Construction Of EP-bifido Pathway In E.coli For Efficient Production Of AcCoA Derivatives

The lower carbon conversion rate of natural metabolic mechanisms limits the economic benefits of the process of converting the substrate into the desired biological product.In the metabolic system of natural microorganisms,glucose is broken down into pyruvic acid by glycolysis such as the EMP pathway,ED pathway,and variants of these pathways.Losing one-third of the carbon atoms to form acetyl-CoA,limiting the theoretical limit of the carbon conversion to 66%.In many other metabolic pathways of microorganisms,such as the pentose phosphate pathway,the similar carbon loss problem is also widespread.To increase the carbon conversion rate,we combined the EMP pathway of E.coli,the PP pathway,and the bifidogenic pathway from B.adolescentis.We designed and constructed an artificial EP-bifido pathway that can use glucose as a substrate to generate some chemicals.The key to the design of EP-bifido is the utilization of the bifunctional phosphoketolase(Fxpk)from Bifidobacterium adolescentis,a bifunctional phosphoketolase Fxpk.Fxpk not only converse F6P into erythrose 4-phosphate(E4P)and acetyl phosphate(AcP),also catalyzes the cleavage of xylulose 5-phosphate(X5P)into glyceraldehyde-3-phosphate(G3P)and AcP.The resulting E4P and G3P can be completely converted to acetyl-CoA via a series of carbon atom rearrangements without carbon loss.Another key to this pathway is the use of diphosphatase,which provides a second irreversible driving force.In the oxidative part of the pentose phosphate pathway(PPP),One molecule of glucose turns into glucose 6-phosphate and undergoes a series of reactions to form ribulose 5-phosphate.,generating one molecule of CO2 and two molecules of NADPH.The pentose phosphate pathway is a highly efficient pathway of reducing power.Its reducing power can be used to production,and Ribulose-5-phosphate,which come form Pentose phosphate pathway,can be an intermediate portion of the carbon rearrangement.We first examined the physiological activity of the engineered bacteria containing the EP-bifido pathway and found that there were fewer byproducts than the control bacteria.The C13 metabolic flux analysis successfully demonstrated the difference between the EP-bifido pathway and control strains.The CO2 production rate of DH5α(pCDF)reached a maximum of 21 mL/h at 7 h,while the maximum CO2 production rate of DH5α.-EP(pFF)at 14 h was only 5.2 mL/h.The engineered bacteria are also relatively low in CO2 rates.We then demonstrated the effectiveness of the EP-bifido pathway via PHB,MVA,and fatty acid Production.Through the EP-bifido pathway,the carbon conversion rate of PHB increased from 26%to 63.7%;the fatty acid increased from 9.17%to 14.36%;and the MVA increased to 64.3%(96%of the maximum theoretical conversion rate).This strategy can be used for the biological production of compounds which takes acetyl-CoA as a precursor,and it can be used in other kinds of microorganisms.