƙashin yanayin da aka yi nazari.A cikin nau'in daji, haɗin P4H da catalysis ya haifar da raguwa a yawan amfanin kwayoyin halitta.Abin sha'awa shine, nau'in ΔputA ya kuma rama asarar ATP da NADH mai alaƙa ta hanyar rage buƙatun makamashi mai ƙarfi a kwatankwacin ƙarancin ɗaukar glucose, maimakon haɓaka ayyukan TCA. zama masu alƙawarin haɓaka haɓakar haɓakar P4H ba kawai dangane da yawan amfanin ƙasa ba, har ma game da ƙimar haɓakar haɓakar halittu da haɓakar proline da yawan amfanin ƙasa akan tushen makamashi.Sakamakon ya nuna cewa, a kan ƙwanƙwasa ƙwanƙwasa, haɗakar da sake zagayowar TCA zuwa proline hydroxylation ta hanyar cosubstrate a-KG ya zama mahimmin mahimmancin mahimmanci da manufa don ƙara haɓaka haɓakar a-KG-dogara biotransformations.