Linking chemistry and genetics in the growing cyanobactin natural products family

Abstract

Ribosomal peptide natural products are ubiquitous, yet relatively few tools exist to predict structures and clone new pathways. Cyanobactin ribosomal peptides are found in ~30% of all cyanobacteria, but the connection between gene sequence and structure was not defined, limiting the rapid identification of new compounds and pathways. Here, we report discovery of four orphan cyanobactin gene clusters by genome mining and an additional pathway by targeted cloning, which represented a tyrosine O-prenylating biosynthetic pathway. Genome mining enabled discovery of five cyanobactins, including peptide natural products from Spirulina supplements. A phylogenetic model defined four cyanobactin genotypes, which explain the synthesis of multiple cyanobactin structural classes and help direct pathway cloning and structure prediction efforts. These strategies were applied to DNA isolated from a mixed cyanobacterial bloom containing cyanobactins.

Figure 1. Prenylagaramide Gene Cluster from Planktothrix agardhii

A. Genetic organization of the pag cluster and sequences of all pag precursor peptides. Black: precursor peptides. Blue: N-terminal protease. Light green: C-terminal protease / macrocyclase. Amino acid sequences of prenylagaramide B and C are boxed, prenylated Tyr residues are highlighted in blue while the actual or predicted C-terminal cleavage site Pro is highlighted in red. Asterisks indicate 100% conservation among variants. Light blue-highlighted sequences indicate the more conserved N- and C-termini of the peptides versus the hypervariable inner region. B. Prenylated tyrosine residues are highlighted in blue. Structures and HRFTMS and IRMPD-MS/MS analysis of prenylagaramide B (Murakami et al., 1999) and C showing the molecular ion and the loss of one isoprenyl group. For 2, the indicated configuration is predicted based upon biogenetic considerations and on the structures of other compounds in the series. See also Table S1 and Figure S1.

Figure 1. Prenylagaramide Gene Cluster from Planktothrix agardhii

A. Genetic organization of the pag cluster and sequences of all pag precursor peptides. Black: precursor peptides. Blue: N-terminal protease. Light green: C-terminal protease / macrocyclase. Amino acid sequences of prenylagaramide B and C are boxed, prenylated Tyr residues are highlighted in blue while the actual or predicted C-terminal cleavage site Pro is highlighted in red. Asterisks indicate 100% conservation among variants. Light blue-highlighted sequences indicate the more conserved N- and C-termini of the peptides versus the hypervariable inner region. B. Prenylated tyrosine residues are highlighted in blue. Structures and HRFTMS and IRMPD-MS/MS analysis of prenylagaramide B (Murakami et al., 1999) and C showing the molecular ion and the loss of one isoprenyl group. For 2, the indicated configuration is predicted based upon biogenetic considerations and on the structures of other compounds in the series. See also Table S1 and Figure S1.

Figure 2. New Pathways by Genome Mining

A. New gene clusters. Black: precursor peptides. Blue: N-terminal protease. Red: Heterocyclase. Light green: C-terminal protease / macrocyclase. Yellow: oxidase. White: transposon. The size of the pathways is not shown to the exact scale. B. Precursor peptide sequences from the new gene clusters. Highlighted sequences indicate the more conserved N- and C-termini of the peptides versus the hypervariable inner region. Residues highlighted in red indicate the predicted C-terminal cleavage site. Asterisks indicate 100% conservation among variants. See also Figure S3.

Figure 3. New cyanobactins from Spirulina supplement powders

A. Genetic organization of the art cluster and sequences of all art precursor peptides. Color codes are the same as in Fig. 1 and Fig. 2. The exact sequences of Arthrospiramides A and B are boxed and the heterocyclized cysteine residues are underlined. B. Chemical structures of four new cyanobactins isolated from Arthrospira spirulina. Below each structure is shown the corresponding FT-MS showing the molecular ion and representative fragment ions verifying the cyclic peptide sequence. The configurational assignments are based upon biogenetic considerations. Assignments are ambiguous adjacent to thiazole residues because these stereocenters are generally chemically labile. See also Table S2 and Figure S4.

Figure 3. New cyanobactins from Spirulina supplement powders

A. Genetic organization of the art cluster and sequences of all art precursor peptides. Color codes are the same as in Fig. 1 and Fig. 2. The exact sequences of Arthrospiramides A and B are boxed and the heterocyclized cysteine residues are underlined. B. Chemical structures of four new cyanobactins isolated from Arthrospira spirulina. Below each structure is shown the corresponding FT-MS showing the molecular ion and representative fragment ions verifying the cyclic peptide sequence. The configurational assignments are based upon biogenetic considerations. Assignments are ambiguous adjacent to thiazole residues because these stereocenters are generally chemically labile. See also Table S2 and Figure S4.

Figure 4. Phylogeny of Cyanobactin Proteins

Maximum likelihood with molecular clock analysis (PROMLK, Phylip) was performed on all conserved cyanobactin proteins. Numbers indicate percentage of bootstrap replicates that support the indicated branch point. Due to the long computational times required, bootstrap numbers varied: PatG: 448; PatF: 892; PatD: 466; PatA: 602; oxidase: 442. Tree branches that were also supported by Maximum Parsimony analysis (MEGA 4.0) and verified using 1000 bootstrap replicates are indicated in blue. Representatives forming different genotypes are highlighted in different colors. “Mat” indicates the protein sequence of the metagenomic DNA fragments isolated by targeted degenerate PCR from a L. majuscula metagenomic bloom sample. See also Figure S6.

Figure 5. Cyanobactin Genotypes and Chemotypes

Cyanobactin biosynthetic pathways can be classified into 4 genotypes that correlate to the resulting chemotypes of natural products. thc products cannot be predicted and are indicated by a question mark. Additional pathways used in this figure include: pat, patellamide; ten, tenuecyclamide; mic, microcyclamide; tru, trunkamide; lyn, lyngbya; tri, trichamide; acy, anacylamide. See also Figure S7.