FK506 biosynthesis is regulated by two positive regulatory elements in Streptomyces tsukubaensis

BMC Microbiology

Hrvoje Petković³  Juan F Martín⁵  Štefan Fujs⁴  Gregor Kosec²  Gregor Kopitar¹  Peter Mrak¹  Carlos Barreiro⁵  Miriam Martínez-Castro⁵  Javier Santos-Aberturas⁵  Tomaž Polak³  Enej Kuščer²  Jaka Horvat⁴  Vasilka Magdevska⁴  Marko Blažič³  Dušan Goranovič⁴ 

[1] Lek Pharmaceuticals d.d., a Sandoz company, Verovškova 57, SI-1526, Ljubljana, Slovenia;Centre of Excellence for Integrated Approaches in Chemistry and Biology of Proteins, (CIPKeBiP), Jamova 39, Ljubljana, Slovenia;Department of Food Science and Technology, Biotechnical Faculty, University of Ljubljana, Jamnikarjeva 101, SI-1000, Ljubljana, Slovenia;Acies Bio d.o.o., Tehnološki Park 21, SI-1000, Ljubljana, Slovenia;Instituto de Biotecnología de León (INBIOTEC), Parque Científico de León, Avenida. Real, No. 1, 24006, León, Spain

关键词: Transcriptional regulator;    Biosynthesis;    Streptomyces tsukubaensis;    Tacrolimus;    FK506;   

Others  :  1221704
DOI  :  10.1186/1471-2180-12-238

 received in 2012-06-28, accepted in 2012-10-03,  发布年份 2012

【 摘 要 】

Background

FK506 (Tacrolimus) is an important immunosuppressant, produced by industrial biosynthetic processes using various Streptomyces species. Considering the complex structure of FK506, it is reasonable to expect complex regulatory networks controlling its biosynthesis. Regulatory elements, present in gene clusters can have a profound influence on the final yield of target product and can play an important role in development of industrial bioprocesses.

Results

Three putative regulatory elements, namely fkbR, belonging to the LysR-type family, fkbN, a large ATP-binding regulator of the LuxR family (LAL-type) and allN, a homologue of AsnC family regulatory proteins, were identified in the FK506 gene cluster from Streptomyces tsukubaensis NRRL 18488, a progenitor of industrial strains used for production of FK506. Inactivation of fkbN caused a complete disruption of FK506 biosynthesis, while inactivation of fkbR resulted in about 80% reduction of FK506 yield. No functional role in the regulation of the FK506 gene cluster has been observed for the allN gene. Using RT-PCR and a reporter system based on a chalcone synthase rppA, we demonstrated, that in the wild type as well as in fkbN- and fkbR-inactivated strains, fkbR is transcribed in all stages of cultivation, even before the onset of FK506 production, whereas fkbN expression is initiated approximately with the initiation of FK506 production. Surprisingly, inactivation of fkbN (or fkbR) does not abolish the transcription of the genes in the FK506 gene cluster in general, but may reduce expression of some of the tested biosynthetic genes. Finally, introduction of a second copy of the fkbR or fkbN genes under the control of the strong ermE* promoter into the wild type strain resulted in 30% and 55% of yield improvement, respectively.

Conclusions

Our results clearly demonstrate the positive regulatory role of fkbR and fkbN genes in FK506 biosynthesis in S. tsukubaensis NRRL 18488. We have shown that regulatory mechanisms can differ substantially from other, even apparently closely similar FK506-producing strains, reported in literature. Finally, we have demonstrated the potential of these genetically modified strains of S. tsukubaensis for improving the yield of fermentative processes for production of FK506.

