Iterative pathway engineering of the diatom Phaeodactylum tricornutum to enhance the biosynthesis of long‐chain polyunsaturated fatty acids using the Cre recombinase‐mediated marker recycling

In the diatom Phaeodactylum tricornutum there are limited selectable markers available for genetic manipulation, which has hampered the biotechnological application of metabolic engineering in this algal species. Here, we developed a bacteriophage P1-derived Cre/loxP recombination system in P. tricornutum, enabling the excision and recycling of the selectable marker. The Sh ble cassette conferring Zeocin resistance, flanked by the loxP sites in the same orientation, could be excised upon expression of the Cre recombinase under the control of the promoter of the nitrate reductase gene, or was encoded on an episome replication vector delivered by bacterial conjugation. An intron was included in the Cre recombinase ORF to prevent self-excision in Escherichia coli. Combining this Cre/loxP system with a multigene assembly method enabled iterative pathway engineering of P. tricornutum to enhance eicosapentaenoic acid (EPA) biosynthesis. The average level of EPA in total fatty acids increased from 25% in wild-type to 31% in the sextuple transgenic lines. Further photoautotrophic cultivations demonstrated the maximal EPA productivity of 3 mg l^-1 d^-1. The Cre/loxP system will be a highly efficient tool to obtain marker-free transgenic algal strains, and should be applicable to engineering of diatoms with complex metabolic pathways to produce valuable metabolites.