Enhancement of Chlorella transformation efficacy by insert fragmentation

Abstract

Chlorella vulgaris, a microalga used to produce recombinant proteins, has several advantages as a bioreactor including ease of post-transitional modifications, low cost of mass culture, and a simple cell structure, and the latter facilitates purification of recombinant proteins. However, the development of efficient transformation methods based on useful selection markers, increased expression, and stability of integrated foreign DNA are still required. In this study, a transformation method for C. vulgaris has been developed in which the wild-type nitrate reductase (NR) gene is knocked out by triple homologous recombination using two overlapping DNA fragments flanked by the upstream and downstream NR fragments, respectively, to enhance the transformation efficiency. Knockout of NR gene enabled screening of transformed cells on plates containing 200 mM KClO3, which is reduced to chlorite, a compound toxic only to wild-type C. vulgaris. Randomly selected cells showed 70 % integration of the target DNA and expression of the foreign protein. We also optimized the culture medium, in which transformed Chlorella reached a density of 108 cells/mL. The integrated DNA was detected after long-term continuous subculture without loss even in the absence of KClO3 selection pressure.