Study Of Regulation Mechanism Of The Biosynthesis Of Amino Acids And Carbon Storages On Curdlan Biosynthesis In Agrobacterium Sp.CGMCC 11546

Curdlan is a neutral water-insoluble exopolysaccharide,which was produced mainly by Agrobacterium spp.under nitrogen-limiting conditions.Because of the unique rheological properties of curdlan,it has enormous potential and widespread applications in the food,biomedicine,construction and water purification industries.However,the molecular mechanism of curdlan biosynthesis and regulation is still not well understood.In this study,the knockout mutants involved in biosynthesis of glutamine,methionine and carbon storages were constructed by homologous recombination to explored the role of amino acid and carbon storages in curdlan biosynthesis in Agrobacterium sp.CGMCC 11546.The analysis of fermentation profiles,transcriptome and metabolomics were performed to clarify the molecular mechanism of that how amino acid and carbon storages influence curdlan biosynthesis.The main results and conclusions are shown as follows:(1)Glutamine synthetase gene glnA plays a vital role in curdlan biosynthesis.In this study,the analysis of curdlan fermentation profiles of?glnA was performed to explore the role of glutamine synthase gene glnA in Agrobacterium sp.CGMCC11546.After 96 h fermentation,the curdlan yield of?glnA mutant of Agrobacterium sp.CGMCC 11546 was decreased by 93.37%compared with that of the wild-type(WT)strain,indicating glnA plays a vital role in the curdlan biosynthesis.Analysis of fermentation profiles illustrated that cell growth and the utilization of sucrose and ammonia were impaired in the?glnA mutant.To further study the molecular mechanism of the effect of glnA on curdlan biosynthesis,transcriptome analysis of the?glnA mutant was conducted compared with the WT strain after 24 h of fermentation.Transcriptome analysis revealed that vatious of genes involved in sucrose transport system,heme biosynthesis and the electron transport chain(ETC),which are essential for carbon utilization and energy generation,were downregulated after 24 h fermentation in the?glnA mutant.Metabolomics analysis indicated flavin adenine dinucleotide(FAD)and adenosine diphosphate(ADP)accumulation in the?glnA mutant,suggesting insufficient energy supply in the?glnA mutant.The addition of dibasic sodium succinate,which was a transport substrate of FAD recyling,could promote the conversion of FAD and FADH₂ and donate electrons to the ETC,thereby partially restored curdlan production by increasing ATP generation.Furthermore,glnA overexpression showed an 18%increase in the curdlan yield of the?glnA mutant compared with that of the WT strain after 96 h fermentation.Taken together,glnA plays a vital role in curdlan biosynthesis through ATP supplying via regulating the expression of genes involved in heme biosynthesis and the ETC.(2)Methionine biosynthesis influence curdlan production.To explore the effect of methionine biosynthesis on curdlan production,?mdeA,?metA and?las I were constructed by the suicide vector p EXG2 in Agrobacterium sp.CGMCC 11546.After 96 h fermentation,analysis of fermentation profiles revealed that the curdlan yield of?mdeA,?metA,?metH and?metZ were significantly lower than that of the WT strain,which was decreasing by 78.92%,77.31%,60.38%,83.89%,respectively.Analysis of fermentation profiles showed that the consumption of ammonia and sucrose of four deletion mutants was impaired and the contents of ATP also decreased in the four deletion mutants.Because FAD and ADP were significantly accumulated in the?metH and?metZ mutants in our previous studies,the addition of a transport substrate of FAD recyling,dibasic sodium succinate,could restore the curdlan production by the four mutants,but did not reach the level of that observed in the WT strain.Furthermore,curdlan biosynthesis was impaired in the WT strain when ethyl potassium malonate was added as an inhibiter to inhibit the conversion of FAD and FADH₂,suggesting that FAD plays an important role in curdlan biosynthesis.Based on the previous study that methionine,glycine and aspartate were insufficient in the?metH and?metZ mutants showed by the metabolomics analysis,methionine,glycine and aspartate were added into the medium to explore the influence of them on curdlan biosynthesis.The addition of methionine,glycine and aspartate improved curdlan production in four mutants especially methionine.In conclusion,the methionine biosynthesis pathway influenced curdlan biosynthesis in Agrobacterium sp.CGMCC11546 by regulating ATP supply from the ETC.(3)Carbon storages have effect on curdlan biosynthesis.Transcriptome analysis of Agrobacterium sp.CGMCC 11546 after 7 h and 24 h curdlan fermentation revealed that genes involved in glycogen biosynthesis were up-regulated while poly-?-hydroxybutyrate(PHB)biosynthesis pathway were down-regulated.To investigate whether glycogen and PHB have effect on curdlan production,?glgC and?glgC-phbC deletion mutants were constructed.The analysis of fermentation profiles of?glgC,?phbC and?glgC-phbC mutants showed that the utilization of sucrose and ammonia were down-regulated,and the curdlan yield were decreased by 53.90%,95.01%and 89.31%respectively,indicating that glycogen and PHB play a vital role in curdlan production,especially PHB.To further study how phbC affects curdlan biosynthesis in Agrobacterium sp.CGMCC 11546,transcriptome and metabolomics analyses were conducted.Transcriptome and metabolomics analysis of?phbC revealed that sucrose metabolism changed significantly,indicating that PHB could affect sugar metabolism to regulate curdlan production.However,the regulation mechanism of how PHB affects the curdlan biosynthesis still unclear,needs to be further studied.In conclusion,this study explored how glutamine,methionine,glycogen and PHB affect curdlan biosynthesis in Agrobacterium sp.CGMCC 11546.These results made a deeper knowledge for the molecular mechanism of curdlan biosynthesis and regulation in Agrobacterium sp.CGMCC 11546,and provided molecular information for the genetic modification of CGMCC 11546.