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Interspecies Complementation of the LuxR Family Pathway-Specific Regulator Involved in Macrolide Biosynthesis
Interspecies Complementation of the LuxR Family Pathway-Specific Regulator Involved in Macrolide Biosynthesis
Journal of Microbiology and Biotechnology. 2016. Feb, 26(1): 66-71
Copyright © 2016, The Korean Society For Microbiology And Biotechnology
  • Received : October 26, 2015
  • Accepted : November 25, 2015
  • Published : February 28, 2016
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About the Authors
SangJoon Mo
Clinical Trial Institute, Biosafety and Validation Center, Dankook University, Choenan 31116, Republic of Korea
joonyoon@ewha.ac.kr
Yeo Joon Yoon
Department of Chemistry and Nano Science, Ewha Womans University, Seoul 03760, Republic of Korea
joonyoon@ewha.ac.kr

Abstract
PikD is a widely known pathway-specific regulator for controlling pikromycin production in Streptomyces venezuelae ATCC 15439, which is a representative of the large ATP-binding regulator of the LuxR family (LAL) in Streptomyces sp. RapH and FkbN also belong to the LAL family of transcriptional regulators, which show greatest homology with the ATP-binding motif and helix-turn-helix DNA-binding motif of PikD. Overexpression of pikD and heterologous expression of rapH and fkbN led to enhanced production of pikromycin by approximately 1.8-, 1.6-, and 1.6-fold in S. venezuelae , respectively. Cross-complementation of rapH and fkbN in the pikD deletion mutant (ΔpikD) restored pikromycin and derived macrolactone production. Overall, these results show that heterologous expression of rapH and fkbN leads to the overproduction of pikromycin and its congeners from the pikromycin biosynthetic pathway in S. venezuelae , and they have the same functionality as the pathwayspecific transcriptional activator for the pikromycin biosynthetic pathway in the ΔpikD strain. These results also show extensive “cross-communication” between pathway-specific regulators of streptomycetes and suggest revision of the current paradigm for pathwayspecific versus global regulation of secondary metabolism in Streptomyces species.
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Sequence alignment of these proteins characterized the LAL-family proteins, which consist of an N-terminal ATP-binding domain and a C-terminal LuxR-type HTH DNA-binding domain. The shaded box signifies ATP- and DNA-binding motifs.
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( A ) pPIKD, pikD from S. venezuelae , pFKBN, fkbN from S. hygroscopicus ATCC 14891, pRAPH, and rapH from S. hygroscopicus NRRL 5491 were constructed into the pSET152 derivative containing the ermE* promoter. The plasmids have a ΦC31 attachment site for genomic interaction, oriT for transformation, and aac(3)IV for selecting transformants in E. coli and Streptomyces sp. ( B ) Comparison of pikromycin production from S. venezuelae , SVPSET152, SVPIKD, SVRAPH, and SVFKBN. Pikromycin was extracted twice with an equal volume of ethyl acetate and then the level of ikromycin production was determined by HPLC. Values are averages of two series of duplicate experiments.
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( A ) Schematic representation of homologous recombination of construct pPKID1 into the genome of S. venezuelae (left). Verification of the in-frame deletion of pikD by Southern blot analysis (right). Lane: M, DIG-labeled DNA molecular weight marker III; 1, genomic DNA of single-crossover pikD mutant; 2, genomic DNA of S. venezuelae ; 3, genomic DNA of double-crossover pikD mutant. ( B ) Pikromycin production was restored by expressing the heterologous LAL-family genes. The pikromycin, methymycin, YC-17, neomethymycin, and narbomycin peaks are indicated by a black circle, black inverted triangle, black lozenge, arrow, and black square, respectively.
Acknowledgements
This work was partially supported by the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT and Future Planning (MISP) (2013R1A2A1A01014230) and High Value-added Food Technology Development Program, Ministry of Agriculture, Food and Rural Affairs, Republic of Korea.
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