Stable transformation of Chlorella: rescue of nitrate reductase-deficient mutants with the nitrate reductase gene

• Published in: Current microbiology Vol. 35; no. 6; pp. 356 - 362
• Main Authors: Dawson, H.N, Burlingame, R, Cannons, A.C
• Format: Journal Article
• Language: English
• Published: New York, NY Springer 01.12.1997; Springer Nature B.V
• Subjects: Algae; Aquatic plants; Bacteriology; Biological and medical sciences; Biology of microorganisms of confirmed or potential industrial interest; Bioreactors; Biotechnology; CHLORELLA; Chlorella sorokiniana; Chlorella vulgaris; Fundamental and applied biological sciences. Psychology; Genes; Genetics; Mission oriented research; Nitrates; Recombinant microorganism; Chlorella; Genetic transformation; Gene; Enzyme; Nitrate reductase; Algae; Chlorophycophyta; Oxidoreductases; Mutation; Thallophyta
• ISSN: 0343-8651; 1432-0991
• DOI: 10.1007/s002849900268

Unicellular green algae, like Chlorella, offer a potentially useful system for the expression of heterologous proteins. However, the development of Chlorella as a bioreactor has been delayed owing to the lack of a stable transformation technique. Here we report on the use of micro-projectile bombardment to introduce the nitrate reductase (NR) gene from Chlorella vulgaris into NR-deficient Chlorella sorokiniana mutants, resulting in stable transformants. The stable transformants were able to grow on nitrate medium after repeated passages between selective and nonselective medium and exhibited inducible nitrate reductase activity comparable to that of wild-type cells. Southern analysis suggests homologous recombination occurs with insertion of the wild type gene into the mutated gene and that the genes of the two Chlorella species used are very similar. Specific RNase protection assays, selecting for a poorly conserved region of the gene, identified the presence of the C. vulgaris NR transcript only in the transformed C. sorkiniana mutant and not in the mutant