Abstract
Purpose
To generate a AfsR-like (AfsR-L) overexpression strain Saccharopolyspora pogona-AfsR-L and investigate its effects on the morphology and metabolism of S. pogona.
Methods
Firstly, we generated the overexpression vector pOJ260-PermE-afsR-L via overlap extension PCR. Then, the recombination strain S. pogona-AfsR-L was constructed via conjugal transfer. To monitor the growth and morphology, mycelia and sporulation were observed. The distinctive proteins and butenyl-spinosyn biosynthesis were investigated by SDS-PAGE, HPLC, and mass spectrometry. And the transcriptional level of afsR-L and other relative functional genes in S. pogona-AfsR-L was analyzed by qRT-PCR. Western blot verified the increased amount of AfsR-L protein in the overexpression strain.
Result
Growth curve and mycelia observation showed that afsR-L overexpression make the stationary phase of S. pogona-AfsR-L longer than that of wild S. pogona by approximate 3 days. Moreover, S. pogona-AfsR-L exhibited a more obvious white phenotype on the solid medium, which means afsR-L overexpression affects the sporulation ability of S. pogona. HPLC analysis revealed that the peak area of the butenyl-spinosyn yield of S. pogona-AfsR-L was 293.6, while that of S. pogona was 250.9. SDS-PAGE analysis showed that the two strains had different whole protein expression profiles, and the distinctive proteins were further identified by LC-MS/MS identification, which showed the possible control mechanism of afsR-L gene in S. pogona.
Conclusion
We concluded that AfsR could directly or indirectly positively regulate the biosynthesis of butenyl-spinosyn and affect the growth features of S. pogona. We envisioned that this result can be expanded to other Streptomyces for strain improvement.
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References
Bierman M, Logan R, O’Brien K, Seno ET, Rao RN, Schoner BE (1992) Plasmid cloning vectors for the conjugal transfer of DNA from Escherichia coli to Streptomyces spp. Gene 116:43–49
Bush MJ, Chandra G, Bibb MJ, Findlay KC, Buttner MJ (2016) Genome-wide chromatin immunoprecipitation sequencing analysis shows that whiB is a transcription factor that cocontrols its regulon with whiA to initiate developmental cell division in Streptomyces. mBio 7(2):e00523-00516
Den Hengst CD, Tran NT, Bibb MJ, Chandra G, Leskiw BK, Buttner MJ (2010) Genes essential for morphological development and antibiotic production in Streptomyces coelicolor are targets of BldD during vegetative growth. Mol Microbiol 78(2):361–379
Elliot MA, Locke TR, Galibois CM, Leskiw BK (2003) BldD from Streptomyces coelicoloris a non-essential global regulator that binds its own promoter as a dimer. FEMS Microbiol Lett 225(1):35–40
Engmark M, Jespersen MC, Lomonte B et al (2017) High-density peptide microarray exploration of the antibody response in a rabbit immunized with a neurotoxic venom fraction. Toxicon 138:151–158
Hahn DR, Gustafson G, Waldron C, Bullard B, Jackson JD, Mitchell J (2006) Butenyl-spinosyns, a natural example of genetic engineering of antibiotic biosynthetic genes. J Ind Microbiol Biotechnol 33(2):94–104
Horinouehi S, Kito M, Nishiyama M, Furuya K, Hug S-K, Miyake K, Beppu T (1990) Primary structure of AfsR, a global regulatory protein for secondary metabolite formation in Streptomyces coelicolor A3(2). Gene (Amsterdam) 95(1):49–56
Huang S, Ding X, Sun Y, Yang Q, Xiao X, Cao Z et al (2012) Proteomic analysis of Bacillus thuringiensis at different growth phases by using an automated online two-dimensional liquid chromatography-tandem mass spectrometry strategy. Appl Environ Microbiol 78(15):5270–5279
