Antenna size reduction and altered division cycles in self-cloned, marker-free genetically modified strains of Haematococcus pluvialis

Abstract The microalga Haematococcus pluvialis ( H. pluvialis ) is widely used for the commercial production of the red high value pigment astaxanthin. The major limitations of this species for supplying cheap natural astaxanthin in large scale cultivation are its slow growth rate and sensitivity to contaminants. We have applied recently developed advanced tools to genetically engineer H. pluvialis , by inserting two genes, the low CO 2 inducible gene lciA (predicted to be a bicarbonate transporter) and mutated nucleic acid binding protein I, nabI * (a constitutively active translational repressor of the LHCII proteins), which were previously reported to enhance biomass productivity when overexpressed in various microalgae. Both genes were successfully integrated into the H. pluvialis genome, either separately or together, using the endogenous norflurazon resistant mutated gene of phytoene desaturase as a selection marker. Successful incorporation and expression of both genes using a linear purified DNA fragment, containing exclusively rearranged lciA , mutated pds and nabI * genes of H. pluvialis , represents to our knowledge the first functional self-cloning approach in microalgae, a breakthrough that can facilitate the widespread use of genetically altered microalgae. Initial detailed characterization of the growth physiology of two transformed strains revealed important information as to the impact of those two genes. Overexpression of the lciA gene alone resulted in alterations in growth physiology and division cycle, but with little impact on biomass productivity. A transformed strain expressing both lciA and mutated nabI * displayed a significant reduction in chlorophyll content, an altered division cycle and possibly enhanced biomass and carotenoid productivity under certain conditions. However, further work and additional progress exploring an array of optimizing options will be required towards creation of modified strains of truly advanced biotechnological potential for commercial applications.