Developing chloroplast and nuclear transformation methods for a fast growing alga
Joanna Szaub and Saul Purton
Structural and Molecular Biology, University College London, UK.
Commercial exploitation of algae requires strains with high rates of biomass production. A search of the literature suggests that the alga with the fastest growth rate yet determined is the thermotolerant species Chlorella sorokiniana with reported doubling times of less than two hours. This value is significantly lower than that reported for other green algae, and for other algal groups including diatoms and cyanobacteria. We have compared three different isolates of C. sorokiniana and identified one that has the best performance with doubling times below three hours and an ability to grow at temperatures as high as 42oC. We are currently optimizing light, temperature and media conditions to achieve the maximum growth rate. In order to exploit this alga's potential as a platform for the synthesis of valuable recombinant products we are developing both nuclear and chloroplast transformation methods. As a first step towards nuclear transformation, we have isolated mutants displaying resistance to the 80S ribosome inhibitor, cycloheximide. Molecular analysis reveals that, in all cases, resistance is associated with a mutation in the nuclear gene (RPL41) encoding ribosomal subunit L41. Moreover, the mutation is associated with a change of a specific proline residue to leucine, serine or threonine, with the P-L change conferring the highest level of resistance. The RPL41 gene from this mutant should provide a suitable dominant marker for transformation of the wild-type strain. For the development of a chloroplast transformation vector we have isolated and sequenced several regions of the chloroplast genome. These have been used to create a plasmid that contains the psbB-clpP region together with an aadA selectable marker within the intergenic region. In order to be functional in C. sorokiniana, the marker is fused to the endogenous psbA promoter/5' UTR element. Biolistic transformation of wild-type cells has results in putative transformant line s resistant to spectinomycin and streptomycin. We are currently checking these lines for the presence of the aadA marker in the chloroplast genome.
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