Design Of PP-bifido Pathway And Its Application In Escherichia Coli And PHB Synthesis

Polyhydroxyalkanoate(PHA)is a type of biopolyeaster synthesized in vivo,PHA can be degraded in the bioactive environment,thus has an extensive application value,and can satisfy people's different requirement of chemicals and primary industries,thereby PHA has a broad application in various domain such as healing drug and agriculture.Poly-3-hydrobutyrate(PHB)is a typical example of PHA.The synthesis of PHB take AcCoA as a precursor and using NADPH to provide reducing power.The general producing process of AcCoA is:glucose generate pyruvate through EMP and pyruvate decarboxylation to AcCoA under the catalyze of pyruvate dehydrogenase complex,but with the decarboxylation of pyruvate it will release CO2,thus led to a waste of carbon source.Our experiment innovatively introduced the f/xpk gene of bifido pathway[1]to synthesize PHB,which can catalyze F6P to form AcP and E4P,the formed E4P can regenerate AcP after the carbon rearrangement process,thus 1mol F6P can genarate 3mol AcCoA without the loss of carbon source,the formed AcP can genarate AcCoA under the catalyze of pta gene to form PHB.But NOG pathway cannot provide the NADPH needed by the synthesis of PHB,we analyzed that the oxidation part of PPP can use lmol glucose to produce 2mol NADPH and generate X5P,the f/xpk gene of NOG pathway have both F6P and X5P activity,it can also catalyze X5P to form AcP and G3P,the formed G3P can generate AcP with the carbon rearrangment,all the process will release only lmol CO2 thus save the carbon source in a certain extent.Through the introduction of f/xpk gene and proper metabolic modification,we constructed the PP-bifido pathway,the metabolic flux through PP-bifido pathway have two directions:one directly generate AcCoA to synthesize PHB under the catalyze of F/Xpk,this way can reach the total save of carbon source;The other one influx to the PPP to generate NADPH for PHB synthesis,and then switch to F/Xpk to partly make a reduced waste of glucose.When the metabolic flux of the two pathway reached a balanced state,it can save carbon source in the greatest extent and not influence PHB synthesis.Thus made the theoretical yield of PHB synthesis increased from 48%to 64%.This article constructed f/xpk and fbp genes to plasmid pCDFtrc which are the coding genes of two irreversible reaction of PP-bifido pathway and transformed it together with PHB producing plasmid pBHR68 to E.coli DH5a.Flask fermentation was shaken at 37? 120rpm,the result shows that the introduction of F/Xpk made the PHB conversion rate increased 1.4 times,reached 30.8%,and successfully proved the introduction of F/Xpk.The PP-bifido pathway needs PPP to provide reducing power to synthesize PHB,but the main glycometabolism of E.coli is EMP,only a small part influx to the PPP pathway,meanwhile,EMP will compete with F/Xpk for the substrate F6P,thus led to the reduced glucose utilization ratio,so we knocked down the major gene pfkA of EMP pathway to redirect metabolic flux and relieve substrate competition.Moreover,in the oxidation section of PPP pathway,intermediate product 6PG will enter into ED pathway to produce G3P and then decarboxylate to AcCoA with CO2 release,so we knocked down the edd gene of ED pathway.With aforementioned metabolic engineering,the PHB conversion rate of obtained DH5aAedd?pfkA/pFF&pBHR68 strain reached 37.5%.Through metabolic engineering of concerned pathway,PHB conversion rate has enhanced further.But we found that when glucose transport through PTS pathway,it will relay phosphoryl group from PEP and genarate pyruvate for glucose phosphprylation,the generated pyruvate will decarbonxylate to AcCoA accompany with release of CO2 thus led to a waste of source.While the phosphoenolpyruvate-independent transport system uses ATP as phosphate donor instead of PEP and will save carbon source.So we knocked out ptsG of PTS pathway to redirect glucose to PEP-independent glucose transport systerm,but inactivition of PTS led to a reduced glucose transport rate thus made a lower growth rate and PHB content.To solve this problem,we modified galP and glk genes of PTS,but how to made expression level of the two genes to a appropriate strength is a tough problem we met.With consulting lots of reference,we found that through modification the RBS of the two genes with MAGE[2]in combination with proper screening method and fermentation examination,we can screen out the strain with a recovered glucose utilizaton rate.RBS of the two genes are designed to be close to the standard SD sequence,transform these synthetic ssDNA with several rounds of electric shock transformation to recombination.Due to a recovered glucose utilization rate will led to a enhanced PHB content and PHB can combine with Nile red dye to show a red colour,the higher PHB content is,the deeper will red colour be.According to that,we have screened out a strain DH5a?edd?pfkA?ptsGGalP,which has a recovered PHB content,only the RBS of galP has changed.With transformation of pFF and pBHR68 plasmids and optimization of fermentation conditions,the strain has a highest PHB conversion rate of 62.2%at 37??150rpm with a baffled flask,2.9 times higher than the control strain without F/Xpk.The innovation point of our experiment lies in the enhancement of glucose utilization rate in PHB production,through innovatively introduction F/Xpk and design of PP-bifido pathway,we saved the carbon source which have been wasted in the traditional PHB production process,thus reduced the production cost.After overexpression of concerned genes,block related competition pathway to redirect metabolic flux and modify glucose transport systerm,the PHB conversion rate enhanced from 21.8%to 62.2%finally.