Isolation and characterization of a nitrate reductase deficient mutant of Chlorella ellipsoidea (Chlorophyta)

Using X-ray mutagenesis and a chlorate-resistance selection method, a nitrate reductase (NR)-deficient mutant of Chlorella ellipsoidea nrm- 4 was isolated, which is incapable of using NO 3 − as a nitrogen source. NR activity was not detected in nrm- 4, suggesting this is a null mutation. Molecular analysis revealed that nrm- 4 has a two base deletion at position 2348–49 of its DNA sequence, which produced a frame-shift mutation. The nrm- 4 mutation is stable and nrm- 4 algae could only use NH 4 + and NO 2 − as nitrogen sources. Expression of a wild-type NR gene could complement this NR-deficient mutant. Furthermore, this transgenic strain was able to grow in NO 3 − medium, when the growth rate of the nrm- 4 strain was equal to that of the wide type alga. The nrm- 4 strain could potentially be used as a bioreactor that uses nitrate as a selectable marker instead of an antibiotic or herbicide.