Ngineering, College of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, PR China. 2Key Laboratory of technique bioengineering (Tianjin University), Ministry of Education, Tianjin 300072, PR China. 3Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin 300072, PR China. Received: 8 March 2014 Accepted: 26 JuneRNA extraction, cDNA synthesis, and real-time qPCR analysis of S. spinosa were performed as described previously [34]. 16S rRNA and rbL13 had been employed to normalize the qPCR information. The primers utilized in qPCR are listed in Table three.Intracellular metabolites employing GC-MS4 mL cell cultures had been mixed with 6 mL cold methanol (-40 ) to arrest metabolism instantaneously. Then, samples have been centrifugated at 3000 g for 3 min. Cell pellets were collected and straight away ground to powder inside a porcelain mortar, which was pre-cooled to -80 , beneath liquid nitrogen for five min. Then 100 mg cell powder was mixed completely with 1 ml -40 50 methanol (methanol/water, 1:1). The samples had been centrifugated at 10000 g for ten min. The supernatants have been collected. Then 10 uL internal common option succinic d4 acid (Sigma, 0.1 mg/ml) was added in to the one hundred uL extract supernatants prior to lyophilization. Just after lyophilization, the derivatization and measurement by GC-MS of these samples have been carried out based on a preceding system [35]. Four biological replicates had been performed for every sample. The identification and quantification of GC-MS peaks were performed employing Agilent software (G1701DA MSD ChemStation ver.Methyl 5-cyanopyrazine-2-carboxylate supplier D.00.00.38).Metabolites involved in Spinosad synthesis determinationShort chain coenzyme A (CoA) in S. spinosa was extracted as described [8]. Acetly-CoA, malonayl-CoA, methylmalonyl-CoA, succinyl-CoA, and propionyl-CoA have been measured by HPLC as described [36]. Pseudoaglycones (PSA), the intermediates of spinosad, was determined by HPLC as described [37].Ethyl 2-diazo-3-oxobutanoate Chemscene Added filesAdditional file 1: Figure S1.PMID:23543429 Various alignments of proteins from Rex family. More file two: Table S1. The intracellular metabolites involved in Carbohydrate metabolism, Energy metabolism, Lipid metabolism, Amino acid metabolism, Nucleotide metabolism and spinosad pathway had been analyzed by GC-MS and HPLC both within the manage group and oxidative condition. Competing interests The authors declare that they’ve no competing interest. Authors’ contributions XMZ and WYL developed the experiments. XMZ and CYX performed each of the experiments and prepared all figures. FLZ and DSL performed mass spectrometry evaluation. XMZ, CYX, CBZ, QGLCY and WYL co-drafted the manuscript. All authors study and authorized the final manuscript. Acknowledgements The authors wish to acknowledge the economic supported by National All-natural Science Foundation of China (No. 31270087 and 21076148), National Essential Standard Research System (No. 2012CB721105), Program for New CenturyReferences 1. Madduri K, Waldron C, Matsushima P, Broughton MC, Crawford K, Merlo DJ, Baltz RH: Genes for the biosynthesis of spinosyns: applications for yield improvement in Saccharopolyspora spinosa. J Ind Microbiol Biotechnol 2001, 27:399?02. 2. Sparks TC, Crouse GD, Durst G: Natural solutions as insecticides: the biology, biochemistry and quantitative structure ctivity relationships of spinosyns and spinosoids. Pest Manag Sci 2001, 57:896?05. 3. Williams T, Valle J, Vi ela E: Will be the naturally derived insecticide Spinosad?compatible with insect all-natural enemies? Biocontrol Sci Tech 2003, 13:459?75.