Giant polyketide synthase enzymes biosynthesize a giant marine polyether biotoxin

Abstract

Prymnesium parvum are harmful haptophyte algae that cause massive environmental fish-kills. Their polyketide polyether toxins, the prymnesins, are amongst the largest nonpolymeric compounds in nature, alongside structurally-related health-impacting "red-tide" polyether toxins whose biosynthetic origins have been an enigma for over 40 years. Here we report the 'PKZILLAs', massive P. parvum polyketide synthase (PKS) genes, whose existence and challenging genomic structure evaded prior detection. PKZILLA-1 and -2 encode giant protein products of 4.7 and 3.2 MDa with 140 and 99 enzyme domains, exceeding the largest known protein titin and all other known PKS systems. Their predicted polyene product matches the proposed pre-prymnesin precursor of the 90-carbon-backbone A-type prymnesins. This discovery establishes a model system for microalgal polyether biosynthesis and expands expectations of genetic and enzymatic size limits in biology.

Fig. 1.. Prymnesin, its source PKZILLA polyketide synthases (PKSs), and other large proteins and PKS systems.

(A) Molecular structure of prymnesin-1 (16). (B) Comparison of polypeptide and coding nucleotide sizes from representative PKSs or computationally summed PKS systems. Blue=PKZILLAs from P. parvum 12B1 (this work). [S]=Computationally summed lengths for independent PKS proteins that participate in the same biosynthetic system. Black dashed lines=Divisions of PKS systems into independent proteins. Red/*=largest known protein (non-PKS) (15). Gold=Representative bacterial PKS systems, including the quinolidomicin (**=previous largest known PKS system) (17) and erythromycin (18) PKSs. Green/***=previous largest genetically studied microalgal PKS (13).

Fig. 2.. Genomic, transcriptomic, and proteomic evidence for the PKZILLAs.

Genomic PKS hotspot loci with gene models and PKS domain and module annotations for (A) PKZILLA-1 and (B) PKZILLA-2. (A, B) Red boxes denote chromosomal locations and relative sizes of the PKZILLA genes. The contiguous log-scale forward-stranded read coverage from the stranded rRNA-depletion RNA-Seq (in gray) is shown across the PKZILLA gene models (in blue). Introns are highlighted with black arrows, while exons are numbered 1–17 for PKZILLA-1 and 1–12 for PKZILLA-2. See fig. S2 for an alternative view and fig. S3, S4 for a detailed view of each intron. The numbered protein-coding exons are colored light blue, medium blue, or dark blue based on whether supporting proteomic peptides from that exon were not detected, detected by protein-multimatch peptide matches alone, or detected by protein-unique plus exon-unique peptide matches, respectively (See Proteomic evidence for the PKZILLAs in Results; fig. S9). Domain and module annotations (starting with the loading module (LM) and module 1 (M1) of PKZILLA-1 and ending with M56 of PKZILLA-2) are shown below the gene models, see key in (C). The bi/tri-modules are boxed in gray and categorized as S2/3M=saturating bi/tri-module, PT2M=pass-through bimodule, and DH2M=dehydrating bimodule. See fig. S6, S7 for non-length-normalized domains.

Fig. 3.. Alignment of PKZILLA PKS modules with the proposed prymnesin biosynthetic precursor.

(A) Structure of the pre-prymnesin biosynthetic precursor (PPBP) inferred from retrobiosynthetic analysis of a structurally compatible poly-epoxide cyclization cascade from the prymnesin-1 aglycone (PA) (16), and its end-to-end alignment with the assembly line modules of the PKZILLA-1/-2 gigasynthase. Select hypothesized reactions are called out in subpanels (B, C, D). See fig S20 for further detail.