Genetic engineering of the green alga Chlorella zofingiensis: a modified norflurazon-resistant phytoene desaturase gene as a dominant selectable marker

The unicellular green alga Chlorella zofingiensis has been proposed as a promising producer of natural astaxanthin, a commercially important ketocarotenoid. But the genetic toolbox for this alga is not available. In the present study, an efficient transformation system was established for C. zofingiensis. The transformation system utilized a modified norflurazon-resistant phytoene desaturase (PDS-L516F, with an leucine-phenylalanine change at position 516) as the selectable marker. Three promoters from endogenous PDS, nitrate reductase (NIT), and ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit (RBCS) genes were tested, with the RBCS promoter demonstrating the highest transformation efficiency. Inclusion of the first intron of the PDS gene further enhanced the efficiency by 91 %. Both particle bombardment and electroporation methods were examined, and the latter gave a fourfold higher transformation efficiency. The introduction of PDS-L516F, which exhibited a 33 % higher desaturation activity than the unaltered enzyme, enabled C. zofingiensis to produce 32.1 % more total carotenoids (TCs) and 54.1 % more astaxanthin. The enhanced accumulation of astaxanthin in transformants was revealed to be related to the increase in the transcripts of PDS, beta-carotenoid ketolase (BKT), and hydroxylase (CHYb) genes. Our study clearly shows that the modified PDS gene is a dominant selectable marker for the transformation of C. zofingiensis and possibly for the genetic engineering of the carotenoid biosynthetic pathway. In addition, the engineered C. zofingiensis might serve as an improved source of natural astaxanthin.