| Poly-y-glutamic acid(y-PGA)is a natural multifunctional biopolymer,due to its various features such as cationic chelation,hygroscopicity,water solubility,non-toxicity and biodegradability,these properties have spawned applications in the areas of food,medicine,water treatment,chemicals and agriculture industries.Glutamic acid is served as the precursor for y-PGA synthesis,which is catalyzed by y-PGA synthase PgsBCAA.Recently years,several metabolic engineering strategies have been developed to improve y-PGA production,however,few researches were focused on the regulation of y-PGA synthesis,and no systematic research has been conducted to analyze the important roles of co-factor generation and energy engineering on the high-level production of y-PGA.The aim of this work is to in-depthly analyze the y-PGA synthesis regulation network in B.licheniformis,and improve y-PGA yield by co-factor regeneration and energy engineering.B.licheniformis WX-02 has been proven as a promising y-PGA producing strain.Firstly,the regulation mechanism of y-PGA synthesis by nitrogen metabolism regulator TnrA has been resolved.Then,regulation mechanism of anaerobic transcription factor Fnr on y-PGA synthesis was analyzed.Furthermore,NAPDH generation pathways were strengthened to improve the y-PGA yield.Finally,the effect of enhanced ATP supply on the synthesis of y-PGA was studied and the production of y-PGA was further increased by combinatorial metabolic engineering.The main conclusions are as follow:1.The metabolic mechanism of transcription factor TnrA regulating y-PGA synthesis in Bacillus licheniformisUnder the condictions of rich and poor nitrogen,through analyze the effects of nitrogen metabolism regulator tnrA deficiency and overexpression on cell growth,our results confirmed that regulator TnrA activate the expression of nitrogen utilization genes under the nitrogen poor condition.Then,the effects of tnrA deficiency and overexpression on y-PGA production were analyzed,and our results implied that TnrA repressed ?-PGA synthesis,and deficiency of tnrA led to a 22%increase of y-PGA production.Transcriptional level assay illustrated that the y-PGA synthase gene cluster pgsBCAA transcriptional level were increased in the tnrA deficient strain WX?tnrA,indicating that y-PGA synthase PgsBCAA was negatively regulated by TnrA.Furthermore,electrophoretic mobility shift assay(EMSA)and enzyme expression assays confirmed that TnrA directly repressed pgsBCAA expression,and the TnrA-binding site "CGTCGTCTTCTGTTACA" in the pgsBCAA promoter was identified by sequence and software analysis.Finally,it was confirmed that the regulation of ?-PGA synthase PgsBCAA by TnrA were highly conserved in other well-studied Bacillus species.Collectively,our results confirmed the important role of TnrA on nitrogen metabolism,and resolved the regulatory mechanism of TnrA on y-PGA synthesis and a new approach that deficiency of tnrA increases y-PGA production.2.The metabolic mechanism of transcription factor Fnr regulating y-PGA synthesis in Bacillus licheniformisFirst,the gene fnr was knocked out in B.licheniformis WX-02,and the y-PGA yields have no significant differences between WX-02 and the fnr deficient strain WX?fnr in the medium without nitrate(BFC medium).However,the y-PGA yield of 8.95 g/L was produced by WXAfnr in the medium with nitrate addition(BFCN medium),decreased by 74%compared to WX-02(34.53 g/L).Then,the fnr complementation strain WX?fnr/pHY-fnr restored the y-PGA synthesis capability,and?-PGA yield was increased by 13%in the Fnr overexpression strain WX/pHY-fnr(39.96 g/L)in BFCN medium,compared to WX/pHY300(35.41 g/L).Furthermore,the transcriptional levels of narK,narG,hmp were increased by 5.41-,4.93-,3.93-fold in WX/pHY-fnr respectively,which led to the increases of nitrate consumption rate and ATP supply for y-PGA synthesis.Collectively,Fnr affects ?-PGA synthesis mainly through manipulating the expression level of nitrate metabolism,and this study provides a novel strategy to improve y-PGA production by overexpression of Fnr.3.Strengthening of NAPDH generation pathway for high-level production of y-PGA in B.licheniformis WX-02NADPH genernation plays an important role in the anabolism of target products.In this research,six genes(Zwf,Gnd,Ghd,PntAB,UdhA and PpnK)encoding the key enzymes in NADPH generation were over-expressed in the y-PGA producing strain B.licheniformis WX-02.Among various recombinants,the strain over-expressing zwf gene(coding for glucose-6-phosphate dehydrogenase),WX/pHY-zwf,produced the highest?-PGA concentration(9.13 g/L),35%improvement compared to the control strain WX-pHY300.However,the growth rates and glucose uptake rates of the mutant WX/pHY-zwf were decreased.The Zwf activity of the zwf over-expression strain increased by 9.28-fold,which led to the improvement of the NADPH generation,and decrease of accumulation of by-products acetoin and 2,3-butanediol.Collectively,these results demonstrated that NADPH generation via over-expression of Zwf is as an effective strategy to improve the y-PGA production in B.licheniformis.4.Enhanced production of Poly-y-glutamic acid by improving ATP supply in metabolically engineered Bacillus licheniformisATP supply plays a vital role in the y-PGA biosynthesis,and the respiration chain was optimized and the Vitreoscilla hemoglobin(VHB),ATP-biosynthetic and nitrate metabolism pathways were strengthened to improve the ATP supply for?-PGA production in this research.(1)Through engineering the respiration chain branches of B.licheniformis,our results confirmed that the elimination of cytochrome bd oxidase branch reduced the maintenance coefficient by 24%,and leading to 22%increase of ATP supply and 19%increase of y-PGA yield.(2)The second approach is to introduce VHB to recombinant B.licheniformis,and ATP concentration and y-PGA yield was increased by 15%and 13%,respectively.(3)In the third approach,the genes purB and adK in ATP-biosynthetic pathway were respectively overexpressed,with the AdK overexpressed strain increased ATP supply and y-PGA yield by 28%and 15%,respectively.(4)Our study also confirmed that the respiratory nitrate reductase,NarGHIJ,is responsible for the conversion of nitrate to nitrite,and assimilatory nitrate reductase NasBC is for conversion of nitrite to ammonia.Both NarGHIJ and NasBC were positively regulated by the two-component system ResD-ResE,and overexpression of NarG,NasC and ResD also improved the ATP supply and the consequent y-PGA yield.Based on the above individual methods,a method of combining the deletion of cydBC gene and overexpression of vgB,adK and resD genes were used to enhance ATP content of the cells to 3.53 ?mol/g DCW,the mutant WX-BCVAR with this enhancement produced 43.81 g/L y-PGA,a 39%improvement compared to wild type strain WX-02.Collectively,our results demonstrate that improving ATP content in B.licheniformis is an efficient strategy to improve y-PGA production. |
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