| Curdlan is a kind of water-insoluble extracellular polysaccharide produced mainly by Agrobacterium strains.Curdlan has unique gel properties,and it is widely used in food industry as thickener,stabilizer and coagulant.It was found that crd R and crd ASC genes of Agrobacterium strains were associated with curdlan synthesis.crd R is a positive regulator of curdlan synthesis.Curdlan synthase catalytic subunit is encoded by crd S,and it is responsible for the transfer of glucose residues from the precursor UDP-glucose to the polysaccharide polymer chain during curdlan synthesis.At present,there are few reports on the regulation mechanism of curdlan synthesis.In order to further improve the understanding of the metabolic regulatory network of curdlan synthesis in Agrobacterium strains.The transposon mutation library of Agrobacterium sp.CA03 was constructed in this study.The genes involved in curdlan synthesis were obtained through the high throughput screening and those genes of mutant strains were identified.Then explore the molecular mechanism of curdlan synthesis in which they are involved were performed.Agrobacterium sp.CA03 was used as recipient and SM10?pir/p SC189 as donor in this study.The ransposon mutation library of CA03 strain was constructed after plasmid p SC189 introduced into CA03 strain.The results showed that the conjugation transfer efficiency was the highest(3.3×10-3)when the ratio of recipient to donor was4:1.The transposon mutation library containing about 3000 mutant strains after screening transconjugants with streptomycin and kanamycin.The transposon insertion sites of nine mutants randomly selected from the mutant library were identified by TAIL-PCR and sequecing.The results showed that the transposon was successfully inserted into the chromosome DNA of Agrobacterium sp.CA03,and the mutation site was random.These results demonstrated that the transposon mutation library has been successfully constructed and can be used to screen genes invovled in curdlan synthesis.The mutants invovled in curdlan synthesis were obtained by high throughput screening of Agrobacterium sp.CA03 transposon mutation library.Then the mutant genes of the mutant strains were identified.After the primary screening of 1380 strains,183 mutants and fifteen mutants were obtained with yield reduction of more than 70%and yield increase of more than 10%respectively.Through the identification and functional analysis of the mutant genes of the mutant strains,it was found that the mutant genes of low yield strains were phb C,gln A,thi G,dks A,pur F,nus A,pp K,met H and met Z.They are mainly involved in cysteine and methionine metabolism,butanoate metabolism,oxidative phosphorylation,nitrogen metabolism,purine metabolism,thiamine metabolism and other metabolic pathways.The mutant genes of high yield strains were Atu3059 and At1D1460-21360,Atu3059 gene encoding Na+/H+antiporter and At1D1460-21360 gene encoding hypothetical proteins.Then we further explored the effects of met Z and met H genes involved in cysteine and methionine metabolism on curdlan synthesis.Firstly,met Z and met H genes knockout strains and complementary strains of Agrobacterium sp.CA03 were constructed.The curdlan production of gene knockout strains and complementary strains were verified by shaking flask fermentation.The results showed that the crude yield of curdlan production of ?met Z was 4.57 g/L,which was 89.09%lower than that of the wild type strain.The crude yield of curdlan production of complementary strain ?met Z::met Z was 22.87 g/L,which was five times higher than that of strain ?met Z.The crude yield of curdlan production of?met H strain was 23.56 g/L,which was 43.77%lower than that of the wild type strain.The crude yield of curdlan production of?met H::met H strain was 34.65 g/L,which was 47.07%higher than that of?met H strain.In addition,it was found that the structure of curdlan produced by?met H was the same as that produced by the wild type strain,but the gel strength and molecular weight of curdlan produced by?met H strain were higher than those of the wild type strain.Compared with the wild type strain,there was no significant difference in growth of ?met Z and?met H,which indicated that the decrease of curdlan production of gene knockout strains was not caused by defective growth.The wild type strain and two complementary strains utilized ammonia nitrogen within 24 h,?met H strain consumed all ammonia nitrogen within 36 h,while ?met Z strain still had some ammonia nitrogen surplus after fermentation 96 h.In addition,the sucrose utilization rate of?met H and ?met Z was 60.08%and 24.63% after 96 h fermentation,respectively,while that of the wild type strain was 79.8%.The results showed that