Metabolic Engineering Of Escherichia Coli LS02T For High Production Of Riboflavin And Flavin Coenzymes

Riboflavin and its active form coenzymes,flavin mononucleotide(FMN)and flavin adenine dinucleotide(FAD),are widely used in feed,food and pharmaceutical industries.This dissertation mainly involves the metabolic engineering of Escherichia coli LS02 T for high-level production of riboflavin and the two flavin coenzymes.First of all,based on the riboflavin-producing strain LS02 T,the influence of the following rational metabolic engineering strategies on the production of riboflavin was studied: First,the modification of the central metabolic pathway.The fbp gene of LS02 T was overexpressed by replacing the promoter with a strong one,resulting in LS31 T.The fermentation results showed that the riboflavin production of LS31 T was almost the same as that of LS02 T and the intracellular concentration of NADPH was increased;Second,the enhancement of the supply of GTP,which was an important precursor for riboflavin synthesis.The negative regulation of Pur R protein on the expression of purine genes is relieved by knocking out the pur R gene.And several purine pathway genes(prs,pur F,gua A,gua B,gmk.ndk)were independently/combinedly overexpressed to enhance the flux of the purine pathway.Among the strains with single gene overexpression,LS33T(overexpressed prs gene)possessed the highest riboflavin production,reaching 663.2 mg/L,which was 7.8% higher than that of the control strain.As for the mutants with multi-gene overexpression,the riboflavin production of LS43T(overexpressed the prs-pur F-gmk-ndk gene)reached 729.7 mg/L,which was the highest of this set of mutants and 18.6% higher than that of the control strain;Third,reducing power rebalance.The availability of NADH was enhanced by overexpressing the sth A gene and knocking out the pnt AB gene and the riboflavin production of the optimal strain LS54 T reached 861.3 mg/L,which was 44.4% higher than the control strain;Four,the modification of the respiratory chain.The inefficient respiratory chain was eliminated by knocking out the ndh and app B genes and the riboflavin production of the optimal strain reached 647.9 mg/L,which was 8.6% higher than that of the control strain;Five,weakening the competitive pathway.Fine-tuning the expression of pur A,rib F,and gua C genes were accomplished by the replacement of the ribosome binding site(RBS)and the optimal mutant LS67T(gua C-DR0.5)with the RBS replacement of the gua C gene produced 658.5 mg/L riboflavin,which was 10.6%higher than the control strain.The modifications beneficial for the improvement of riboflavin production were further combined,and the riboflavin yield of the optimal strain LS72 T in shake flask fermentation reached 1.3 g/L.The riboflavin production of LS72 T in a 5 L fermentor reached 21 g/L after 58 h fementation with a yield of 110 mg riboflavin/g glucose.In order to further efficiently convert riboflavin into flavin coenzymes FMN and FAD,the plasmid p5C-RF-rib F was constructed to co-overexpress the riboflavin operon and the rib F gene(encoding bifunctional riboflavin kinase/FMN adenyltransferase,RFK/FMNAT)and then it was transformed into strain LS02,resulting in EF4 strain.The riboflavin,FMN and FAD production of EF4 reached 160.4 mg/L,145 mg/L and239 mg/L,respectively.Then,to further increase the production of FMN and FAD and their proportion in total flavin,the modular engineering method was launched.The riboflavin operon(module 1)and rib F gene(module 2)was modularly expressed.Various dosages of module expression were achieved through chromosomal integration and expression plasmids with different copy numbers.The gene dosage of module 1and module 2 was between 2-20 and 5-20 respectively.Among this set of mutants,EF7(Module 1: Module 2 = 5:20)performed the best and its production of FAD,FMN and riboflavin in shake flask fermentation reached respectively 324.1,171.6 and 83.7 mg/L and the conversion rate of riboflavin reached 78%.Then,EF7 strain was fed-batch cultured in a 5 L fermentor and the FAD titer reached 1.8 g/L,which was the highest level of FAD produced by microbial fermentation at present.At the same time,there was 0.87 g/L of FMN and 0.6 g/L of riboflavin in the fermentation broth.Then,the bifunctional enzyme RFK/FMNAT was modified.Several FMNAT inactivated mutant strains were screened out through the error-prone PCR method.These mutants could overproduce riboflavin and FMN without accumulation of FAD in the culture broth.Next,the mutated rib F gene was expressed according to the optimal module ratio(module 1: module 2 = 5:20),and the resulting optimal strain EF12 produced 586.1mg/L of FMN and 194.2 mg/L of riboflavin,without accumulation of FAD in shake flask fermentation and the cr-RF value was more than 71%.The FMN production of EF12 in the 5 L fermentor reached 1017.5 mg/LThe research results of this dissertation have significantly improved the production and yield of riboflavin,and provided a highly competitive producer for the current industrial production of riboflavin.In addition,E.coli strain with high production of FMN and FAD was constructed on the basis of riboflavin overproducing strain,which which laid a preliminary foundation for low-cost production of the two coenzymes through fermentation.