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Characterization of the Jomthonic Acids Biosynthesis Pathway and Isolation of Novel Analogues in Streptomyces caniferus GUA-06-05-006A.

ABSTRACT: Jomthonic acids (JAs) are a group of natural products (NPs) with adipogenic activity. Structurally, JAs are formed by a modified ?-methylphenylalanine residue, whose biosynthesis involves a methyltransferase that in Streptomyces hygroscopicus has been identified as MppJ. Up to date, three JA members (A?C) and a few other natural products containing ?-methylphenylalanine have been discovered from soil-derived microorganisms. Herein, we report the identification of a gene (jomM) coding for a putative methyltransferase highly identical to MppJ in the chromosome of the marine actinobacteria Streptomyces caniferus GUA-06-05-006A. In its 5' region, jomM clusters with two polyketide synthases (PKS) (jomP1, jomP2), a nonribosomal peptide synthetase (NRPS) (jomN) and a thioesterase gene (jomT), possibly conforming a single transcriptional unit. Insertion of a strong constitutive promoter upstream of jomP1 led to the detection of JA A, along with at least two novel JA family members (D and E). Independent inactivation of jomP1, jomN and jomM abolished production of JA A, JA D and JA E, indicating the involvement of these genes in JA biosynthesis. Heterologous expression of the JA biosynthesis cluster in Streptomyces coelicolor M1152 and in Streptomyces albus J1074 led to the production of JA A, B, C and F. We propose a pathway for JAs biosynthesis based on the findings here described.

SUBMITTER: Garcia-Salcedo R

PROVIDER: S-EPMC6117699 | biostudies-literature | 2018 Jul

REPOSITORIES: biostudies-literature

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Characterization of the Jomthonic Acids Biosynthesis Pathway and Isolation of Novel Analogues in Streptomyces caniferus GUA-06-05-006A.

García-Salcedo Raúl R Álvarez-Álvarez Rubén R Olano Carlos C Cañedo Librada L Braña Alfredo F AF Méndez Carmen C de la Calle Fernando F Salas José A JA

Marine drugs 20180731 8

Jomthonic acids (JAs) are a group of natural products (NPs) with adipogenic activity. Structurally, JAs are formed by a modified β-methylphenylalanine residue, whose biosynthesis involves a methyltransferase that in Streptomyces hygroscopicus has been identified as MppJ. Up to date, three JA members (A⁻C) and a few other natural products containing β-methylphenylalanine have been discovered from soil-derived microorganisms. Herein, we report the identification of a gene (jomM) codi ...[more]

PMID: 30065171

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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.

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.

Dataset Information

Characterization of the Jomthonic Acids Biosynthesis Pathway and Isolation of Novel Analogues in Streptomyces caniferus GUA-06-05-006A.

Publications

Characterization of the Jomthonic Acids Biosynthesis Pathway and Isolation of Novel Analogues in <i>Streptomyces caniferus</i> GUA-06-05-006A.

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