title: A novel high-throughput and quantitative method based on visible color shifts for screening Bacillus subtilis THY-15 for surfactin production.
abstract: A novel chromatic visible screening method using bromothymol blue (BTB) as a color indicator and cetylpyridinium chloride (CPC) as a mediator was constructed to obtain the high titer surfactin-producing strains. The reliability and quantification accuracy of color shift were also confirmed. Regular chromatic responses from faint yellow-green to dark green and bright blue reflected the different ranges of surfactin concentrations. Moreover, the quantitative accuracy of surfactin quantification in the range of 100-500 mg/L was verified by reverse-phase high-performance liquid chromatography (RP-HPLC) using different fermentation supernatant samples. Using this CPC-BTB method, a superior surfactin producer, Bacillus subtilis THY-15, was successfully screened. The producer's surfactin (Srf) titer reached 1240 mg/L. RP-HPLC analysis of THY-15 revealed four surfactin isoforms. As identified by amino acid analysis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis, the isoforms of surfactin in fraction 1, 2 and 4 had the same circular peptide sequence of Glu-Leu-Leu-Val-Asp-Leu-Leu but different iso-C13, C14 and C15 fatty acid chains, but the isoform in fraction 3 possessed a special peptide sequence of Glu-Val-Leu-Leu-Asp-Leu-Val.
authors.affiliation: Key Laboratory for Industrial Biocatalysis of the Ministry of Education, Department of Chemical Engineering, Tsinghua University, Beijing, 100084, China.
authors.forename: Huan
authors.initials: H
authors.lastname: Yang
authors.forename: Huimin
authors.initials: H
authors.lastname: Yu
authors.forename: Zhongyao
authors.initials: Z
authors.lastname: Shen
mesh_terms: D000596:Amino Acids / Q000032:analysis; D001412:Bacillus subtilis / Q000302:isolation & purification* / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D005227:Fatty Acids / Q000378:metabolism; D005285:Fermentation; D057166:High-Throughput Screening Assays / Q000379:methods*; D006863:Hydrogen-Ion Concentration; D055666:Lipopeptides / Q000032:analysis / Q000096:biosynthesis*; D010456:Peptides, Cyclic / Q000032:analysis / Q000096:biosynthesis*; D015203:Reproducibility of Results; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D053719:Tandem Mass Spectrometry
chemical_list: D000596:Amino Acids; D005227:Fatty Acids; D055666:Lipopeptides; D010456:Peptides, Cyclic