Gene Cloning, Expression And Molecular Evolution Of Glycerol Dehydratase

1,3-propanediol (1,3-PD) is an important material for chemicalindustry, and it can be used for not only a good solvent, antifreeze,protect reagent, but also a monomer in the synthesis of polyesters for usein carpet and textile fibers. 1, 3-PD production by biological methodshows many advantages over the chemical one in that the formerconsumes less energy, produces less pollution, and shows higherefficiency. Therefore, there is much interest in researches on thebiological method. Glycerol dehydratase is the key and rate-limitedenzyme in this process, so it is very important to study. The genes ofglycerol dehydratase and other correlative enzymes were cloned andhigh-expressed in E.coli, and the properties of recombinant enzymes werestudied systematically. Then, glycerol dehydratases were evolved through rational and irrational design. The 3D structures of correspondingenzymes were successful constructed by homology modeling,furthermore, the relations between 3D structures and enzymes propertieswere studied in this paper. The main results out of this investigation wereas follows:1 Gene cloning, expression of glycerol dehydratase and correlativeenzymes and purification, properties and 3D structures ofcorresponding recombinant enzymes(1) The dhaBCE gene from Citrobacter freundii was cloned and high-expressed. The property of purified recombinant enzyme was determined,and the 3D structure of glycerol dehydratase was successful constructedby homology modeling, furthermore, the probable active site wasforecasted.(2) The gldABC gene from Klebsiella pneumoniae was cloned andhigh-expressed. The property of purified recombinant enzyme wasdetermined, and the 3D structure of glycerol dehydratase was successfulconstructed by homology modeling, furthermore, the probable active sitewas forecasted.(3) A segment of DNA was successfully cloned and expressed from themetagenome DNA, and this gene was confirmed as glycerol dehydratasegene. 3D structure information of this enzyme was obtained by homologymodeling, Asp366 changed to Gly336, and this site was one of the six amino acids which binds with substrate 1,2-PD in the active site, andcompared to other glycerol dehydratase, the residues hydrogen-bonded to1,2-PD were changed, as probably led to no-activity of this glyceroldehydratase.(4) The dhaT gene from C. freundii was cloned and high-expressed. Theproperty of purified recombinant 1, 3-propanediol dehydrogenase wasdetermined, and the 3D structure of glycerol dehydratase was successfulconstructed and analyzed by homology modeling.(5) dhaBCE and dhaT genes of C. freundii were connected by over-lapPCR. These two genes were successfully co-expressed as a polycistron inE.coli, and 1,3-PD was determined by HPLC and Gas chromatography.(6) The genes dhaF and dhaG from C. freundii were cloned andco-expressed, and the property of the recombinant enzyme showed: thereactivate factor could not only reactivate glycerol dehydratase of C.freundii, but also cross-reactivate glycerol dehydratase of K. pneumoniaeand Clostridium priferingens. On the contrary, it was not effective withrespect to the corresponding enzyme of Clostridium pasteurianum. At last,the 3D structure of this reactivate factor was successfully constructed byhomology modeling.(7) The genes gdrA and gdrB from K. pneumoniae were cloned andco-expressed, and the property of the recombinant enzyme was as follow:the reactivate factor could not only reactivate glycerol dehydratase of K. pneumoniae, but also cross-reactivate glycerol dehydratase of C. freundiiand Clostridium priferingens. On the contrary, it could not reactivate thecorresponding enzyme of Clostridium pasteurianum. At last, the 3Dstructure of this reactivate factor was successfully constructed byhomology modeling.2 High throughput screening method of glycerol dehydrataseWe have developed a highly-efficient high throughput screeningmethod for detection of novel mutation towards pH stability and thermalstability in GDHt based on a color reaction through a two-layer agar inthe ordinary Petri Dish. This method can greatly reduce the effect ofbackground, and this screening technique is successful.3 Directed evolution of glycerol dehydratase (irrational design)(1) Family shuffling was carried out using five genes encoding glyceroldehydratase based on the integrative fragment methods using DNaseI andrestriction enzymes. Though mutation enzyme having excellent propertyhad not been obtained, sequence analysis showed the gene of mutationenzyme was recombined greatly by this family shuffling method. Thisresearch is beneficial to the advanced study of glycerol dehydratasefamily shuffling.(2) The gene of glycerol dehydratase from K. pneumoniae was directedevolved by error-prone PCR, and two mutations (GDHtKP5-2 andGDHtKP6-1) whose properties improved obviously were obtained. The half-times of GDHtKP6-1 at pH6.0 and 45℃were 16 and 6 times morethan those of GDHtKP. The 3D structure of wild and mutation enzymeswere successful constructed by homology modeling, furthermore, therelations between 3D structures and enzymes properties were studiedprimarily.4 Rational design of glycerol dehydratase(1) The genes encodingα,βandγsubunits of C. freundii and K.pneumoniae and metagenome were cloned, respectively, and sixheteroenzymes were obtained by swapping the a subunit genes of thethree glycerol dehydratase based on the rational design of energyminimization. The pH and thermal stability and Vrnax of someheteroenzymes were improved, and some showed no activity. The acidstability of GDHt(CK) and GDHt(KC) were improved in some degree, atthe same time, the hetheroenzyme GDHt(KU) containedβ-γsubunit ofthe no activity metagenome glycerol dehydratase obtained functional andhighly stable. Furthermore, the 3D structures of three glyceroldehydratase and six heteroenzymes were constructed by homologymodeling, and the effect of 3D structure on the enzyme property wasanalyzed primarily.(2) The genes encoding glycerol dehydratase of K. pneumoniae wassite-mutated through PoPMusic rational design. The pH and thermalstability and V_max of mutational enzyme were improved, the pH stability of GDHtKP525 was improved about 1.5～2 times; and the alkalinestability of GDHtKP60 was improved too. Furthermore, the 3D structuresof wild and mutational enzymes were successfully constructed byhomology modeling, and the effect of 3D structure on the enzymeproperty was analyzed primarily.(3) The genes encoding glycerol dehydratase of C. freundii wassaturation-mutated through ProSa rational design. The properties of somemutational enzymes were improved. The Km values of mutation 283-2-1and 283-3-3 increased, especially, the Km value of 283-2-1 was about 2～3times than that of GDHtCF. On the contrary, the Km value of mutational297-6-12 was decreased obviously, and was about 1/3～5 of that of wildenzyme. All properties of mutational enzyme 297-6-11 did not changedexcept the V_max value, which was about 2～3 times than that of wildenzyme. However, the properties of mutations about siteα222 were notimproved ever. Furthermore, the 3D structures of wild and mutationalenzymes were successfully constructed by homology modeling, and theeffect of 3D structure on the enzyme property was analyzed primarily.To our knowledge, this article is the first systematic study toimprove the property of glycerol dehydratase successfully by rational andirrational design evolution. This study is beneficial to understanding therelationship between 3D structures and properties of glycerol dehydratase,as well as the researches of producing 1,3-propanediol highly by the gene engineering strain.