| As an carbohydrate,sugars provide important nutrients and energy for organisms.As a monosaccharide,glucose is the most widely used in industrial production.The transport and metabolism of glucose by cells restrict the growth of bacterial cells and the yield of target products,thereby affecting the cost and efficiency of industrial production.Rhodobacter sphaeroides is rich in a variety of nutritious medicinal ingredients and has a high potential for application in the fields of food and medicine,but the bacteria grows slowly in the glucose matrix.At present,there have been relevant researches on the glucose metabolism of Rhodobacter sphaeroides,but its transport mechanism is still unclear.In this paper,Rhodobacter sphaeroides 2.4.1 was used as the research object to elucidate the glucose transmembrane transport mechanism and increase the glucose metabolism rate.The main research results are as follows:(1)Search and functional prediction of glucose transmembrane transport related genes.By searching the whole genome sequence of Rhodobacter sphaeroides 2.4.1 in the NCBI database,two genes involved in glucose transmembrane transport were found:glk and fruA,encoding glucokinase(Glk)of non-phosphotransfer system and EIIBC of phosphotransfer system,respectively.(2)Functional characterization of the two genes glk and fruA were studied.From the phenotypic and shake flask fermentation analysis of the knockout strains ?glk,?fruA,?glk?fruA and wild-type,it turned out that,there was no significant difference between?fruA and wild type,while ?glk and ?glk?fruA showed weaker growth in the SMM plate with glucose as the sole carbon source.Shake flask fermentation results showed that wild-type grew faster in the early phase of logarithmic growth,while ?fruA grew fastest of all and the growth of ?glk and ?glk?fruA was significantly inhibited in the middle and later phases of logarithmic growth.The phenomenon indicated that the transmembrane transport of glucose from Rhodobacter sphaeroides required a combination of PTS and non-PTS systems,in which Glk played a key role in transmembrane transport.(3)Exploration of other transmembrane transport pathways of glucose.The mutant strain AglkAfruA could still grow slowly in the medium with glucose as the sole carbon source,suggesting that there were other transmembrane transport pathways.Bioinformatics analysis revealed that when glucose entered the periplasmic space of Rhodobacter sphaeroides 2.4.1,it was likely to produce gluconate through the action of glucose dehydrogenase(encoded by Rsp-2673)and then entered cells.The mutants ?Rsp-2673,?fru?ARsp-2673,?glk?fruA?Rsp-2673 and ?glk?Rsp-2673 were obtained through seamless knockout and after that the analysis of phenotype and shake flask fermentation were performed with wild-type strain.The results showed that the effect of this pathway on the glucose transmembrane transport of Rhodobacter sphaeroides was weak,with the?fruA?Rsp-2673 biomass being the largest.(4)Enhancement of glucose transmembrane transport and metabolism.The?fru?Rsp-2673 was used as the starting strain to construct the overexpression strains of the glk,edd,glkE.coli and galPE.coli genes.The results showed that overexpression of glk to strengthen the non-PTS system,the growth of the strain was significantly inhibited;overexpression of edd to strengthen the ED pathway,the glucose metabolism rate of the strain was increased but it grew slowly in the early stages;overexpression of galPE.coli to strengthen the transmembrane transport,the glucose metabolism rate of the strain wasgreatly increased and its biomass was larger.Finally,analysis of shake flask fermentation of?fru?ARsp-2673-GalP/OP showed that its biomass increased by about 70%and sugar consumption rate increased by about 65%compared with the wild type. |
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