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. 2011 Aug;193(16):4214-23.
doi: 10.1128/JB.05062-11. Epub 2011 Jun 10.

Amino acid precursor supply in the biosynthesis of the RNA polymerase inhibitor streptolydigin by Streptomyces lydicus

Cristina Gómez  1 , Dina H Horna, Carlos Olano, Martina Palomino-Schätzlein, Antonio Pineda-Lucena, Rodrigo J Carbajo, Alfredo F Braña, Carmen Méndez, José A Salas
Affiliations

Amino acid precursor supply in the biosynthesis of the RNA polymerase inhibitor streptolydigin by Streptomyces lydicus

Cristina Gómez et al. J Bacteriol. 2011 Aug.

Abstract

Biosynthesis of the hybrid polyketide-nonribosomal peptide antibiotic streptolydigin, 3-methylaspartate, is utilized as precursor of the tetramic acid moiety. The three genes from the Streptomyces lydicus streptolydigin gene cluster slgE1-slgE2-slgE3 are involved in 3-methylaspartate supply. SlgE3, a ferredoxin-dependent glutamate synthase, is responsible for the biosynthesis of glutamate from glutamine and 2-oxoglutarate. In addition to slgE3, housekeeping NADPH- and ferredoxin-dependent glutamate synthase genes have been identified in S. lydicus. The expression of slgE3 is increased up to 9-fold at the onset of streptolydigin biosynthesis and later decreases to ∼2-fold over the basal level. In contrast, the expression of housekeeping glutamate synthases decreases when streptolydigin begins to be synthesized. SlgE1 and SlgE2 are the two subunits of a glutamate mutase that would convert glutamate into 3-methylaspartate. Deletion of slgE1-slgE2 led to the production of two compounds containing a lateral side chain derived from glutamate instead of 3-methylaspartate. Expression of this glutamate mutase also reaches a peak increase of up to 5.5-fold coinciding with the onset of antibiotic production. Overexpression of either slgE3 or slgE1-slgE2 in S. lydicus led to an increase in the yield of streptolydigin.

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Figures

Fig. 1.
Fig. 1.
Proposed pathway for the biosynthesis of streptolydigin (compound 1). The incorporation of glutamate, in the form of 3-methylaspartate, to generate the tetramic acid lateral side chain is shown in thick lines. M7, PKS module 7; KS, ketosynthase; ATa, acyl transferase specific for malonyl-CoA; ACP, acyl carrier protein; KR*, inactive ketoreductase; C, condensation domain; A', SlgN1 adenylation domain; A, SlgN2 adenylation domain; PCP, peptidyl carrier protein.
Fig. 2.
Fig. 2.
(A) Scheme representing the replacement in the chromosome of the wild-type slgE3 gene by a version mutated through the insertion of an apramycin resistance cassette. (B) UPLC analysis of mutant SLME3. (C) UPLC analysis of mutant SLME3 complemented by pEM4TslgE3. (D) Scheme representing the replacement of the wild-type slgE1-E2 genes by one mutated through the insertion of an apramycin resistance cassette. (E) UPLC analysis of mutant SLME1E2 and MS analysis of compounds 2 and 3. (F) UPLC analysis of mutant SLME1E2 complemented by pEM4TslgE1E2. aac(3)IV, apramycin resistance gene; hyg, hygromycin resistance gene; bla, β-lactamase gene; 1, streptolydigin; 2, streptolydigin B; 3, streptolydigin C; AU, arbitrary units.
Fig. 3.
Fig. 3.
(A) Structures of streptolydigin B (compound 2) and novel derivative streptolydigin C (compound 3) showing the proposed origin of tetramic acid lateral side chain from glutamate (thick lines). C, condensation domain; A′, SlgN1 adenylation domain; A, SlgN2 adenylation domain; PCP, peptidyl carrier protein. (B) Antibiotic activity of streptolydigin (area 1) and streptolydigin C (area 3) against S. albus. Each paper disk was soaked with 2 μg of the corresponding compound. Control without antibiotic (area C).
Fig. 4.
Fig. 4.
Effect of slgE3 or slgE1-slgE2 overexpression in S. lydicus on streptolydigin production. Cultures were performed on R5A solid medium, and streptolydigin production was determined by high-pressure liquid chromatography analysis. Experiments were run in triplicate. C, S. lycidus/pEM4T; E3, S. lydicus/pEM4TslgE3; E1/E2, S. lydicus/pEM4TslgE1E2.
Fig. 5.
Fig. 5.
(A) Scheme representing PCR amplification of glutamate synthase gluS-α, gluS-β, and gluS-FD from the chromosome of S. lydicus NRRL2433. (B) Sequence alignments of NADPH-dependent glutamate synthase α subunits showing the conserved regions used for designing degenerated the oligoprimers HEI-I, HEI-J, and HEI-F. GluS-α, S. lydicus NRRL2433; SAV6189, S. avermitilis MA-4680; SCO2026, S. coelicolor A3(2); SACE3998 and SACE5742, Saccharopolyspora erythraea NRRL2338. (C) Sequence alignments of NADPH-dependent glutamate synthase β subunits showing the conserved regions used for designing degenerated oligoprimer HEI-C. GluS-β, S. lydicus NRRL2433; SAV6190 and SAV6258, S. avermitilis MA-4680; SCO1977 and SCO2025, S. coelicolor A3(2); SACE3997 and SACE5741, Saccharopolyspora erythraea NRRL2338. (D) Sequence alignments of ferredoxin-dependent glutamate synthase showing the conserved regions used for designing degenerated oligoprimers HEI-FR1 and HEI-FR2. SlgE3 and GluS-FD, S. lydicus NRRL2433; SAV954 and SAV1232, S. avermitilis MA-4680; SSEG09946; S. sviceus ATCC 29083; SSAG01435, Streptomyces sp. strain Mg1. aa, amino acids.
Fig. 6.
Fig. 6.
(A) Growth and streptolydigin production by S. lydicus wild-type strain in R5A medium. Growth was monitored by measuring the absorbance at 600 nm for determining DNA content by the diphenylamine assay. Streptolydigin production was spectrophotometrically quantified at 360 nm by peak area integration (⋄). Experiments were run in triplicate. The arrows show time points when total RNA was isolated. (B) Transcriptional analysis of S. lydicus GSs (slgE3, gluS-α, gluS-β, and gluS-FD) and GM (slgE2) by RT-PCR. RNA samples were obtained, during growth in R5A liquid medium, at 12, 24, 48, and 72 h from S. lydicus wild type and at 72 h from S. lydicus SLME3 and SLME1E2. (C) Quantification of gene expression by qRT-PCR. The hrdB gene was used as an internal control to quantify the relative expression of target genes. The expression level of each gene at 12 h was taken as the calibrator. Error bars were calculated from three independent determinations of mRNA abundance in each sample.
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