【 授权许可】

   
2012 Goranovič et al.; licensee BioMed Central Ltd.

【 图 表 】

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【 参考文献 】

• [1]Thomson AW: FK-506 enters the clinic. Immunol Today 1990, 11(2):35-36.
• [2]Wallemacq PE, Reding R: FK506 (tacrolimus), a novel immunosuppressant in organ transplantation: clinical, biomedical, and analytical aspects. Clin Chem 1993, 39(11 Pt 1):2219-2228.
• [3]Meingassner JG, Stutz A: Immunosuppressive macrolides of the type FK 506: a novel class of topical agents for treatment of skin diseases? J Invest Dermatol 1992, 98(6):851-855.
• [4]Easton JB, Houghton PJ: Therapeutic potential of target of rapamycin inhibitors. Expert Opin Ther Targets 2004, 8(6):551-564.
• [5]Graziani EI: Recent advances in the chemistry, biosynthesis and pharmacology of rapamycin analogs. Nat Prod Rep 2009, 26(5):602-609.
• [6]McDaniel R, Welch M, Hutchinson CR: Genetic approaches to polyketide antibiotics. 1. Chem Rev 2005, 105(2):543-558.
• [7]Motamedi H, Shafiee A: The biosynthetic gene cluster for the macrolactone ring of the immunosuppressant FK506. Eur J Biochem 1998, 256(3):528-534.
• [8]Motamedi H, Cai SJ, Shafiee A, Elliston KO: Structural organization of a multifunctional polyketide synthase involved in the biosynthesis of the macrolide immunosuppressant FK506. Eur J Biochem 1997, 244(1):74-80.
• [9]Shafiee A, Cameron PM, Boulton DA, Kaplan L, Motamedi H: Methylating enzyme from Streptomyces MA6858. United States Patent Office. US5264355, Filed 2.7.1992, Issued 23.11.1993
• [10]Motamedi H, Shafiee A, Cai SJ, Streicher SL, Arison BH, Miller RR: Characterization of methyltransferase and hydroxylase genes involved in the biosynthesis of the immunosuppressants FK506 and FK520. J Bacteriol 1996, 178(17):5243-5248.
• [11]Mo S, Kim DH, Lee JH, Park JW, Basnet DB, Ban YH, Yoo YJ, Chen SW, Park SR, Choi EA, Kim E, Jin YY, Lee SK, Park JY, Liu Y, Lee MO, Lee KS, Kim SJ, Kim D, Park BC, Lee SG, Kwon HJ, Suh JW, Moore BS, Lim SK, Yoon YJ: Biosynthesis of the allylmalonyl-CoA extender unit for the FK506 polyketide synthase proceeds through a dedicated polyketide synthase and facilitates the mutasynthesis of analogues. J Am Chem Soc 2011, 133(4):976-985.
• [12]Goranovič D, Kosec G, Mrak P, Fujs S, Horvat J, Kuščer E, Kopitar G, Petković H: Origin of the allyl group in FK506 biosynthesis. J Biol Chem 2010, 285(19):14292-14300.
• [13]Zhuo Y, Zhang W, Chen D, Gao H, Tao J, Liu M, Gou Z, Zhou X, Ye BC, Zhang Q, Zhang S, Zhang LX: Reverse biological engineering of hrdB to enhance the production of avermectins in an industrial strain of Streptomyces avermitilis. Proc Natl Acad Sci U S A 2010, 107(25):11250-11254.
• [14]Martin JF, Liras P: Engineering of regulatory cascades and networks controlling antibiotic biosynthesis in Streptomyces. Curr Opin Microbiol 2010, 13(3):263-273.
• [15]Wietzorrek A, Bibb M: A novel family of proteins that regulates antibiotic production in streptomycetes appears to contain an OmpR-like DNA-binding fold. Mol Microbiol 1997, 25(6):1181-1184.
• [16]De Schrijver A, De Mot R: A subfamily of MalT-related ATP-dependent regulators in the LuxR family. Microbiology 1999, 145:1287-1288.
• [17]Rascher A, Hu Z, Viswanathan N, Schirmer A, Reid R, Nierman WC, Lewis M, Hutchinson CR: Cloning and characterization of a gene cluster for geldanamycin production in Streptomyces hygroscopicus NRRL 3602. FEMS Microbiol Lett 2003, 218(2):223-230.
• [18]Bibb MJ: Regulation of secondary metabolism in streptomycetes. Curr Opin Microbiol 2005, 8(2):208-215.
• [19]Demain AL, Adrio JL: Strain improvement for production of pharmaceuticals and other microbial metabolites by fermentation. Prog Drug Res 2008, 65(251):253-289.
• [20]Kuščer E, Coates N, Challis I, Gregory M, Wilkinson B, Sheridan R, Petković H: Roles of rapH and rapG in positive regulation of rapamycin biosynthesis in Streptomyces hygroscopicus. J Bacteriol 2007, 189(13):4756-4763.
