Genome Mining and Evolutionary Analysis Reveal Diverse Type III Polyketide Synthase Pathways in Cyanobacteria.

Cyanobacteria are prolific producers of natural products, including polyketides and hybrid compounds thereof. Type III polyketide synthases (PKSs) are of particular interest, due to their wide substrate specificity and simple reaction mechanism, compared with both type I and type II PKSs. Surprisingly, only two type III PKS products, hierridins, and (7.7)paracyclophanes, have been isolated from cyanobacteria. Here, we report the mining of 517 cyanobacterial genomes for type III PKS biosynthesis gene clusters. Approximately 17% of the genomes analyzed encoded one or more type III PKSs. Together with already characterized type III PKSs, the phylogeny of this group of enzymes was investigated. Our analysis showed that type III PKSs in cyanobacteria evolved into three major lineages, including enzymes associated with 1) (7.7)paracyclophane-like biosynthesis gene clusters, 2) hierridin-like biosynthesis gene clusters, and 3) cytochrome b5 genes. The evolutionary history of these enzymes is complex, with some sequences partitioning primarily according to speciation and others putatively according to their reaction type. Protein modeling showed that cyanobacterial type III PKSs generally have a smaller active site cavity (mean = 109.035 Å3) compared with enzymes from other organisms. The size of the active site did not correlate well with substrate size, however, the "Gatekeeper" amino acid residues within the active site were strongly correlated to enzyme phylogeny. Our study provides unprecedented insight into the distribution, diversity, and molecular evolution of cyanobacterial type III PKSs, which could facilitate the discovery, characterization, and exploitation of novel enzymes, biochemical pathways, and specialized metabolites from this biosynthetically talented clade of microorganisms.

Engineering mammalian cells to produce plant-specific N-glycosylation on proteins.

Protein N-glycosylation is an essential and highly conserved posttranslational modification found in all eukaryotic cells. Yeast, plants and mammalian cells, however, produce N-glycans with distinct structural features. These species-specific features not only pose challenges in selecting host cells for production of recombinant therapeutics for human medical use but also provide opportunities to explore and utilize species-specific glycosylation in design of vaccines. Here, we used reverse cross-species engineering to stably introduce plant core α3fucose (α3Fuc) and ß2xylose (ß2Xyl) N-glycosylation epitopes in the mammalian Chinese hamster ovary (CHO) cell line. We used directed knockin of plant core fucosylation and xylosylation genes (AtFucTA/AtFucTB and AtXylT) and targeted knockout of endogenous genes for core fucosylation (fut8) and elongation (B4galt1), for establishing CHO cells with plant N-glycosylation capacities. The engineering was evaluated through coexpression of two human therapeutic N-glycoproteins, erythropoietin (EPO) and an immunoglobulin G (IgG) antibody. Full conversion to the plant-type α3Fuc/ß2Xyl bi-antennary agalactosylated N-glycosylation (G0FX) was demonstrated for the IgG1 produced in CHO cells. These results demonstrate that N-glycosylation in mammalian cells is amenable for extensive cross-kingdom engineering and that engineered CHO cells may be used to produce glycoproteins with plant glycosylation.