Functional Study Of Corynebacterium Glutamicum ABC Transporter NCgl0909-NCgl0910

The ATP-binding cassette(ABC)transporters are kind of indispensable membrane proteins that can transport substances across cell membranes.In many bacteria,ABC transporters are involved in the absorption of nutrients,the secretion of bacterial toxins,and the pumping of antibiotics from cells.Several ABC transporters have been found to play an important role in the uptake and absorption of aromatic compounds by C.glutamicum.In the preliminary study,by analyzing the differences in transcriptome data between wild-type strain and engineering strain expressing exogenous proteins,an ABC transporter co-coded by NCgl0909 and NCgl0910 was discovered.Through bioinformatics analysis,we found this transporter had a high sequence similarity with ABC transporters that transport aromatic compounds in other species.According to conserved domain analysis,it is speculated that the gene NCgl0908 upstream of this transporter may encode a laccase capable of oxidizing phenols and aromatic compounds;the NCgl0909 gene may encode a nucleotide binding domain with the function of binding and hydrolyzing ATP;NCgl0910 gene encodes a transmembrane domain for substrate recognition and transport.The gene cluster composed of these three genes may be involved in the transportation and degradation of laccase substrate in C.glutamicum.Then,the transcription level of C.glutamicum under the induction of laccase substrates was detected by qRT-PCR,It was found that the transcription levels of NCgl0908 gene and NCgl0910 gene were up-regulated,which was up to 5.76 times and 3.93 times.In order to analyze the function of NCgl0909-NCgl0910 transporter,we cloned,expressed and purified NCgl0908 protein?NCgl0909 protein and NCgl0910 protein in E.coli respectively.The detection of ABTS as a substrate revealed that NCgl0908 protein showed laccase activity,indicating that NCgl0908 could decompose aromatic compounds in C.glutamicum.By Ultramicro ATPase assay kit,it was found that NCgl0909 protein showed ATPase activity,indicating that NCgl0909 may bind and hydrolyze ATP to provide energy for the transport of substances across the membrane.Through isothermal titration calorimetry(ITC),we found that NCgl0910 recombinant protein can bind to three typical laccase substrates(ABTS?SGZ?DMP),indicating that NCgl0910 can recognize specific substrates.Using liquid chromatography-mass spectrometry technology,it was found that the overexpression strain of NCgl0909 and NCgl0910 improved the ability of C.glutamicum to take up laccase substrates,indicating that this transporter can transport specific substrates.Finally,in order to explore the effects of these two genes on energy metabolism and exogenous protein expression,the growth characteristics,intracellular ATP content and EGFP expression of gene knockout?overexpression strains and wild-type strain were determined.The results showed that the NCgl0909 knockout strain could increase the intracellular ATP content and the expression of EGFP.This result is also consistent with the conclusion of the previous transcriptome data that NCgl0909 is related to energy metabolism.This reporter reveals that NCgl0909-NCgl0910 is a new ABC transporter transporting laccase substrates in C.glutamicum.The preliminary results obtained will provide a reference for exploring the mechanism of C.glutamicum involved in the transport of aromatic compounds,and also lays a theoretical foundation for the host transformation of C.glutamicum expression system.