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Global and pathway-specific transcriptional regulations of pactamycin biosynthesis in Streptomyces pactum.

ABSTRACT: Pactamycin, a structurally unique aminocyclitol natural product isolated from Streptomyces pactum, has potent antibacterial, antitumor, and anti-protozoa activities. However, its production yields under currently used culture conditions are generally low. To understand how pactamycin biosynthesis is regulated and explore the possibility of improving pactamycin production in S. pactum, we investigated the transcription regulations of pactamycin biosynthesis. In vivo inactivation of two putative pathway-specific regulatory genes, ptmE and ptmF, resulted in mutant strains that are not able to produce pactamycin. Genetic complementation using a cassette containing ptmE and ptmF integrated into the S. pactum chromosome rescued the production of pactamycin. Transcriptional analysis of the ?ptmE and ?ptmF strains suggests that both genes control the expression of the whole pactamycin biosynthetic gene cluster. However, attempts to overexpress these regulatory genes by introducing a second copy of the genes in S. pactum did not improve the production yield of pactamycin. We discovered that pactamycin biosynthesis is sensitive to phosphate regulation. Concentration of inorganic phosphate higher than 2 mM abolished both the transcription of the biosynthetic genes and the production of the antibiotic. Draft genome sequencing of S. pactum and bioinformatics studies revealed the existence of global regulatory genes, e.g., genes that encode a two-component PhoR-PhoP system, which are commonly involved in secondary metabolism. Inactivation of phoP did not show any significant effect to pactamycin production. However, in the phoP::aac(3)IV mutant, pactamycin biosynthesis is not affected by external inorganic phosphate concentration.

SUBMITTER: Lu W

PROVIDER: S-EPMC6258269 | biostudies-literature | 2018 Dec

REPOSITORIES: biostudies-literature

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Global and pathway-specific transcriptional regulations of pactamycin biosynthesis in Streptomyces pactum.

Lu Wanli W Alanzi Abdullah R AR Abugrain Mostafa E ME Ito Takuya T Mahmud Taifo T

Applied microbiology and biotechnology 20181001 24

Pactamycin, a structurally unique aminocyclitol natural product isolated from Streptomyces pactum, has potent antibacterial, antitumor, and anti-protozoa activities. However, its production yields under currently used culture conditions are generally low. To understand how pactamycin biosynthesis is regulated and explore the possibility of improving pactamycin production in S. pactum, we investigated the transcription regulations of pactamycin biosynthesis. In vivo inactivation of two putative p ...[more]

PMID: 30276712

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Project description:Milbemycins, a group of 16-membered macrolides with potent anthelminthic and insecticidal activity, are produced by several Streptomyces and used widely in agricultural, medical and veterinary fields. Milbemycin A3 and A4, the main components produced by Streptomyces bingchenggensis, have been developed as an acaricide to control mites. The subsequent structural modification of milbemycin A3/A4 led to other commercial products, such as milbemycin oxime, lepimectin and latidectin. Despite its importance, little is known about the regulation of milbemycin biosynthesis, which has hampered efforts to enhance milbemycin production via engineering regulatory genes.milR, a regulatory gene in the milbemycin (mil) biosynthetic gene cluster of S. bingchenggensis, encodes a large ATP-binding regulator of the LuxR family (LAL family), which contains an ATPase domain at its N-terminus and a LuxR-like DNA-binding domain at the C-terminus. Gene disruption and genetic complementation revealed that milR plays an important role in the biosynthesis of milbemycin. ?-glucuronidase assays and transcriptional analysis showed that MilR activates the expression of the milA4-E operon and milF directly, and activates the other mil genes indirectly. Site-directed mutagenesis confirmed that the ATPase domain is indispensable for MilR's function, and particularly mutation of the conserved amino acids K37A, D122A and D123A, led to the loss of MilR function for milbemycin biosynthesis. Overexpression of an extra copy of milR under the control of its native promoter significantly increased production of milbemycin A3/A4 in a high-producing industrial strain S. bingchenggensis BC04.A LAL regulator, MilR, was characterized in the mil gene cluster of S. bingchenggensis BC04. MilR could activate milbemycin biosynthesis through direct interaction with the promoter of the milA4-E operon and that of milF. Overexpression of milR increased milbemycin A3/A4 production by 38 % compared with the parental strain BC04, suggesting that genetic manipulation of this activator gene could enhance the yield of antibiotics.

Project description:BackgroundStreptomyces albus J1074 produces glycosylated antibiotics paulomycin A, B and E that derive from chorismate and contain an isothiocyanate residue in form of paulic acid. Paulomycins biosynthesis pathway involves two glycosyltransferases, three acyltransferases, enzymes required for paulic acid biosynthesis (in particular an aminotransferase and a sulfotransferase), and enzymes involved in the biosynthesis of two deoxysugar moieties: D-allose and L-paulomycose.ResultsInactivation of genes encoding enzymes involved in deoxysugar biosynthesis, paulic acid biosynthesis, deoxysugar transfer, and acyl moieties transfer has allowed the identification of several biosynthetic intermediates and shunt products, derived from paulomycin intermediates, and to propose a refined version of the paulomycin biosynthesis pathway. Furthermore, several novel bioactive derivatives of paulomycins carrying modifications in the L-paulomycose moiety have been generated by combinatorial biosynthesis using different plasmids that direct the biosynthesis of alternative deoxyhexoses.ConclusionsThe paulomycins biosynthesis pathway has been defined by inactivation of genes encoding glycosyltransferases, acyltransferases and enzymes involved in paulic acid and L-paulomycose biosynthesis. These experiments have allowed the assignment of each of these genes to specific paulomycin biosynthesis steps based on characterization of products accumulated by the corresponding mutant strains. In addition, novel derivatives of paulomycin A and B containing L-paulomycose modified moieties were generated by combinatorial biosynthesis. The production of such derivatives shows that L-paulomycosyl glycosyltransferase Plm12 possesses a certain degree of flexibility for the transfer of different deoxysugars. In addition, the pyruvate dehydrogenase system form by Plm8 and Plm9 is also flexible to catalyze the attachment of a two-carbon side chain, derived from pyruvate, into both 2,6-dideoxyhexoses and 2,3,6-trideoxyhexoses. The activity of the novel paulomycin derivatives carrying modifications in the L-paulomycose moiety is lower than the original compounds pointing to some interesting structure-activity relationships.

Dataset Information

Global and pathway-specific transcriptional regulations of pactamycin biosynthesis in Streptomyces pactum.

Publications

Global and pathway-specific transcriptional regulations of pactamycin biosynthesis in Streptomyces pactum.

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