A Study on two Transporters Important for Carbon Partitioning in C. reinhardtii

Abstract

Microalgae are potential sources of energy and high-value materials. Microalgae platform has advantages for not only reducing greenhouse gases but also converting inorganic carbon to organic carbon as carbohydrates or lipids. I approached forward and reverse genetics methods to test the ability of microalgae as a potential platform. First, I created a mutant pool of strain CC-503 of the model green microalga Chlamydomonas reinhardtii, by random insertion of an antibiotic resistance gene (AphVII), and screened the pool for lines that showed altered carbon metabolism. I identified a mutant that mutated in CrMEX1, a putative Maltose Exporter-Like protein 1 (Cre12.g486600.t1.2). The mutant had reduced levels of CrMEX1 expression and, similarly to the Arabidopsis mex1 knockout mutant, which over-accumulated starch granules in the chloroplast. The mutant’s lipid levels were slightly higher than those of the wild type, and its initial growth kinetics were not significantly different from those of the wild type, but the mutant culture did not reach the same high cell density as the wild type in acetate-containing culture medium under continuous light. These results suggest that CrMEX1 encodes a maltose transporter protein and that export of photoassimilates from chloroplasts is necessary for normal Chlamydomonas growth, even under continuous light with an ample supply of carbon in the form of acetate. Second, I found CrABCA2, an ER-localized Chlamydomonas ABCA transporter as a homologous gene of Arabidopsis ABCA9, reported as an ER-localized acyl-CoA and fatty acid transporter (Kim et al., 2013) in the Chlamydomonas. I used phylogenetic analysis to isolated CrABCA2, one of the closest genes with AtABCA9 among five ABCA genes in Chlamydomonas genome. CrABCA2 expression and protein level were highly induced by nitrogen starvation, which is TAG biosynthesis inducing condition. crabca2 knockout mutants could not accumulate TAGs like wild type level; in contrast, CrABCA2 overexpressing lines accumulate more TAGs compared to parental lines. I found CrABCA2 localized to the ER, known to the site of TAG synthesis with membrane fraction and immunogold assay on transmission electron microscopy (TEM). I have demonstrated two organic carbon transporter in Chlamydomonas involved in carbon partitioning. This study could help to understand how Chlamydomonas transport photoassimilates and storage lipids. In addition, I suggest that improvement of carbon partitioning process in microalgae could reach to the future platform for bioenergy and valuable bio-product production.