title: Flavin mononucleotide (FMN)-based fluorescent protein (FbFP) as reporter for promoter screening in Clostridium cellulolyticum.
abstract: Conventional methods for screening promoters in anaerobic bacteria are generally based on detection of enzymatic reactions and thus usually complicated or strain specific. Therefore a more efficient and universal method will be valuable. Here, using cellulolytic bacteria Clostridium cellulolyticum H10 as a model, we employed an oxygen-independent flavin-based fluorescent protein (FbFP) derived from Pseudomonas putida as a quantitative reporter for the screening of promoter via monitoring fluorescence intensity. The stability and reliability of FbFP fluorescence were proven by the high correlation (R(2)=0.87) between fluorescence intensity and abundance of FbFP. Moreover, two endogenous promoters with exceptional performance were identified and characterized, including a constitutive promoter p3398 and an inducible promoter p1133. Compared to the existing reporter systems widely used in clostridia, this FbFP-based method is more rapid, intuitive and versatile, and the endogenous promoters reported here should enrich the synthetic biology toolbox for this and related organisms.
authors.affiliation: Single-Cell Center, CAS Key Laboratory of Biofuels, Shandong Key Laboratory of Energy Genetics, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, Shandong 266101, China; University of Chinese Academy of Sciences, Beijing 100049, China.
authors.forename: Lin
authors.initials: L
authors.lastname: Teng
authors.affiliation: Key Laboratory of Horticulture Science for Southern Mountainous Region, Ministry of Education, College of Horticulture and Landscape Architecture, Southwest University, Chongqing 400716, China.
authors.forename: Kun
authors.initials: K
authors.lastname: Wang
authors.affiliation: Single-Cell Center, CAS Key Laboratory of Biofuels, Shandong Key Laboratory of Energy Genetics, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, Shandong 266101, China. Electronic address: xujian@qibebt.ac.cn.
authors.forename: Jian
authors.initials: J
authors.lastname: Xu
authors.affiliation: Single-Cell Center, CAS Key Laboratory of Biofuels, Shandong Key Laboratory of Energy Genetics, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, Shandong 266101, China. Electronic address: xucg@qibebt.ac.cn.
authors.forename: Chenggang
authors.initials: C
authors.lastname: Xu
mesh_terms: D046973:Ruminiclostridium cellulolyticum / Q000235:genetics* / Q000378:metabolism; D005486:Flavin Mononucleotide / Q000235:genetics / Q000378:metabolism*; D017930:Genes, Reporter; D008163:Luminescent Measurements / Q000379:methods*; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism*; D011401:Promoter Regions, Genetic
chemical_list: D008164:Luminescent Proteins; D005486:Flavin Mononucleotide
keywords: Clostridium cellulolyticum; Constitutive; FbFP; Inducible; Promoter screening