Development of an oxygen-independent flavin mononucleotide-based fluorescent reporter system in Clostridium beijerinckii and its applications.

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Journal of biotechnology, ISSN: 1873-4863, Vol: 265, Page: 119-126

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Seo, Seung-Oh, Lu, Ting, Jin, Yong-Su, Blaschek, Hans P
Elsevier BV
Biochemistry, Genetics and Molecular Biology, Immunology and Microbiology
article description
Clostridium beijerinckii is a predominant solventogenic clostridia with great attraction for renewable liquid biofuel and biochemical production. Metabolic engineering and synthetic biology can be employed to engineer the strain toward desirable phenotypes. However, current limited information such as promoter strength and gene regulation may hinder the efficient engineering of strain. To investigate genetic information and complex cellular bioprocesses of C. beijerinckii, in vivo fluorescence reporter system can be employed. In general, green fluorescence protein (GFP) and relative analogs have been widely used as real-time reporters. However, GFP-family proteins require molecular oxygen for fluorescence maturation. Considering a strict anaerobic growth condition of clostridia, an oxygen-independent fluorescence reporter such as a flavin mononucleotide-based fluorescent protein (FbFP) can be used as an alternative fluorescence reporter. In this study, we synthesized and expressed the codon-optimized FbFP gene for C. beijerinckii (CbFbFP) based on the nucleotide sequence of Bacillus subtilis YtvA variant EcFbFP in C. beijerinckii NCIMB 8052 wild-type. The protein expression and in vivo fluorescence of CbFbFP in C. beijeirnckii were confirmed under an anaerobic growth condition. Through the fluorescence-activated cell sorting (FACS), we isolated the bright cells from the heterogenous population of the C. beijerinckii cells expressing the CbFbFP. Several mutations were found in the isolated plasmid which may be responsible for the high-level expression of CbFbFP in C. beijerinckii. The mutant plasmid and CbFbFP reporter were further applied for strain selection, real-time fluorescence measurement, population analysis, and metabolic engineering in this study.

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