Biosynthetic mechanism of the yellow pigments in the marine bacterium Pseudoalteromonas sp. strain T1lg65

Abstract

The Pseudoalteromonas genus marine bacteria have attracted increasing interest because of their abilities to produce bioactive metabolites. The pigmented Pseudoalteromonas group encodes more secondary metabolite biosynthetic gene clusters (BGCs) than the non-pigmented group. Here, we report a yellow pigmented bacterium Pseudoalteromonas sp. strain T1lg65, which was isolated from a mangrove forest sediment. We showed that the yellow pigments of T1lg65 belong to the group of lipopeptide alterochromides. Further genetic analyses of the alterochromide BGC revealed that the yellow pigments are biosynthesized by aryl-polyene synthases and nonribosomal peptide synthases. Within the gene cluster, altA encodes a tyrosine ammonia acid lyase, which catalyzes synthesis of the precursor 4-hydroxycinnamic acid (4-HCA) from tyrosine in the alterochromide biosynthetic pathway. In addition, altN, encoding a putative flavin-dependent halogenase, was proven to be responsible for the bromination of alterochromides based on gene deletion, molecular docking, and site mutagenesis analyses. In summary, the biosynthetic pathway, precursor synthesis, and bromination mechanism of the lipopeptide alterochromides were studied in-depth. Our results expand the knowledge on biosynthesis of Pseudoalteromonas pigments and could promote the development of active pigments in the future.IMPORTANCEThe marine bacteria Pseudoalteromonas spp. are important biological resources because they are producers of bioactive natural products, including antibiotics, pigments, enzymes, and antimicrobial peptides. One group of the microbial pigments, alterochromides, holds a great value for their novel lipopeptide structures and antimicrobial activities. Previous studies were limited to the structural characterization of alterochromides and genome mining for the alterochromide biosynthesis. This work focused on the biosynthetic mechanism for alterochromide production, especially revealing functions of two key genes within the gene cluster for the alterochromide biosynthesis. On the one hand, our study provides a target for metabolic engineering of the alterochromide biosynthesis; on the other hand, the 4-HCA synthase AltA and brominase AltN show potential in the biocatalyst industry.

Keywords: Pseudoalteromonas; alterochromides; biosynthesis; halogenation.

Fig 1

Chemical structure and mass spectrum for the alterochromide molecules. (A) Mass spectrum of the wild-type strain cultivated in 2216E medium revealed two highest peaks with mass-to-charge ratio (m/z) of 792.54 and 806.57, and the sample exhibited a golden yellow color. (B) Mass spectrum of the wild-type strain cultivated in 2216E medium with exogenous bromides revealed four highest peaks with m/z of 870.53, 886.53, 950.46, and 964.45, and the sample exhibited a yellow–brown color. (C) Potential molecular results and their m/z values for alterochromides. The B-form molecule has an isoleucine as the C-terminal residue, while the B′-form molecule has a leucine as the C-terminal residue.

Fig 2

Alterochromide biosynthetic gene cluster in Pseudoalteromonas sp. strain T1lg65. Comparison of the alterochromide biosynthetic gene clusters of Pseudoalteromonas sp. strain JCM 20779 and Pseudoalteromonas sp. strain T1lg65. FAS: fatty acid synthase; NRPS: non-ribosomal peptide synthetase. Homology comparison of the gene clusters between Pseudoalteromonas sp. strain JCM 20779 and Pseudoalteromonas sp. strain T1lg65 can be found in Table S1.

Fig 3

Colony colors and the extracted ion chromatogram for the wild-type (WT) and mutant strains. (A) Extracted ion chromatogram of WT, with L-tyrosine, p-coumaric acid, alterochromide, bromoalterochromide, and dibromoalterochromide present. (B) Extracted ion chromatogram of ΔaltA (the altA-deleted mutant), with only L-tyrosine present. (C) Extracted ion chromatogram of ΔaltB (the altB-deleted mutant), with L-tyrosine and p-coumaric acid present. (D) Extracted ion chromatogram of ΔaltCD (the altCD-deleted mutant), with L-tyrosine and p-coumaric acid present. (E) Extracted ion chromatogram of ΔaltJ (the altJ-deleted mutant), with L-tyrosine, p-coumaric acid, alterochromide, bromoalterochromide, and dibromoalterochromide present. (F) Extracted ion chromatogram of ΔaltK (the altK-deleted mutant), with L-tyrosine and p-coumaric acid present. Each of the aforementioned samples was obtained after overnight cultivation in 2216E medium with exogenous addition of KBr (1 g/L).

Fig 4

Characterization of tyrosine ammonia acid lyase AltA. (A) SDS-PAGE analyses of the heterologous expression of AltA and its expression level. The AltA protein possesses a molecular weight of 59.53 kDa. (B) Analyses of the substrate specificity of AltA using HPLC. (C) Determination of the optimal temperature for the enzyme activity of AltA. (D) Determination of the optimal pH for the enzyme activity of AltA. The optimal temperature and pH for the catalytic activity of AltA is 40°C and 11, respectively.

Fig 5

Effect of the AltN halogenase on production and halogenation of alterochromides. (A) The ΔaltN strain without KBr. (B) The ΔaltN strain with KBr. (C) The ΔaltN^c strain without KBr. (D) The ΔaltN^c strain with KBr. Pigment extracts and the corresponding MS spectrums of the ΔaltN and ΔaltN^c strains cultured in 2216E media with or without KBr are shown.

Fig 6

Characterization of the structure and function of the AltN halogenase. (A) Global diagram of the substrate–protein binding interaction associated with an FAD-binding domain and a substrate-binding domain. (B) Schematic representation of the FAD–protein interaction, with the dashed lines indicating hydrogen bonding. (C) Schematic representation of the substrate–protein interactions, with the dashed lines indicating hydrogen bonding. (D) Magnified view of the key interaction sites, where the distance between the active center of the substrate-binding domain and metastable complex is 10.05 Å, and exogenous bromide ions bind to the FAD-binding domain through a channel of 3.85 Å in length. (E) Secondary metabolite component profile of the mutant strains, where blue represents the presence of the product and white represents the absence of the product. The key amino acid residue of Lys73 (K73) was changed to other amino acid residues (K73X where X refers to one of other amino acid residues) through the saturation mutagenesis technique. Alterochromides B/B′: Alt-B/B′; alterochromide C: Alt-C; bromoalterochromides B/B′: bromoAlt-B/B′; bromoalterochromide C: bromoAlt-C; dibromoalterochromides B/B′: dibromoAlt-B/B′.

Fig 7

Proposed biosynthetic pathway for the alterochromide production in Pseudoalteromonas sp. strain T1lg65. R: aryl-polyene starter unit; C: condensation domain; A: adenylation domain; T: thiolation domain; E: epimerization domain; TE: thioesterase domain; and ACP: acyl carrier protein.