Exploration Of Heterologous Expression And Assembly Effect Of Main Components Of Microalgae Pyrenoid

Rubisco is a rate limiting key enzyme for carbon fixation in photosynthesis.Its catalytic reaction makes inorganic carbon(Ci)in nature enter the Carbon cycle of the biosphere,but the catalytic efficiency is low,and the reaction is Sexual inhibition by O2 competition.Therefore,some organisms have evolved a CO2 concentration mechanism(CCM)to make up for this defect.The Pyrenoid of eukaryotic microalgae is mainly composed of Rubisco and linked proteins,which is the core of its biophysical CCM.Compared to biochemical CCMs,they are more easily expressed and utilized by heterologous systems.Heterogeneous system research on Rubisco assembly conditions and construction of liquid-liquid phase separation(LLPS)structure of Pyrenoid are of great significance for humans to understand CO2 fixation mechanism,improve crop yield and improve global climate.First of all,we explored the assembly function of A.thaliana cofactor to Rubisco from C.reinhardtii in E.coli prokaryotic expression system,and the interaction between CrEPYC1 and AtRubisco.The result is that the cofactors of A.thaliana cannot assist in the correct folding of CrLSU and the formation of L8 catalytic core,nor can they mediate the binding of CrSSU to AtL8 to form a hybrid Rubisco.EPYC1 can interact with CrSSU alone to form a LLPS structure that can be observed by transmission electron microscopy,but its interaction with AtSSU is unstable.Secondly,we attempted to predict and screen some cofactors that may be involved in the assembly of CrRubisco,but the currently selected cofactors are not sufficient to assist in the assembly of CrRubisco in the prokaryotic system,so further research and screening are needed.Compared to the Green-type Rubisco,the βE-βF ring on the Red-type Rubisco small subunit can replace the cofactors to recruit LSUs to form the Rubisco holoenzyme.We explored the biogenic conditions of Rubisco in E.coli using P.tricornutum and P.purpureum as experimental subjects.The results showed that they both can achieve heterologous construction with the assistance of the E.coli chaperone protein EcGroES/EL,and the biogenic requirements of P.purpureum are simpler.To address the incompatibility of red Rubisco chaperone proteins expressed in plant chloroplasts,we predicted the sequences of the chaperone proteins Cpn60 and co-chaperone proteins Cpn10 and Cpn20 in P.purpureum and validated their functions.The results showed that the P.purpureum chaperone protein cannot be soluble expressed in E.coli,which is different from that of C.reinhardtii.However,co-expression with the E.coli chaperone protein can preliminarily verify that Cpn10 and Cpn20 can function as the RED-type Rubisco functional group of the chaperone protein in vitro folding and in vivo assembly experiments.