Screening And Regulation Mechanism Of Two-component Systems Of Agrobacterium Related To Curdlan Biosynthesis

Curdlan is a linear neutral extracellular polysaccharide linked byβ-（1,3）-glycosidic bonds produced by Agrobacterium sp.under nitrogen-limited conditions.Curdlan is colorless and odorless,and has unique rheological and water retention properties,so it is often used as a food additive in the food industry.Curdlan also has anti-tumor,anti-inflammatory and other immune activities,so it has a promising future in the pharmaceutical field.At present,the mechanism of Curdlan metabolic pathway and the two-component systems Ntr BC regulating Curdlan biosynthesis through sensing nitrogen limiting signals are well understood,but whether other two-component systems can regulate Curdlan is not well known.In this study,new genes of two-component systems related to the Curdlan synthesis were screened and knockout.Analysing the metabolic curve,transcriptome and metabolomics,then construction of complementary strain and overexpression strain to explore the mechanism of two-component systems’genes which is related to Curdlan synthesis.The main conclusions are shown as follows:（1）In this study,based on the whole genome of Agrobacterium sp.CGMCC 11546and the transcriptome data during the growth phase and Curdlan production phase,we screened 11 pairs of candidate genes of two-component systems which is related to Curdlan synthesis.The knockout strainΔgene0716-gene0717 showed the most significant decrease in Curdlan yield.The gel strength,fracture displacement and molecular weight of Curdlan produced by knockout mutantΔgene0716-gene0717 were32.33%,22.98%and 49.99%lower than those of the wild strain,respectively.The results of both FTIR spectrum and ¹³C NMR spectrum of both of them were similar and consistent with the previous reports,indicating that knockout of gene0716-gene0717didn’t affect the Curdlan’s molecular structure.（2）In this study,the knockout strainΔgene0717 andΔgene0716 were constructed to reveal their effects on the Curdlan synthesis,respectively.The metabolic curves showed that the p H changes and nitrogen utilization between the wild strain and the knockout strains have no difference.When Curdlan begin to pruduce,both living cells number and sucrose utilization of knockout strainΔgene0716 andΔgene0716-gene0717 are lower than those of the wild strain.It results in a decrease in Curdlan yield.Compared with the wild strain,the knockout strainΔgene0716 showed a greater decrease in Curdlan yield than the knockout strainΔgene0717.It probably because the response regulator Gene0716 was activated not only by the sensor Gene0717,but also by other histidine kinase sensors.The soluble polysaccharide content of the knockout strains was reduced but not significantly different from that of the wild strain,suggesting that the reduced yield of Curdlan in the knockout strain was not caused by an increased production on soluble polysaccharide.（3）Transcriptome results showed that there were 70 significant expressed down-regulated genes inΔgene0716-gene0717 compared with the wild strain,and four of them were related to energy synthesis pathway.There are no up-regulated genes.It was speculated that some of the genes related to energy synthesis were affected by the deletion of gene0716-gene0717,thus the knockout strain had impaired energy synthesis.This resulted in a lower yield of Curdlan.Metabolomic results showed a significant decrease in glucose-6-P content,indicating that the ability of cells to uptake and utilize of carbon sources decreased.It is also led to a decrease in the flow of glucose-6-P to the glucose synthesis and TCA cycle and then a decrease in the synthesis of the glucose precursor UDP-glucose and ATP,resulting in a lack of both substrate and energy supply for Curdlan synthesis.As a result,the production of Curdlan decreased.The ATP content of knockout strainΔgene0716 andΔgene0716-gene0717 was lower 21.54%and 17.05%than wild strain,respectively.This result proved that the gene0716 affects energy synthesis.（4）In this study,we constructed complement strains and overexpression strains of two-component systems gene0716,gene0717 and gene0716-gene0717.Compared with the knockout strain,the Curdlan yield of complement strains were increased by 39.89%,24.74%and 51.97%,respectively,but were still lower than the wild strain.This indicates that the complement of genes of the two-component systems can only partially restore the Curdlan synthesis ability of the knockout strains.This may be due to the differences in the copy number and expression levels of the gene between the plasmid and chromosome,thus resulting in different Curdlan yields.when gene0716 gene overexpression was induced inΔgene0716 at 0 h,the yield of Curdlan induced at 0 h was basically the same as that of the wild strain,while the 18 h induction was much lower than that of the wild strain.Therefore,it is speculated that the gene0716 starts to function at the cell growth stage and continues to affect the synthesis of Curdlan.Whether at 0 h or 18 h gene0717 overexpression inΔgene0717,the yield of Curdlan in the knockout strain could not restore its ability to produce Curdlan.In contrast,when gene0717 overexpression in WT at 18 h,the yield of Curdlan was 19.65%higher than that of WT.The above results indicate that the transcription factor Gene0716 plays a major role in the two-component systems Gene0716-gene0717,but enhancing the expression of gene0717 can improve the yield of Curdlan in wild type.In conclusion,a novel two-component systems gene gene0716-gene0717 related to Curdlan was identified by genome-wide analysis,transcriptome analysis and gene knockout.Metabolic curve analysis,transcriptome and metabolome analysis were used to explore the mechanism of gene0716-gene0717 regulating Curdlan synthesis.The function of the gene0716-gene0717 was verified by gene complement and overexpression.The results of this study extend the understanding of the regulatory mechanism of Curdlan synthesis and provide a new theoretical basis for genetic modification of Curdlan producing strains.