Physiological Function Of Membrane Protein RHOGL009301 Involved In Transport Of Benzoate In Rhodococcus Sp.R04

Rhodococcus sp.R04 was isolated from oil-contami-nated soil in northern China.Strain R04 was able to biodegrade biphenyl and other aromatic compounds.Strain R04 degraded biphenyl through the 2,3-dioxygenase pathway and produced intermediate metabolites in this process,such as HOPDA,benzoate.These metabolites were excreted firstly,and then into the body.Transcriptase sequencing of R04 showed that six genes were up-regulated in the culture of biphenyl,and the highest expression gene was RHOGL009301.These genes were thought to encode membrane proteins,but the physiological function of RHOGL009301 was not clear.It was probable that RHOGL009301 was involved in transport.The first biodegrade step of aromatic compounds was transmembrane into the cell by transport proteins.The intermediate metabolites in this process also need the membrane protein to transmembrane in and out the cell.Benzoate was an important intermediate metabolite in biodegradation of various aromatic compounds.It was widely used in food,pharmaceutical,chemical,plastics and dyestuff industry.The research on microbial degradation and transport mechanism of benzoic acid has important effect on human survival and health.In this study,the deficient strain R04?MP was obtained by knocked out and the growth and metabolism of wild strain and deficient strain were compared to determine the relationship between the physiological function of the membrane protein and the transport of benzoate.The biomass of the deficient strain when grown on biphenyl and benzoate was significantly decreased,the growth rate was slowed down and the stationary phase was delayed.The final biomass of wild strain and deficient strain was significantly different with the increase of the concentration of biphenyl and benzoate.HPLC analysis showed that the deficient strain R04?MP could biodegrade biphenyl and the concentration of internal biphenyl did not change obviously.Therefore,the membrane protein was not involved in transport biphenyl.The external benzoate decreased slowly and the internal benzoate increased slowly when the deficient strain R04?MP grown on benzoate.The membrane protein was involved in transport benzoate.Genome sequencing of R04 proved that the genes of benzoate transport protein BenE and BenQ were found in R04 genome.The deficient strain R04?MP only reduced the transport of benzoate and slowed down the degradation rate of benzoate and biphenyl,but the metabolism of benzoate did not stop and strain R04 did not stop growing.The membrane protein RHOGL009301 of R04 was successful expressed in Rhodococcus erythropolis.The biomass of Re-9301 after induced was significantly increased when grown on biphenyl and benzoate.The expression of membrane protein RHOGL009301 increased the transport of internal and external benzoate and accelerated the degradation of benzoate.Benzoate was the intermediate metabolite of biphenyl degradation,so it also accelerated the degradation of biphenyl.The physiological function of the membrane protein RHOGL009301 is involved in transport of benzoate.The membrane protein is one of the transport proteins involved in transport of benzoate.The RHOGL009301 gene sequence is different and the protein sequence is only low similarity(only 13%)compared with the benzoate transport protein reported in the literature.We can conclude that the membrane protein is a novel benzoate transport protein.