Abstract
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.
Highlights
Increase in greenhouse gas emissions and associated global warming has led to growing interest in the development and production of biofuels
The presence or absence of the loxP_cUMPS_loxP sequence in the genomes of 436 transformants was examined by PCR with the primer set, loxPF and loxPR, and forty transformants were shown to contain the intact loxP_cUMPS_loxP sequence (Supplementary Fig. S2b)
The copy number of cDNA of the uridine monophosphate synthase gene (cUMPS) was determined by quantitative real-time PCR
Summary
Increase in greenhouse gas emissions and associated global warming has led to growing interest in the development and production of biofuels. Strain improvement to increase microalgal lipid productivity would be required. Studies on this subject are still at very preliminary stages[2]. Efforts for crop improvement without introducing foreign DNA have been made to avoid public concern. These methods are referred to as “self-cloning” in microorganisms and “cisgenesis and intragenesis” in plants. The frequency of transformants containing the intact expression units of the two genes was very low (less than 10% of Ura+); and second, the transgene expression was not always high probably due to transgene silencing This problem could be overcome if the two expression cassettes are introduced sequentially, each time selecting the best transformant(s). Current self-cloning system with only one selectable marker gene is not applicable to such sequential introduction of transgenes
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