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 NO₃ ^- 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 NH₄ ⁺ and NO₂ ^- 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 NO₃ ^- 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.