Sammendrag
Diatoms are a group of eukaryotic microalgae of high ecological importance, contributing to up to 20%
of oxygen production worldwide. They are of commercial interest for potential biotechnological applica-
tions, e.g. for use as fish feed in aquaculture due to their lipid profile or for bio-remediation of wastewa-
ter. Pigment-binding proteins of the light harvesting antenna are inserted into the thylakoid membrane
via the chloroplast signal recognition particle pathway. Previous studies in plants and green algae have
shown that knockout of genes of this pathway can result in strains with a truncated light harvesting
antenna. These strains are valuable for biotechnological approaches as they may grow better in dense
bio-reactor cultures. Two isoforms of the Albino3 insertase Alb3, named Alb3a and Alb3b, had been
identified in the model diatom Phaeodactylum tricornutum. Knockout of Alb3a was attempted, but no bi
-allelic loss-of-function mutant could be obtained, indicating that Alb3a is an essential gene. In contrast,
knockout of Alb3b resulted in a pronounced change of coloration of the cells from brown to green and
a strong decrease in the main light-harvesting antenna proteins was observed. However, growth rates
were found to be reduced. Thus, further genes involved in this pathway need to be identified. There-
fore, we are employing a proximity labeling approach to identify interaction partners of the two Alb3
proteins. Phaeodactylum tricornutum strains expressing fusion proteins of the respective Alb3 isoform
and the peroxidase APEX2 were generated. These strains were then acclimated to medium light condi-
tions, followed by a shift to low light conditions to induce Alb3 activity. Upon addition of the membrane-
permeable substrate biotin-phenol, APEX2 biotinylates proteins in close proximity, which are then affini-
ty purified and identified by mass spectrometry. Potential interaction partners identified will then be
confirmed and characterized by generation of knockout strains.
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