• [21]Wu K, Chung L, Revill WP, Katz L, Reeves CD: The FK520 gene cluster of Streptomyces hygroscopicus var. ascomyceticus (ATCC 14891) contains genes for biosynthesis of unusual polyketide extender units. Gene 2000, 251(1):81-90.
• [22]Won SJ, Yu JY, Jin KH, Kyoung SS: Method for promoting production of FK506 by introducing an fkbN gene encoding transcription regulator derived from Streptomyces hygroscopicus var. ascomyceticus ATCC 14891 strain. Korean Intellectual Property Office. KR100800233, Filed 05. 02. 2007, Issued 25. 01. 2008
• [23]Won SJ, Yu JY, Jin KH, Kyoung SS: Method for promoting production of FK506 by introducing fkbR1 gene encoding FK520 transcription regulator derived from Streptomyces sp. Korean Intellectual Property Office. KR100800222, Filed 05.02. 2007, Issued 25. 01. 2008
• [24]Molnar I, Aparicio JF, Haydock SF, Khaw LE, Schwecke T, Konig A, Staunton J, Leadlay PF: Organisation of the biosynthetic gene cluster for rapamycin in Streptomyces hygroscopicus: analysis of genes flanking the polyketide synthase. Gene 1996, 169(1):1-7.
• [25]Henikoff S, Wallace JC, Brown JP: Finding protein similarities with nucleotide sequence databases. Methods Enzymol 1990, 183:111-132.
• [26]Walker JE, Saraste M, Runswick MJ, Gay NJ: Distantly related sequences in the alpha- and beta-subunits of ATP synthase, myosin, kinases and other ATP-requiring enzymes and a common nucleotide binding fold. EMBO J 1982, 1(8):945-951.
• [27]Kosec G, Goranovič D, Mrak P, Fujs S, Kuščer E, Horvat J, Kopitar G, Petković H: Novel chemobiosynthetic approach for exclusive production of FK506. Metab Eng 2012, 14(1):39-46.
• [28]Mo S, Yoo YJ, Ban YH, Lee SK, Kim E, Suh JW, Yoon YJ: Roles of fkbN in positive regulation and tcs7 in negative regulation of FK506 biosynthesis in Streptomyces sp. strain KCTC 11604BP. Appl Environ Microbiol 2012, 78(7):2249-2255.
• [29]Shirling EB, Gottlieb D: Methods for characterization of Streptomyces species. Int J Syst Bacteriol 1966, 16(3):313-340.
• [30]Kieser T, Bibb MJ, Buttner MJ, Chater KF, Hopwood DA: Practical Streptomyces genetics. Norwich, United Kingdom: The John Innes Foundation; 2000.
• [31]Sambrook J, Russell DW: Molecular Cloning: A Laboratory Manual. 3rd edition. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory; 2001.
• [32]Paget MS, Chamberlin L, Atrih A, Foster SJ, Buttner MJ: Evidence that the extracytoplasmic function sigma factor sigmaE is required for normal cell wall structure in Streptomyces coelicolor A3(2). J Bacteriol 1999, 181(1):204-211.
• [33]Margulies M, Egholm M, Altman WE, Attiya S, Bader JS, Bemben LA, Berka J, Braverman MS, Chen YJ, Chen Z, Dewell SB, Du L, Fierro JM, Gomes XV, Godwin BC, He W, Helgesen S, Ho CH, Irzyk GP, Jando SC, Alenquer ML, Jarvie TP, Jirage KB, Kim JB, Knight JR, Lanza JR, Leamon JH, Lefkowitz SM, Lei M, Li J, et al.: Genome sequencing in open microfabricated high density picoliter reactors. Nature 2005, 437(7057):376-380.
• [34]Bibb MJ, Findlay PR, Johnson MW: The relationship between base composition and codon usage in bacterial genes and its use for the simple and reliable identification of protein-coding sequences. Gene 1984, 30(1–3):157-166.
• [35]Ishikawa J, Hotta K: FramePlot: a new implementation of the frame analysis for predicting protein-coding regions in bacterial DNA with a high G + C content. FEMS Microbiol Lett 1999, 174(2):251-253.
• [36]Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ: Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Research 1997, 25(17):3389-3402.
• [37]Jeanmougin F, Thompson JD, Gouy M, Higgins DG, Gibson TJ: Multiple sequence alignment with Clustal X. Trends Biochem Sci 1998, 23(10):403-405.
• [38]Hong B, Phornphisutthimas S, Tilley E, Baumberg S, McDowall KJ: Streptomycin production by Streptomyces griseus can be modulated by a mechanism not associated with change in the adpA component of the A-factor cascade. Biotechnol Lett 2007, 29(1):57-64.
