Figure 1.
Structures of echinomycin, thiocoraline,triostin A, SW-163C, QXC and HQA.
QXC: quinoxaline-2-carboxylic acid; HQA: 3-hydroxyquinaldic acid. Echinomycin and triostin A have QXC as their aromatic precursor during biosynthesis while thiocoraline and SW-163C have HQA as the aromatic precursor.
Figure 2.
Organization of echinomycin biosynthetic gene cluster and echinomycin biosynthetic pathway in S. griseovariabilis.
(A) Scheme of the biosynthetic gene cluster of echinomycin in S. griseovariabilis. The transcription direction of each gene was indicated by arrow. qui6 and qui7 are NRPS genes encoding the NRPS responsible for biosynthesis of the NRP backbone of echinomycin. To clarify the domains contained in Qui6 and Qui7, the scheme of the protein products encoded by qui6 and qui7 were situated rightly below the corresponding genes. Since the transcription directions of both qui6 and qui7 were reversed, their protein products were also exhibited inversely. From N terminal to C terminal, the domains contained were C, A, M, T, C, A, M, T, TE in Qui6 and C, A, T, E, C, A, T in Qui7. C: condensation-domain; A: adenylation-domain; T: peptidyl carrier protein-domain; M: methylation-domain; E: epimerase-domain; TE: thioesterase-domain. (B) Echinomycin biosynthetic pathway in S. griseovariabilis. FabC was an ACP engaged in fatty acid biosynthesis and its encoding gene was outside the gene cluster.
Figure 3.
Proposed biosynthetic pathway for QXC produced by
S. griseovariabilis. The biosynthesis of QXC is a part of that of echinomycin. L-Trp: L-tryptophan; L-Trp-S-Qui18: L-tryptophanyl-S-Qui18; (2S,3S) hTrp-S-Qui18: (2S,3S) β-hydroxytryptophanyl-S-Qui18; (2S,3S) hTrp: (2S,3S) β-hydroxytryptophan; (2S,3R) hNFK: (2S,3R) N-formyl-β-hydroxykynurenine; QXC: quinoxaline-2-carboxylic acid.
Table 1.
Deduced functions of the ORFs in the echinomycin biosynthetic gene cluster from S. griseovariabilis and nucleotide sequence alignment with echinomycin biosynthetic gene cluster from S. lasaliensis and triostin A biosynthetic gene cluster from S. triostinicus.
Figure 4.
Q-TOF-MS analysis of the amino acid loading activity of Qui18.
(A) Holo-Qui18 was used as the enzyme, L-tryptophan was tested as substrate; (B) The same as (A) except Qui5 was supplemented (C) Coexpression of Qui5 and holo-Qui18 was used as the enzyme, D-tryptophan was tested as substrate; (D) The same as (C) except the tested substrate was L-phenylalanine; (E) The same as (C) except the tested substrate was L-tryptophan; (F) MS result of the peak I, II and III, which respectively correspond to holo-Qui18, L-tryptophan loaded holo-Qui18 and Qui5; (G) Illustration of the protein molecule corresponding to I, II and III. The molecular weight change from I to II was so small relatively to the molecular weight of I that the retention time for I and II were nearly the same.
Figure 5.
LC-MS analysis of the hydroxylation activity of Qui15.
Before the hydroxylation reaction, all the samples were incubated with coexpression of Qui5 and holo-Qui18 for 2 hours to ensure the loading of L-tryptophan other than the negative control of (A). (A) L-tryptophan wasn’t incubated with coexpression of Qui5 and holo-Qui18 and hence all L-tryptophan was free instead of the loaded form; (B) L-tryptophan was incubated with coexpression of Qui5 and holo-Qui18 form but then no Qui15 was supplemented; (C) After the incubation of L-tryptophan and coexpression of Qui5 and holo-Qui18 for 2 hours, Qui15 was supplemented and an additional 60 min incubation was adopted; (D) the same as (C), except the additional incubation time was 120 min; (e) (2S,3S) β-hydroxytryptophan standard sample. The peak I is L-tryptophan and the peak II is β-hydroxytryptophan.
Figure 6.
Phylogenetic tree representing evolutionary relationships of the tryptophan 2,3-dioxygenase family.
S. griseovariabilis is the strain from which the gene cluster was reported by us and it’s an echinomycin producer. S. triostinicus, S. lasaliensis, micromonospora sp. ML1 and S. sp. SNA15896 respectively produce triostin A, echinomycin, thiocoraline and SW-163. The above five strains are underlined. The apparent evolutionary distances among these family members were shown.
Figure 7.
LC-MS comparison of echinomycin standard to the fermentation products from wild-type S. griseovariabilis and qui17::aadA mutant.
The peak I is echinomycin; WT: wild-type S. griseovariabilis; ZC1: S. griseovariabilis ZC1 mutant; ECM: echinomycin standard.
Figure 8.
LC-MS analysis of the reaction catalyzed by Qui17.
‘No protein’ represents the sample of negative control lacking Qui17.The reaction was terminated after incubation of 30 min, 60 min, 90 min respectively. The peaks I and II are (2S,3S) β-hydroxytryptophan and (2S,3R) N-formyl-β-hydroxykynurenine. The chemical structure of each compound is shown.