Karnicar K, Drobnak I, Petek M, Magdevska V, Horvat J, Vidmar R et al (2016) Integrated omics approaches provide strategies for rapid erythromycin yield increase in Saccharopolyspora erythraea. Microb Cell Factories 15:93
Kim I-K, Lee C-J, Kim M-K, Kim J-M, Kim J-H, Yim H-S et al (2006) Crystal structure of the DNA-binding domain of BldD, a central regulator of aerial mycelium formation in Streptomyces coelicolor A3(2). Mol Microbiol 60(5):1179–1193
Kirst HA (2010) The spinosyn family of insecticides: realizing the potential of natural products research. J Antibiot 63(3):101–111
Lee P-C, Umeyama T, Horinouchi S (2002) afsS is a target of AfsR, a transcriptional factor with ATPase activity that globally controls secondary metabolism in Streptomyces coelicolor A3(2). Mol Microbiol 43(6):1413–1430
Lewer P, Hahn DR, Karr LL, Duebelbeis DO, Gilbert JR, Crouse GD et al (2009) Discovery of the butenyl-spinosyn insecticides: novel macrolides from the new bacterial strain Saccharopolyspora pogona. Bioorg Med Chem 17(12):4185–4196
Li L, Zheng G, Chen J, Ge M, Jiang W, Lu Y (2017) Multiplexed site-specific genome engineering for overproducing bioactive secondary metabolites in actinomycetes. Metab Eng 40:80–92
Li L, Rang J, He H et al (2018) Impact on strain growth and butenyl-spinosyn biosynthesis by overexpression of polynucleotide phosphorylase gene in Saccharopolyspora pogon. Appl Microbiol Biotechnol 102:8011–8021
Liu Y, Sun G, Zhong Z, Ji L, Zhang Y, Zhou J et al (2017) Overexpression of AtEDT1 promotes root elongation and affects medicinal secondary metabolite biosynthesis in roots of transgenic Salvia miltiorrhiza. Protoplasma 254(4):1617–1625
Luo Y, Ding X, Xia L, Huang F, Li W, Huang S et al (2011) Comparative proteomic analysis of Saccharopolyspora spinosa SP06081 and PR2 strains reveals the differentially expressed proteins correlated with the increase of spinosad yield. Proteome Sci 9:40
Luo Y, Zhang L, Barton K, Zhao H (2015) Systematic identification of a panel of strong constitutive promoters from Streptomyces albus. ACS Synth Biol 4(9):1001–1010
Maharjan S, Oh TJ, Lee HC, Sohng JK (2008) Heterologous expression of metK1-sp and afsR-sp in Streptomyces venezuelae for the production of pikromycin. Biotechnol Lett 30(9):1621–1626
Millar NS, Denholm I (2007) Nicotinic acetylcholine receptors: targets for commercially important insecticides. Invertebr Neurosci 7(1):53–66
Muller S, Pflock M, Schar J, Kennard S, Beier D (2007) Regulation of expression of atypical orphan response regulators of Helicobacter pylori. Microbiol Res 162:1–14
Nurmohamed S, Vincent HA, Titman CM, Chandran V, Pears MR, Du D et al (2011) Polynucleotide phosphorylase activity may be modulated by metabolites in Escherichia coli. J Biol Chem 286(16):14315–14323
Olano C, Lombo F, Mendez C, Salas JA (2008) Improving production of bioactive secondary metabolites in actinomycetes by metabolic engineering. Metab Eng 10(5):281–292
Parajuli N, Viet HT, Ishida K, Tong HT, Lee HC, Liou K et al (2005) Identification and characterization of the afsR homologue regulatory gene from Streptomyces peucetius ATCC 27952. Res Microbiol 156(5–6):707–712
Reeves AR, Brikun IA, Cernota WH, Leach BI, Gonzalez MC, Weber JM (2006) Effects of methylmalonyl-CoA mutase gene knockouts on erythromycin production in carbohydrate-based and oil-based fermentations of Saccharopolyspora erythraea. J Ind Microbiol Biotechnol 33(7):600–609
Santos-Beneit F, Rodríguez-García A, Sola-Landa A et al (2009) Cross-talk between two global regulators in Streptomyces: PhoP and AfsR interact in the control of afsS, pstS and phoRP transcription. Mol Microbiol 72(1):53–68
