Engineering Riboflavin Biosynthesis Pathway in <em>Escherichia coli</em> for Riboflavin Production

Related Articles

Abstract

A 3.5 kb rib operon of Bacillus subtilis 168 was amplified by PCR and cloned into different copy number of plasmids, including pSC101, p15A and pBR322. The resulting plasmids, pSC101-BSrib, p15A-BSrib and pBR322-BSrib, were transformed into Escherichia coli K-12 MG1655. The results showed that, in shaking-flask fermentation, the riboflavin titer was higher as the plasmid copy number increased. Then the fermentation conditions of E. coli K-12 MG1655 ECX3 were optimized. It was founded that the optimum culturing conditions for producing riboflavin were adding 0.1 mmol·L^-1 IPTG and incubating at 42 ℃. Under above condition, strain ECX3 produced 251.4 mg·L^-1 riboflavin in LB medium with 10 g·L^-1 glucose. Finally, we decreased bifunctional riboflavin kinase/FMN adenylyltransferase (encoded by ribF gene) activity to reduce the conversion of riboflavin to FMN and FAD by a scarless gene manipulation method. The final riboflavin production strain ECX4 produced 292.3 mg·L^-1 riboflavin in shaking-flask experiments.

	Service

	Email this article
	Add to my bookshelf
	Add to citation manager
	Email Alert
	RSS
	Articles by authors
	Xu Zhibo
	Lin Zhenquan
	Chen Tao

Keywords： riboflavin； Escherichia coli； Bacillus subtilis； rib operon； riboflavin kinase/FMN adenylyltransferase

Received 2014-04-01;

About author:

Cite this article:

Xu Zhibo, Lin Zhenquan, Chen Tao.Engineering Riboflavin Biosynthesis Pathway in Escherichia coli for Riboflavin Production[J] Chemcial Industry and Engineering, 2016,V33(2): 85-90

[1] Massey V. The chemical and biological versatility of riboflavin[J]. Biochem Soc Trans, 2000, 28(4): 283-296
[2] Koizumi S, Yonetani Y, Maruyama A, et al. Production of riboflavin by metabolically engineered Corynebacterium ammonia genes[J]. Appl Microbiol Biotechnol, 2000, 53(6): 674-679
[3] Stahmann K P, Revuelta J L, Seulberger H. Three biotechnical processes using Ashbya gossypii, Candida famata, or Bacillus subtilis compete with chemical riboflavin production[J]. Appl Microbiol Biotechnol, 2000, 53(5): 509-516
[4] Peberdy J F. Biology of industrial microorganisms[C]//Demain NAS Ed.. Biology of Penicillins. Menlo Park: Benjamin-Cummings, 1985
[5] Zhang F, Rodriguez S, Keasling J D. Metabolic engineering of microbial pathways for advanced biofuels production[J]. Curr Opin Biotechnol, 2011, 22(6): 775-783
[6] Bacher A, Eberhardt S, Fischer M, et al. Biosynthesis of vitamin b2 (riboflavin)[J]. Annu Rev Nutr, 2000, 20: 153-167
[7] Sklyarova S A, Kreneva R A, Perumov D A, et al. The characterization of internal promoters in the Bacillus subtilis riboflavin biosynthesis operon[J]. Genetika, 2012, 48(10): 967-974
[8] Mack M, van Loon A P, Hohmann H P. Regulation of riboflavin biosynthesis in Bacillus subtilis is affected by the activity of the flavokinase/flavin adenine dinucleotide synthetase encoded by ribC[J]. J Bacteriol, 1998, 180(4): 950-955
[9] Kuhlman T E, Cox E C. Site-Specific chromosomal integration of large synthetic constructs[J]. Nucleic Acids Res, 2010, 38(6): e92
[10] Goswami R S. Targeted gene replacement in fungi using a split-marker approach[J]. Methods Mol Biol, 2012, 835: 255-269
[11] Bandyopadhyay D, Chatterjee A K, Datta A G. Effect of cadmium, mercury and copper on partially purified hepatic flavokinase of rat[J]. Mol Cell Biochem, 1997,167(1/2): 73-80
[12] Bradford M M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[J]. Anal Biochem, 1976, 72: 248-254
[13] Hümbelin M, Griesser V, Keller T, et al. GTP cyclohydrolase II and 3,4-dihydroxy-2-butanone 4-phosphate synthase are rate-limiting enzymes in riboflavin synthesis of an industrial Bacillus subtilis strain used for riboflavin production[J]. J Ind Microbiol Biotechnol, 1999, 22(1): 1-7