Abstract
The model diatom Phaeodactylum tricornutum is an attractive candidate for synthetic biology applications. Development of auxotrophic strains of P. tricornutum would provide alternative selective markers to commonly used antibiotic resistance genes. Here, using CRISPR/Cas9, we show successful editing of genes in the uracil, histidine, and tryptophan biosynthetic pathways. Nanopore long-read sequencing indicates that editing events are characterized by the occurrence of large deletions of up to ~ 2.7 kb centered on the editing site. The uracil and histidine-requiring phenotypes can be complemented by plasmid-based copies of the intact genes after curing of the Cas9-editing plasmid. Growth of uracil auxotrophs on media supplemented with 5-fluoroorotic acid and uracil results in loss of the complementing plasmid, providing a facile method for plasmid curing with potential applications in strain engineering and CRISPR editing. Metabolomic characterization of uracil auxotrophs revealed changes in cellular orotate concentrations consistent with partial or complete loss of orotate phosphoribosyltransferase activity. Our results expand the range of P. tricornutum auxotrophic strains and demonstrate that auxotrophic complementation markers provide a viable alternative to traditionally used antibiotic selection markers. Plasmid-based auxotrophic markers should expand the range of genome engineering applications and provide a means for biocontainment of engineered P. tricornutum strains.
Highlights
The model diatom Phaeodactylum tricornutum is an attractive candidate for synthetic biology applications
Uracil-requiring mutants were generated by random mutagenesis that resulted in the identification of the bi-functional uridine monophosphate synthase (PtUMPS) gene predicted to catalyze the conversion of orotate into uridine monophosphate (UMP)[22]
The PHATR_3140 gene, hereafter called PtPRA-PH/CH, encodes a predicted bifunctional protein that shares sequence similarity with the bacterial protein HisIE, and its plant counterpart HISN225,26. These proteins possess two functional domains that are homologous to the phosphoribosyl-ATP pyrophosphohydrolase (PRA-PH) and phosphoribosyl-AMP cyclohydrolase (PRA-CH) enzymes, respectively
Summary
The model diatom Phaeodactylum tricornutum is an attractive candidate for synthetic biology applications. The uracil and histidine-requiring phenotypes can be complemented by plasmid-based copies of the intact genes after curing of the Cas9-editing plasmid. P. tricornutum is diploid, meaning that Cas9-edited cells must be carefully screened to determine if knockouts are monoallelic or biallelic and exhibit loss of heterozygosity These plasmid-based tools and synthetic pathways are currently maintained by available antibiotic-based selections, including zeocin, phleomycin, nourseothricin, and blasticidin-S and their resistance genes, Sh ble, nat, and bsr[12,13,14,15]. We used a plasmid-based editing strategy to generate knockouts in the uracil, histidine, and tryptophan biosynthesis pathways of P. tricornutum and show for the first time that plasmid-based copies of the intact PtUMPS and PtPRA-PH/CH genes can complement the uracil- and histidine-requiring phenotypes, respectively. Our results suggest a simple methodology to cure plasmids from uracil auxotrophs to enable strain and genome engineering
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