title: Differential proteomic profiling reveals regulatory proteins and novel links between primary metabolism and spinosad production in Saccharopolyspora spinosa.
abstract: BACKGROUND
  Saccharopolyspora spinosa is an important producer of antibiotic spinosad with clarified biosynthesis pathway but its complex regulation networks associated with primary metabolism and secondary metabolites production almost have never been concerned or studied before. The proteomic analysis of a novel Saccharopolyspora spinosa CCTCC M206084 was performed and aimed to provide a global profile of regulatory proteins.
  
  
  RESULTS
  Two-dimensional-liquid chromatography-tandem mass spectrometry (LC-MS/MS) identified 1090, 1166, 701, and 509 proteins from four phases respectively, i.e., the logarithmic growth phase (T1), early stationary phase (T2), late stationary phase (T3), and decline phase (T4). Among the identified proteins, 1579 were unique to the S. spinosa proteome, including almost all the enzymes for spinosad biosynthesis. Trends in protein expression over the various time phases were deduced from using the modified protein abundance index (PAI), revealed the importance of stress pathway proteins and other global regulatory network proteins during spinosad biosynthesis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis followed by one-dimensional LC-MS/MS identification revealed similar trend of protein expression from four phases with the results of semi-quantification by PAI. qRT-PCR analysis revealed that 6 different expressed genes showed a positive correlation between changes at translational and transcriptional expression level. Expression of three proteins that likely promote spinosad biosynthesis, namely, 5-methyltetrahydropteroyltriglutamate-homocysteine S-methyltransferase (MHSM), glutamine synthetase (GS) and cyclic nucleotide-binding domain-containing protein (CNDP) was validated by western blot, which confirmed the results of proteomic analysis.
  
  
  CONCLUSIONS
  This study is the first systematic analysis of the S. spinosa proteome during fermentation and its valuable proteomic data of regulatory proteins may be used to enhance the production yield of spinosad in future studies.
authors.forename: Qi
authors.initials: Q
authors.lastname: Yang
authors.affiliation: Hunan Provincial Key Laboratory of Microbial Molecular Biology-State Key laboratory Breeding Base of Microbial Molecular Biology, College of life Science, Hunan Normal University, Changsha 410081, China. dingxuezhi@hunnu.edu.cn.
authors.forename: Xuezhi
authors.initials: X
authors.lastname: Ding
authors.forename: Xuemei
authors.initials: X
authors.lastname: Liu
authors.forename: Shuang
authors.initials: S
authors.lastname: Liu
authors.forename: Yunjun
authors.initials: Y
authors.lastname: Sun
authors.forename: Ziquan
authors.initials: Z
authors.lastname: Yu
authors.forename: Shengbiao
authors.initials: S
authors.lastname: Hu
authors.forename: Jie
authors.initials: J
authors.lastname: Rang
authors.forename: Hao
authors.initials: H
authors.lastname: He
authors.forename: Lian
authors.initials: L
authors.lastname: He
authors.forename: Liqiu
authors.initials: L
authors.lastname: Xia
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D004338:Drug Combinations; D005974:Glutamate-Ammonia Ligase / Q000235:genetics / Q000378:metabolism; D018942:Macrolides / Q000737:chemistry / Q000378:metabolism*; D053858:Metabolic Networks and Pathways; D008780:Methyltransferases / Q000235:genetics / Q000378:metabolism; D020543:Proteome / Q000032:analysis / Q000378:metabolism*; D040901:Proteomics; D016930:Saccharopolyspora / Q000254:growth & development / Q000378:metabolism*; D053719:Tandem Mass Spectrometry
chemical_list: D001426:Bacterial Proteins; D004338:Drug Combinations; D018942:Macrolides; D020543:Proteome; D008780:Methyltransferases; C022725:5-methyltetrahydropteroyltriglutamate-homocysteine S-methyltransferase; D005974:Glutamate-Ammonia Ligase; C415329:spinosad