Functional Identification Of Superoxide Dismutases And Key Enzymes In The Gentisate Pathway From Haloferax Sp.D1227

Heterologous expression of a number of superoxide dismutases?SODs?from bacteria,yeast or plants have been reported to improve the salt tolerance of the host,but few from archaea.In particular,no SODs from archaea has been reported to be expressed in bacteria to improve their salt tolerance.Haloferax sp.D1227 is a haloarchaeal strain,from which no SOD has been reported.Previous studies indicated that this strain was able to grow on several aromatic acids and degrade them via gentisate pathway.Nevertheless,the complete pathway has not been reported at molecular level.The current study includes two parts.The first one is the study on the superoxide dismutases from strain D1227.?1?Two putative SOD encoding genes from strain D1227 were found and designated sodA and sodB.When sodA and sodB were heterologous expressed in both E.coli BL21?DE3?and environmental pollutant para-nitrophenol?PNP?utilizer Burkholderia sp.SJ98 respectively,they both exhibited SOD activity.For SodA,the specific activities were 21.07±0.02 U/mg and 84.56±0.16 U/mg in cell extracts of strains BL21[pET-28a-sodA]and SJ98[pBBR-sodA],respectively,while the specific activity of purified protein SodA_D1227 from E.coli was 179.46±3.43 U/mg.For SodB,the specific activities were32.69±0.08 U/mg and 125.90±0.07 U/mg in cell extracts of strains BL21[pET-28a-sodB]and SJ98[pBBR-sodB],respectively,while the purified SodB_D1227 showed specific activity of 73.24±0.76 U/mg.?2?Strain SJ98[pBBR-sodA]grew well in M9 containing 500 mmol/L NaCl with glucose as the carbon source,but its control strain SJ98[pBBR1MCS-2]almost lost its growth ability.When PNP was used as the carbon source,strain SJ98[pBBR-sodA]still had a normal growth and degradation ability with PNP,while this ability of strain SJ98[pBBR1MCS-2]was almost lost.?3?In the medium without NaCl or with 250 mmol/L NaCl,the recombinant strain SJ98[pBBR-sodB]was significantly superior to wild-type strain in both growth and degradation when 2-chloro-4-nitrophenol?2C4NP?was used as the carbon source.?4?In this study,according to the data of qPCR and analysis of enzyme activity,it was proved that the transcription of the catabolic gene pnpA was not enhanced by the expression of the exogenous superoxide dismutase SodB_D1227.?5?The structural analysis of SodA_D1227 and SodB_D1227 simulated by Phyre 2showed that they both have the typical structural characteristics of the Fe/Mn-SOD family.The second part is the study of the key enzymes involved in aromatic compounds catabolism via gentisate in strain D1227.Previous studies reported that strain D1227 was able to utilize several aromatic acids via gentisate pathway.In our lab,gentisate 1,2-dioxygenase?GDO?and maleylpyruvate hydrolase?MPH?in strain D1227 were functional identified.In this study:?1?Through bioinformatics analysis,a possible fumarylpyruvate hydrolase?FPH?HagE1,and two possible FPHs or maleylpyruvate isomerases?MPIs?HagE2 and HagE3 were found and respectively expressed in E.coli Rosetta and archaeal host Haloferax volcanii H1424.But unfortunately,they did not exhibit their corresponding enzyme activity.?2?According the previous reports of 3-hydroxybenzoic acid?3HBA?degradation via gentisate by bacteria,we initially presumed the transformation of 3HBA to gentisate was catalyzed by 3-hydroxybenzoate-6-monooxygenase?3HBA-6-MO?in archaeal strain D1227.In this study,three putative 3HBA-6-MOs HagH,HagX1 and HagX2 were found and expressed in the archaeal host H1424,but no activity was detected.Therefore,it is likely that the initial conversion of3HBA to gentisate was not catalyzed by monooxygenase in archaeal strain D1227,which may be different from those found in bacteria.?3?Three putative key enzymes,involved in conversion of 3HBA to gentisate in order,were putative 3-hydroxybenzoyl-coenzyme A?3HBA-CoA?ligase HagL,3HBA-CoA hydroxylase HagH and gentisate-CoA thioesterase HagT.The aforementioned three proteins,HagL,HagH and HagT,were all expressed into a single E.coli Rosetta.The results of the biotransformation showed that,after 9 hours incubation,the concentration of 3HBA decreased about 85%by resting cells of Rosetta[pET-28a-hagLHT].The cell extract of strain H1424 containing HagL was able to use six aromatic acids?benzoic acid,3-hydroxybenzoic acid,3-phenylpropionic acid,phenylacetic acid,4-hydroxybenzoic acid and cinnamic acid?as its substrates,and HagL probably is an aromatic acid-CoA ligase with a broad substrate range.?4?We found that strain D1227could use phenylacetic acid?PAA?for growth,which was not reported before.Furthermore,after a ten-day cultivation,the value of OD₆₀₀ of the bacteria was 5 folds of the initial one.This study provides a potential feasibility for the use of archaeal SODs in transforming bacteria to adapt to the degradation of organic pollutants in a high salinity environment.Meanwhile,it also provides preliminary evidences for us to further explore the molecular mechanism of aromatic acids degradation by archaea.