Riboflavin,an important vitamin utilized in pharmaceutical products and as a feed additive,is mainly produced by metabolically engineered bacterial fermentation.However,the reliance on antibiotics in the production pr...Riboflavin,an important vitamin utilized in pharmaceutical products and as a feed additive,is mainly produced by metabolically engineered bacterial fermentation.However,the reliance on antibiotics in the production process leads to increased costs and safety risks.To address these challenges,an antibiotic-free Escherichia coli riboflavin producer was constructed using metabolic engineering approaches coupled with a novel plasmid stabilization system.Initially,competitive pathways and feedback inhibition were attenuated to enhance the metabolic flux towards riboflavin.Key genes in the purine pathway were overexpressed to boost the availability of riboflavin precursors.Subsequently,a plasmid stabilization system based on toxin was screened and characterized,achieving a plasmid retention rate of 84.9%after 10 days of passaging.Finally,transcriptomic analysis at the genome-wide level revealed several rate-limiting genes,including pgl,gnd,and yigB,which were subsequently upregulated,leading to a 26%improvement in riboflavin production.With optimization of the culture medium,the final strain allowed the production of 11.5 g/L of riboflavin with a yield of 90.4 mg/g glucose in 5 L bioreactors without antibiotics.These strategies can be extended to other plasmid-based riboflavin derivative production systems.展开更多
The probiotic bacterium Escherichia coli Nissle 1917(EcN)holds significant promise for use in clinical and biological industries.However,the reliance on antibiotics to maintain plasmid-borne genes has overshadowed its...The probiotic bacterium Escherichia coli Nissle 1917(EcN)holds significant promise for use in clinical and biological industries.However,the reliance on antibiotics to maintain plasmid-borne genes has overshadowed its benefits.In this study,we addressed this issue by engineering the endogenous cryptic plasmids pMUT1 and pMUT2.The non-essential elements were removed to create more stable derivatives pMUT1NR△and pMUT2HBC△.Synthetic promoters by integrating binding motifs on sigma factors were further constructed and applied for expression of Bacteroides thetaiotaomicron heparinaseⅢand the biosynthesis of ectoine.Compared to traditional antibiotic-dependent expression systems,our newly constructed antibiotic-free expression systems offer considerable advantages for clinical and synthetic biology applications.展开更多
基金supported by the National Key Research and Devel-opment Program of China(No.2021YFC2100900,2022YFA0912200)the National Natural Science Foundation of China(No.32071470,32300063)+1 种基金the Natural Science Foundation of Jiangsu Province(No.BK20221080,No.BK20210464)the Research Program of State Key Laboratory of Food Science and Resources,Jiangnan University(No.SKLF-ZZB-202408).
文摘Riboflavin,an important vitamin utilized in pharmaceutical products and as a feed additive,is mainly produced by metabolically engineered bacterial fermentation.However,the reliance on antibiotics in the production process leads to increased costs and safety risks.To address these challenges,an antibiotic-free Escherichia coli riboflavin producer was constructed using metabolic engineering approaches coupled with a novel plasmid stabilization system.Initially,competitive pathways and feedback inhibition were attenuated to enhance the metabolic flux towards riboflavin.Key genes in the purine pathway were overexpressed to boost the availability of riboflavin precursors.Subsequently,a plasmid stabilization system based on toxin was screened and characterized,achieving a plasmid retention rate of 84.9%after 10 days of passaging.Finally,transcriptomic analysis at the genome-wide level revealed several rate-limiting genes,including pgl,gnd,and yigB,which were subsequently upregulated,leading to a 26%improvement in riboflavin production.With optimization of the culture medium,the final strain allowed the production of 11.5 g/L of riboflavin with a yield of 90.4 mg/g glucose in 5 L bioreactors without antibiotics.These strategies can be extended to other plasmid-based riboflavin derivative production systems.
基金financially supported by the National Natural Science Foundation of China(32370066,32000058)the Fundamental Research Funds for the Central Universities(JUSRP622003)+1 种基金National First-class Discipline Program of Light Industry Technology and Engineering(QGJC20230202)the Postgraduate Research&Practice Innovation Program of Jiangsu Province(KYCX23_2487).
文摘The probiotic bacterium Escherichia coli Nissle 1917(EcN)holds significant promise for use in clinical and biological industries.However,the reliance on antibiotics to maintain plasmid-borne genes has overshadowed its benefits.In this study,we addressed this issue by engineering the endogenous cryptic plasmids pMUT1 and pMUT2.The non-essential elements were removed to create more stable derivatives pMUT1NR△and pMUT2HBC△.Synthetic promoters by integrating binding motifs on sigma factors were further constructed and applied for expression of Bacteroides thetaiotaomicron heparinaseⅢand the biosynthesis of ectoine.Compared to traditional antibiotic-dependent expression systems,our newly constructed antibiotic-free expression systems offer considerable advantages for clinical and synthetic biology applications.
