Genetic Studies On Hyperthermophilic Thermotoga Spp.

The search for sustainable,and renewable sources of energy requires a collective effort to meet the future energy demands and to reduce the harmful effects of climate change.In this regard,biofuels derived from biomass such as lignocelluloses have been widely discussed as an efficient energy source in recent decades.The major bottleneck in lignocellulosic biomass utilization is the hydrolysis of different biomass constituents into fermentable sugars.The hydrolysis of lignocellulosic biomass by biological means has turned up as an eco-friendly,sustainable,and renewable method;in particular,the hyperthermostable organisms have unique potential for lignocellulosic biomass conversion because they can work at high process temperatures,and are often tolerant to harsh industrial conditions.The Thermotoga species can grow up to 90℃,and can produce an array of enzymes to degrade carbohydrate polymers,which makes them excellent candidates for efficient biomass degradation and also a source of hyperthermophilic enzymes.To broaden the large-scale industrial applications of hyperthermostable enzymes,numerous strategies have been employed to improve their yield and thermostability which includes optimization of the growth conditions,genetic and chemicals modifications,and development of efficient expression regulation systems.Besides,the efficient conversation of lignocellulosic biomass requires synergistic action of cellulases and hemicellulases(mannanases and xylanases).Previously,hemicellulases in particular mannanases did not receive much attention;as the pretreatment methods removed the hemicellulosic part of the biomass.However,with the development of green-pretreatment methods where the hemicellulosic part remains intact;commercial development of the hemicellulases has become an important goal for efficient utilization of LcB.In the current study,the high-level expression of the TM1752 gene which was annotated as cellulase gene encoding protein Cel5B;from Thermotoga maritima was attained in E.coli using heat shock ’pHsh’ vector.The recombinant protein was purified and biochemically characterized.Surprisingly,the purified enzyme exhibited specific activities of 416 and 215 U/mg on substrates galactomannan and carboxymethyl cellulose,which is the highest among hyperthermophilic mannanases.However,the putative enzyme did not show sequence homology with any of the previously reported mannanases and;therefore,the enzyme was identified as bifunctional mannanase and cellulase and renamed as Man/Cel5B.Interestingly,Co2+ions increased the enzyme activity of Man/Cel5B up to 200%,therefore the enzyme characterization was attained in the presence of 1 mM Co2+ions.Man/Cel5B exhibited maximum activity at 85℃ and pH 5.5;and depicted high thermal and pH stability as it retained more than 50%activity after 5 h of incubation at 85℃,and retained up to 80%activity after incubation for 1 h at pH 5-8.The Km and Vmax of Man/Cel5B were observed to be 4.5 mg/mL and 769 U/mg,respectively,for galactomannan.Thin layer chromatography depicted that the locust bean gum could be efficiently degraded to mannobiose,mannotriose,and other mannooligosaccharides by Man/Cel5B.Furthermore,the degradation of sawdust with Man/Cel5B resulted in the production of 80 μM of reducing sugars after 12 h treatment.All these characteristics together suggest that Man/Cel5B has attractive applications for future food,feed and biofuel industries.In order to be cost-competitive with fossil fuels,every effort should be made to make biomass degrading enzymes more efficient and more economically feasible.Besides relatively low stability and high cost of enzyme production,one of the major problems with soluble enzymes is their recovery from the reaction mixture after completion of the catalytic process;which makes the process very expensive.Immobilization of enzymes on/inside the solid matrix is an efficient technique to counteract these limitations.In this regard;Man/Cel5B was immobilized on the glutaraldehyde activated chitosan beads which resulted in 73.3%and 71.8%protein loading efficiency and immobilization yield respectively.The Fourier Transform Infrared Spectroscopy analysis confirmed the immobilization of Man/Cel5B on chitosan beads.The immobilized Man/Cel5B was biochemically characterized and the properties were compared with the soluble Man/Cel5B with no added Co2+ions.The optimum temperature of immobilized Man/Cel5B increased from 85℃ to 95℃;however,the optimum pH remained unchanged.Besides,the thermodynamics of locust bean gum hydrolysis by both free and immobilized Man/Cel5B was evaluated;which revealed a decrease in the activation energy of immobilized enzyme.Moreover,the thermostability of the immobilized enzyme increased to a great extent,the enzyme was able to retain>65%of its activity after 5 h of incubation at 85℃ compared to 55%of the free enzyme.At higher temperatures,the free enzyme lost activity more rapidly compared to the immobilized enzyme.The immobilized enzyme retained more than 60%and 50%of activity after 5 h of incubation at 90℃ and 95℃,respectively.However,the free enzyme could only retain 49 and 26%of initial enzyme activity after 5 h at 90℃ and 95℃,respectively.The immobilized enzyme could also be used 15 times and retained 54%of its activity.The Thermotoga spp.are great candidates for biomass degradation biofuel fermentation after a strain is genetically modified.To fully unlock the biotechnological potential of these organisms;genetic manipulation is a desirable approach.In the last part of this study,a gene knockout system for glms gene deletion in Thermotoga neapolitana was developed;using double cross over homologous recombination.A gene targeting suicide vector "pTs2" was constructed that contains a Chloramphenicol gene resistance cassette,a homologous recombinant region upstream and downstream of glms gene,and lacks the origin of replication for T.neapolitana.The plasmid was transformed into T.neapolitana using the ultrasonic sonoporation method.Different time intervals were tested,where exposure of the cells to ultrasonic sonoporation for 40 sec(at 40 kHz)was found to be the optimum time duration for transformation of DNA into T.neapolitana.Under positive selection the plasmid was found to integrate by first cross over homologous recombination in T.neapolitana genome downstream of the glms gene.The resulting merodiploid mutants were cultivated in the presence and absence of glucosamine to select for the colonies that underwent second cross over and lost the plasmid backbone together with glms gene.The second cross over mutants were able to grow only in the presence of glucosamine.The gene knockout system constructed in the current study will open the way to a systematic,genetic approach for the elucidation of unknown gene functions in T.neapolitana.This system can also be used for gene knockins to construct efficient biomass degrading Thermotoga strain.In conclusion,a novel bifunctional mannanase and cellulase was characterized from Thermotoga maritima that could effectively degrade locust bean gum into mannobiose,mannotriose and higher mannooligosaccharides.Besides,this enzyme could also degrade different agricultural wastes into fermentable sugars.Therefore,this enzyme has a great potential to be used in the biofuel and food industry.The immobilization of the Man/Cel5B could enhance the thermostability of the enzyme and also the enzyme was able to be reused which enhances the biotechnological application of the enzyme.Furthermore,a two-step homologous recombination method was constructed for the knock out of glms gene in Thermotoga neapolitana.This method can be used for the genetic manipulation of Thermotoga spp.in future for enhanced biomass degradation.Also,this method can also be used for the knock in of TM1752 gene encoding Man/Cel5B gene in Thermotoga neapolitana to enhance the biomass degradation capability of Thermotoga neapolitana.