the utilization rates of ammonia nitrogen and sucrose of ?met Z and?met H strains decreased,which affected the synthesis of curdlan.We further analyzed how met Z and met H genes affect the metabolic regulation network of curdlan synthesis by transcriptomics and metabonomics.The results showed that 197 differentially expressed genes were identified in both two knockout strains compared with those of the wild type strain.These differentially expressed genes were mainly classified into oxidative phosphorylation,sulfur metabolism,cysteine and methionine metabolism,and valine,leucine and isoleucine degradation pathways.The knockout of met Z and met H genes had less effect on the gene expression of glucose metabolism pathway in the synthesis of curdlan by Agrobacterium,and the expression of many genes involved in the electron transport chain was down-regulated.The results showed that the regeneration of energy ATP in electron transfer chain was affected in ?met Z and?met H strains,and the energy supply was insufficient in the synthesis of curdlan,which led to the decrease of curdlan production.met H and met Z genes involved in cysteine and methionine metabolism.the expression of methionine synthase gene and methionine synthesis gene were down-regulated in ?met Z and?met H strains.The results showed that the knockout of met H and met Z genes affected the synthesis of methionine,and then affected the synthesis of methanethiol.It is speculated that methanethiol may be involved in the synthesis of curdlan.In addition,many genes involved in sugar ABC transporter are down-regulated in ?met Z strain,which may affect the sugar transport of ?met Z strain,and then affect the sugar utilization rate,and finally affect the synthesis of curdlan.The results were consistent with the decrease of sugar utilization rate of knockout strains in the metabolic curve analysis.Compared with the wild type strain, ?met Z and?met H strains had lower utilization rate of sugar,which led to the accumulation of carbohydrate metabolites.In addition,the accumulation of intermediate products in TCA cycle weakened the TCA cycle in ?met Z and?met H strains.At the same time,the accumulation of FAD indicates that the synthesis of FADH2 is blocked,which indicates that the reduction power is weakened in the electron transfer chain,which leads to the decrease of energy synthesis.In addition,the expression of ATP synthase in the electron transport chain was down-regulated,which weakened the conversion of ADP to ATP,and finally led to the accumulation of ADP.Therefore,it is speculated that the lack of energy supply of ATP may lead to a significant reduction in the synthesis of curdlan,resulting in the decrease of the yield of curdlan in knockout strains.In addition,it was found that L-methionine in ?met Z and?met H strains was significantly lower than that in wild type train by metabolome analysis of amino acid metabolism.The results showed that met Z and met H genes were involved in the synthesis and metabolism of L-methionine.At the same time,it was found that the content of C6-HSL in the fermentation broth of ?met Z strain decreased significantly.It has been reported that C6-HSL involved in the synthesis of extracellular polysaccharides.The results showed that met Z and met H genes affected the synthesis of quorum sensing signaling molecule C6-HSL by affecting the synthesis of L-methionine,and finally affected the synthesis of curdlan.These results suggest that the knockout of met Z and met H genes lead to the synthesis of curdlan is seriously defective.Suggest that met Z and met H genes contributed to the metabolic regulation of curdlan synthesis,and play an important role in the process of curdlan synthesis.In a word,the CA03 transposon mutation library of Agrobacterium was constructed,and eleven genes involved in curdlan synthesis were obtained by screening and identification of mutant strains.It was found that met Z and met H genes classified into cysteine and methionine metabolism played an important role in curdlan synthesis.After met Z and met H genes knockout,the utilization rate of ammonia nitrogen and sucrose decreased,sugar metabolism,energy synthesis and L-methionine synthesis were impaired.The substrate utilization and energy supply were insufficient in the process of curdlan synthesis,which eventually led to the decreased yield of curdlan production.In this subject,new genes involved in curdlan synthesis were obtained,and the molecular mechanism of curdlan synthesis which were involved in was studied.It improved our knowledge of curdlan metabolic regulation network in Agrobacterium.It provides theoretical support for increasing curdlan yield by molecular biological methods. |
PDF Full Text Request