The Functional And Applicational Research On Molecular Chaperones Originated From Hyperthermophilic Archaea Pyrococcus Furiosus

Synthetic biology is the design and construction of new biological parts,devices,systems or the re-design of existing natural biological systems for useful purposes based on certain disciplines and existing knowledge,and bestow novel functions on engineered cells.Currently,researchers have proposed to construct extremophiles regulatory gene element to improve industrial and agricultural microbial resistance against adverse environment,simplify the production process,enhance production efficiency and reduce costs.Hyperthermophilic archaea Pyrococcus furiosus thrives in extreme environment,overexpressing chaperones is one of the main mechanisms of this organism to cope with extreme environment.Molecular chaperones are defined as a functional class of unrelated families of proteins that assist the non-covalent assembly of other polypeptide-containing structures in vivo,but are not the components of these assembled structures when they are performing their normal biological functions.P.friosus owns the simplified molecular chaperone system,only three main chaperones are demostrated so far:prefoldin,hsp60 and shsp.Our previous studies revealed that prefoldin could help Escherichia coli surviving at 50? and shsp could protect enzymes from thermal aggregation.Therefore,we constructed a series of hyperthermophilic chaperone"elements" and investigated their effects on helping E.coli fighting environmental stress as well as their other biological functions.Firstly,the effect of P.furiosus chaperone on the soluble expression of heterogenous protein in E.coli was studied.The extracellular a-amylase from hyperthermophilic archaea P.furiosus(PFA)has prominent enzyme properties.When compared with the thermostable a-amylase commonly used in the market,PFA exhibits higher optimal temperature(100?)and thermostability,lower optimal pH(5.5)and does not require Ca2+ for its activity and thermostability.These properties make PFA more suitable for industrial requirments.Due to the inherent difficulty to cultivate P.furiosus,sufficient production of this enzyme can only be achieved using recombinant expression.However,recombinant PFA protein expresses as inclusion bodies in E.coli.Resaerchers have tried many methods to express PFA in its soluble form,but these methods have resulted in poor yields of total soluble recombinant enzyme.We tried to use homogenous molecular chaperones to promote the soluble expression of PFA and improve its enzymatic activity,we constructed a series of recombinant plasmids encoding the chaperone genes of P.furiosus and co-expressed them with PFA gene in E.coli.We measured the PFA enzymatic activity and performed protein electrophoresis to analyze whether chaperones originated from P.furiosus could promote the soluble expression of PFA in E.coli.It indicated that all chaperones from P.furiosus could promote the soluble expression of PFA with different extents,and prefoldin was most effective on the soluble expression of PFA.We also found that improving the expression level of chaperones could further promote the soluble fraction of PFA as well as the enzymatic activity.Osmolytes treatment could further enhance the soluble fraction of PFA,whereas it had no effect on improving enzymatic activity.We also investigated the effect of chaperones from two kinds of organism:prefoldin from P.furiosus and GroEL/ES system from E.coli on the soluble expression of PFA.It turned out that GroEL/ES to some extent could promote the soluble expression of PFA as well as the enzymatic activity,whereas this system was less effective than prefoldin on the soluble expression of PFA.Secondly,the effect of hyperthermophilic archaea P.furiosus chaperone system on helping E.coli coping with mistranslated proteins which inducing by aminoglycoside and defending oxidative environment was investigated.Maintaining translational fidelity is critical in all three domains of life.A slight decrease in translational fidelity leads to a severe neurological disorder in mice.Severe mistranslation leads to growth arrest or cell death in bacteria and respiratory defects in yeast.Mistranslated proteins are susceptible to misfolding and forming protein aggregates.Protein misfolding and aggregation are strongly associated with multiple human neuropathies such as Alzheimer's disease and Parkinson's disease.It has potentials for treating human desease by investgating how to reduce the protein aggaregates caused by protein misfolding.Streptomycin(Str)targets the 30S ribosome subunits to induce amino acid misincorporation.The accumulation of mistranslated proteins induces intracellular protein aggregation and further causes function defect or even