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We previously developed a self-cloning system that introduces cDNA of the uridine monophosphate synthase gene ( cUMPS ) of  Coccomyxa  sp. strain Obi as a selectable marker into uracil-auxotrophic mutants (Ura − ) of the same alga. Here, we developed a Cre/ loxP -based system for the removal of  cUMPS  flanked by directly repeated  loxP  sites from the  Coccomyxa  genome using the intracellular delivery of purified Cre recombinase to generate an Ura −  strain that was used as a host for second-round transformation using  cUMPS  as the selection marker. Employing this marker–gene-recycling system,  Coccomyxa  strains devoid of foreign DNA except the 34-bp  loxP  sequence, which overexpressed an acyl-(acyl-carrier-protein) thioesterase gene, and a type-2 diacylglycerol acyltransferase gene, were constructed by the sequential introduction of two expression cassettes for the respective genes. One of the resulting strains showed 1.4-fold higher lipid productivity than the wild-type strain. This method will be applicable to other eukaryotic microalgae to create marker-free transgenic strains.

Metabolic engineering using iterative self-cloning to improve lipid productivity in Coccomyxa