• [39]Kolling R, Lother H: AsnC: an autogenously regulated activator of asparagine synthetase A transcription in Escherichia coli. J Bacteriol 1985, 164(1):310-315.
• [40]Schell MA: Molecular biology of the LysR family of transcriptional regulators. Annu Rev Microbiol 1993, 47:597-626.
• [41]Magdevska V, Gaber R, Goranovič D, Kuščer E, Boakes S, Duran Alonso MB, Santamaria RI, Raspor P, Leadlay PF, Fujs S, Petković H: Robust reporter system based on chalcone synthase rppA gene from Saccharopolyspora erythraea. J Microbiol Methods 2010, 83(2):111-119.
• [42]Flett F, Mersinias V, Smith CP: High efficiency intergeneric conjugal transfer of plasmid DNA from Escherichia coli to methyl DNA-restricting streptomycetes. FEMS Microbiol Lett 1997, 155(2):223-229.
• [43]Tunca S, Barreiro C, Sola-Landa A, Coque JJ, Martin JF: Transcriptional regulation of the desferrioxamine gene cluster of Streptomyces coelicolor is mediated by binding of DmdR1 to an iron box in the promoter of the desA gene. FEBS J 2007, 274(4):1110-1122.
• [44]Bikandi J, San Millan R, Rementeria A, Garaizar J: In silico analysis of complete bacterial genomes: PCR, AFLP-PCR and endonuclease restriction. Bioinformatics 2004, 20(5):798-799.
• [45]Boos W, Shuman H: Maltose/maltodextrin system of Escherichia coli: transport, metabolism, and regulation. Microbiol Mol Biol Rev 1998, 62(1):204-229.
• [46]Wilson DJ, Xue Y, Reynolds KA, Sherman DH: Characterization and analysis of the PikD regulatory factor in the pikromycin biosynthetic pathway of Streptomyces venezuelae. J Bacteriol 2001, 183(11):3468-3475.
• [47]Aparicio JF, Molnar I, Schwecke T, Konig A, Haydock SF, Khaw LE, Staunton J, Leadlay PF: Organization of the biosynthetic gene cluster for rapamycin in Streptomyces hygroscopicus: analysis of the enzymatic domains in the modular polyketide synthase. Gene 1996, 169(1):9-16.
• [48]Brautaset T, Sekurova ON, Sletta H, Ellingsen TE, Strøm AR, Valla S, Zotchev SB: Biosynthesis of the polyene antifungal antibiotic nystatin in Streptomyces noursei ATCC 11455: analysis of the gene cluster and deduction of the biosynthetic pathway. Chem Biol 2000, 7(6):395-403.
• [49]He W, Lei J, Liu Y, Wang Y: The LuxR family members GdmRI and GdmRII are positive regulators of geldanamycin biosynthesis in Streptomyces hygroscopicus 17997. Arch Microbiol 2008, 189(5):501-510.
• [50]Stragier P, Richaud F, Borne F, Patte JC: Regulation of diaminopimelate decarboxylase synthesis in Escherichia coli. I. Identification of a lysR gene encoding an activator of the lysA gene. J Mol Biol 1983, 168(2):307-320.
• [51]Maddocks SE, Oyston PC: Structure and function of the LysR-type transcriptional regulator (LTTR) family proteins. Microbiology 2008, 154(Pt 12):3609-3623.
• [52]Wilkinson CJ, Hughes-Thomas ZA, Martin CJ, Bohm I, Mironenko T, Deacon M, Wheatcroft M, Wirtz G, Staunton J, Leadlay PF: Increasing the efficiency of heterologous promoters in actinomycetes. J Mol Microbiol Biotechnol 2002, 4(4):417-426.
• [53]Martinez-Castro M, Barreiro C, Romero F, Fernandez-Chimeno RI, Martin JF: Streptomyces tacrolimicus sp. nov., a low producer of the immunosuppressant tacrolimus (FK506). Int J Syst Evol Microbiol 2011, 61(Pt 5):1084-1088.
• [54]Salehi-Najafabadi Z, Barreiro C, Martinez-Castro M, Solera E, Martin JF: Characterisation of a gamma-butyrolactone receptor of Streptomyces tacrolimicus: effect on sporulation and tacrolimus biosynthesis. Appl Microbiol Biotechnol 2011, 92(5):971-984.
• [55]Chater KF, Chandra G: The use of the rare UUA codon to define "expression space" for genes involved in secondary metabolism, development and environmental adaptation in streptomyces. J Microbiol 2008, 46(1):1-11.
• [56]Chen D, Zhang Q, Cen P, Xu Z, Liu W: Improvement of FK506 production in Streptomyces tsukubaensis by genetic enhancement of the supply of unusual polyketide extender units via utilization of two distinct site-specific recombination systems. Appl Environ Microbiol 2012, 78:5093-5103.