cell death.Many evidences reveal that erroneous protein synthesis induces the overexpression of molecular chaperones,implying that chaperones may assist the refolding of mistranslated proteins.In order to investigate whether chaperone systems from hyperthermophilic archaea could help host cells inhibiting protein aggregation,we overexpressed P.furiosus chaperones in E.coli and treated cells with different Str concentrations.It indicated that prefoldin and hsp60 assisted E.coli fighting against Str from different aspects.The overexpression of prefoldin could facilitate cell growth,rescue membrane potential and reduce the intracellular ROS concentration.While overexpression of hsp60 facilitated cell growth and promoted cell survival.We also found that prefoldin and hsp60 could suppressed protein aggregation caused by Str treatment effectively.In addition,we investigated the effect of chaperone system GroEL/ES originated from E.coli on reducing the toxicity caused by Str.It showed that the protection of GroEL/ES on cells exposure to Str decreased with the Str concentration and the protein aggregation content increased with Str concentration and the treatment time especially chaperones themselves.Besides,we investigated the effect of chaperones from P.furiosus and E.coli on helping E.coli defending oxidative environment.The results showed that all chaperones tested in this paper could help E.coli coping with oxidants menadione and plumbagin with different extents.Hsp60 originated from P.furiosus was most effective on helping host strain fighting against oxidative environment.Finally,the effect of chaperones from hyperthermophilic archaea P.fiuriosus on the bioconversion of indigo was explored.P450s catalyze the oxidation of a broad array of endogenous and exogenous organic substrates,they are best known for breaking down xenobiotics-including pharmaceuticals-in humans,but also defend insects against plant toxins,and are involved in the synthesis of antibiotics in bacteria and fungi.The current demand for P450 chemistry lies primarily within the pharmaceutical field,especially for the application in metabolite synthesis(pharmaceuticals)such as artemisinin and amorpha-4,11-diene biosynthesis.P450 BM3 is isolated from Bacillus megaterium,its P450 domain(BMP)is fused to a reductase domain(BMR).This peculiar fusion architecture confers to the enzyme an efficient electron transfer rate and a high catalytic turnover.In addition,the enzyme can be easily expressed as recombinant proteins in E.coli with high solubility and it only requires NADPH and oxygen to function.Thus,P450 BM3 has been a target of mutagenesis studies and synthetic biology.Protein engineering studies of P450 BM3 reveal that mutants can be endowed with new substrate scopes and can exhibit region-and enantio-selectivity on new substrates and can be engineered to be highly selective and active toward new substrates.In our study,site-directed mutagenesis was performed on cytochromes P450 BM3:A74G,F87V,L188Q and D168H.This mutant could use indole as substrate and catalyze it to indigo,but the indigo production was very low.In order to figure out whether molecular chaperones could enhance the production of indigo or not,we co-expressed chaperones from P.furiosus with P450 BM3 in E.coli to investigate whether chaperones could increase the production of indigo.The results showed that the production of strains overexpressing prefoldin was higher than others,and increasing expression level of chaperones could not further increase the indigo production.We also investigated the effect of chaperones from two kinds of organism:prefoldin from P.furiosus and GroEL/ES system from E.coli on the indigo bioconversion.It indicated that GroEL/ES could further promote the indigo production when compared with prefoldin.We also found that increasing indole concentration could not improve the production and even reduce the production.By CO-reduced difference spectrum of P450 BM3 and in vitro enzymatic activity experiments,we found that the enzymatic activity of strains overexpressing P450 BM3 alone was high whereas its intracellular indigo production was low.It meant that enzyme level was not the restrictive factor of indigo production.Therefore we performed preliminary research on the indigo formation mechanism.It turned out that intracellular NADPH/NADP+ ratio may affect the indigo production.The NADPH/NADP+ ratio of strains co-expression prefoldin and GroEL/ES was relatively higher than that of P450 BM3 alone,and higher NADPH/NADP+ ratio was benefit for the indigo formation.We also found that GroEL/ES improved the indigo production from two aspects:regulating the NADPH/NADP+ ratio and promoting the soluble fraction of P450 BM3.Prefoldin,however,could not increase the soluble fraction of P450 BM3.