Giant polyketide synthase enzymes in the biosynthesis of giant marine polyether toxins

Abstract

Prymnesium parvum are harmful haptophyte algae that cause massive environmental fish kills. Their polyketide polyether toxins, the prymnesins, are among the largest nonpolymeric compounds in nature and have biosynthetic origins that have remained enigmatic for more than 40 years. In this work, we report the "PKZILLAs," massive P. parvum polyketide synthase (PKS) genes that have evaded previous detection. PKZILLA-1 and -2 encode giant protein products of 4.7 and 3.2 megadaltons that have 140 and 99 enzyme domains. Their predicted polyene product matches the proposed pre-prymnesin precursor of the 90-carbon-backbone A-type prymnesins. We further characterize the variant PKZILLA-B1, which is responsible for the shorter B-type analog prymnesin-B1, from P. parvum RCC3426 and thus establish a general model of haptophyte polyether biosynthetic logic. This work 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) Map of historical or ongoing major P. parvum or Prymnesium spp. fish-killing blooms: England (49, 50), Netherlands (51), Israel (6, 52, 53), Texas (5, 54, 55), Scandinavia (4), and Oder River (3, 37). (B) Molecular structure of prymnesin-1 (31). (C) Molecular structure of prymnesin-B1 (56). (D) Comparison of polypeptide and coding nucleotide sizes from representative PKSs or computationally summed PKS systems. Blue indicates PKZILLAs from P. parvum 12B1 (this work); [S] indicates computationally summed lengths for independent PKS proteins that participate in the same biosynthetic system; black dashed lines indicate divisions of PKS systems into independent proteins; red and * indicate the largest known protein (non-PKS) (18); gold indicates representative bacterial PKS systems, including the quinolidomicin (** indicates the previous largest known PKS system) (42) and erythromycin (57) PKSs; and green and *** indicate the previous largest genetically studied microalgal PKS (16). AA, amino acid.

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

(A to C) 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, and exons are numbered 1 to 17 for PKZILLA-1 and 1 to 12 for PKZILLA-2. See fig. S2 for an alternative view and figs. S3 and S4 for a detailed view of each intron. The numbered protein-coding exons are colored light blue, medium blue, or dark blue on the basis of 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 section “Proteomic evidence for the PKZILLAs” in the text and fig. S10). 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- and trimodules are boxed in gray and categorized as S2/3M (saturating bi- and trimodule), PT2M (pass-through bimodule), and DH2M (dehydrating bimodule). See figs. S6 and S7 for non–length-normalized domains. KRn, N-terminal KR subdomain; KRc, C-terminal KR subdomain; KR*, catalytically novel or inactive KR.

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

(A to C) PKZILLA-1 snapshot assembly line reactions at (bi)modules M6, M21/M22, and M30/M31 that depict FLX-catalyzed α-hydroxylation, reducing nonelongation “pass-through”, and dual methylation-saturation reactions. Dotted lines indicate omitted contiguous PKZILLA protein sequence. (B) PKZILLA-2 snapshot assembly line reactions at modules M41, M50, and M56 that depict 2,4-dienoyl-ACP reduction, transamination, and decarboxylative desulfation reactions. (C) Proposed structures of the PKZILLA-compatible pre-prymnesin-1 biosynthetic precursor, its corresponding epoxy-pre-prymnesin-1 intermediate, and the prymnesin-1 aglycone (31). The unassigned starter R group in PPBP is presently unknown yet responsible for providing the terminal acetylene group. For simplicity, all oxidative modifications are depicted post assembly line. The snapshot reactions in panels (A) and (B) are correspondingly boxed in (C). See fig. S21 for further detail. Me, methyl.

Fig. 4.. Comparative genomics, biosynthesis, and chemistry of the A- and B-type prymnesins.

(A) Synteny plot of PKZILLA-1 from P. parvum 12B1 (top) and PKZILLA-B1 from P. parvum RCC3426 (bottom) showing a 15-kbp deletion and other modifications relative to the 12B1 sequence. (B) Domain organization of PKZILLA-B1 modules M16B to M21B and its assembly line biosynthesis of the differentiating C45–C55 fragment. Inactive domains are colored gray. (C) Partial structure and proposed conversion of pre–prymnesin-B1 via epoxy-pre–prymnesin-B1 to prymnesin-B1. Because the C46 stereocenter in prymnesin-B1 has not yet been established (56), the configuration of the C45-C46 alkene is not drawn. Full structures are shown in fig. S23. (D) Corresponding partial structures and biosynthesis of prymnesin-1 and intermediates highlighting the structure region that is distinct from the B1 series shown in (C). The omitted structure regions are identical between the A- and B-type prymnesins as supported by their respective PKZILLA gigasynthases. See Fig. 1 for full chemical structures.