Sun D, Zhu J, Chen Z, Li J, Wen Y (2016) SAV742, a novel AraC-family regulator from Streptomyces avermitilis, controls avermectin biosynthesis, cell growth and development. Sci Rep 6:36915
Tanaka A, Takano Y, Ohnishi Y, Horinouchi S (2007) AfsR recruits RNA polymerase to the afsS promoter: a model for transcriptional activation by SARPs. J Mol Biol 369:322–333
Tang Y, Xia L, Ding X, Luo Y, Huang F, Jiang Y (2011) Duplication of partial spinosyn biosynthetic gene cluster in Saccharopolyspora spinosa enhances spinosyn production. FEMS Microbiol Lett 325(1):22–29
Tatarko M, Romeo T (2001) Disruption of a global regulatory gene to enhance central carbon flux into phenylalanine biosynthesis in Escherichia coli. Curr Microbiol 43(1):26–32
Virginie Molle WJP, Findlay KC, Buttner MJ (2000) WhiD and WhiB, homologous proteins required for different stages of sporulation in Streptomyces coelicolor A3(2). J Bacteriol 182(5):1286–1295
Wang W, Wang H, Hu H, Peng H, Zhang X (2015) Overexpression of afsR and optimization of metal chloride to improve lomofungin production in Streptomyces lomondensis S015. J Microbiol Biotechnol 25(5):672–680
Xu J, Zhang J, Zhuo J et al (2017) Activation and mechanism of a cryptic oviedomycin gene cluster via the disruption of a global regulatory gene, adpA, in Streptomyces ansochromogenes. J Biol Chem 292(48):19708–19720
Yang Q, Ding X, Liu X, Liu S, Sun Y, Yu Z et al (2014) Differential proteomic profiling reveals regulatory proteins and novel links between primary metabolism and spinosad production in Saccharopolyspora spinosa. Microb Cell Factories 13(1):27
Yang Q, Li Y, Yang H, Rang J, Tang S, He L et al (2015) Proteomic insights into metabolic adaptation to deletion of metE in Saccharopolyspora spinosa. Appl Microbiol Biotechnol 99(20):8629–8641
Yoshihiro Mouri KK, Fujita A, Tezuka T, Ohnishi Y (2017) Regulation of sporangium formation by BldD in the rare actinomycete Actinoplanes missouriensis. J Bacteriol 199(12):e00840-00816
Zhang Y, He H, Liu H, Wang H, Wang X, Xiang W (2016) Characterization of a pathway-specific activator of milbemycin biosynthesis and improved milbemycin production by its overexpression in Streptomyces bingchenggensis. Microb Cell Factories 15(1):152
Zhu Z, Li H, Yu P, Guo Y, Luo S, Chen Z et al (2017) SlnR is a positive pathway-specific regulator for salinomycin biosynthesis in Streptomyces albus. Appl Microbiol Biotechnol 101(4):1547–1557
Funding
This work was supported by funding from the National Natural Science Foundation of China (31770106), the National Basic Research Program (973) of China (2012CB722301), the Major Research Projects in Hunan Province (2017NK1030), and the Cooperative Innovation Center of Engineering and New Products for Developmental Biology of Hunan Province (20134486).
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L. L. and S. Y. developed the bacterial recombinant and performed bacterial strain isolation. Y. S., L. G., and J. T. performed HPLC. J. R., X. D., and Z. L. performed LC-MS/MS and data analysis. L. G., H. H., Z. Y., and S. P. performed Total RNA isolation and qRT-PCR analysis. L. G. and H. H. performed Western blot. L. L., H. H., Y. S., and L. X. designed the study and wrote the draft of manuscript. L. G. and Y. S. modified and corrected this manuscript. All authors discussed the results and approved the final manuscript.
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Li, L., Gong, L., He, H. et al. AfsR is an important regulatory factor for growth and butenyl-spinosyn biosynthesis of Saccharopolyspora pogona. Ann Microbiol 69, 809–818 (2019). https://doi.org/10.1007/s13213-019-01473-8
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DOI: https://doi.org/10.1007/s13213-019-01473-8