Increased riboflavin production by knockout of 6-phosphofructokinase I and blocking the Entner–Doudoroff pathway in Escherichia coli
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To construct an Escherichia coli strain capable of producing riboflavin with high titer and yield.
A low copy number plasmid pLS01 containing a riboflavin operon under the control of a constitutive promoter was constructed and introduced into Escherichia coli MG1655. Subsequently, the pfkA, edd and ead genes were disrupted, and the resulting strain LS02T produced 667 mg riboflavin/l in MSY medium supplied with 10 g glucose/l in flask cultivation. In a fed-batch process, riboflavin production of the strain reached 10.4 g/l with a yield of 56.8 mg riboflavin/g glucose.
To our knowledge, this is the first report of engineered E. coli strains that can produce more than 10 g riboflavin/l in fed-batch cultivation, indicating that E. coli has potential for riboflavin production.
KeywordsEscherichia coli Fed-batch fermentation Metabolic engineering Riboflavin
The authors thank Dr. Thomas E. Kuhlman and Dr. Zhenquan Lin for kindly providing some of the plasmids used in this study. This work was supported by the National 973 Project (2012CB725203), and National High-tech R&D Program of China (2012AA022103, 2012AA02A702).
Supplementary Table 1—Primers used in this study.
Supplementary Figure 1—pLS01 map.
Supplementary Figure 2—Time profiles of cell growth, glucose consumption and riboflavin production of LS02T cultivated in LBG, M9 and MS media.