Recently developed transgenic techniques to explore and exploit the metabolic potential of microalgae present several drawbacks associated with the delivery of exogenous DNA into the cells and its subsequent integration at random sites within the genome. Here, we report a highly efficient multiplex genome-editing method in the diatom Phaeodactylum tricornutum, relying on the biolistic delivery of CRISPR-Cas9 ribonucleoproteins coupled with the identification of two endogenous counter-selectable markers, PtUMPS and PtAPT. First, we demonstrate the functionality of RNP delivery by positively selecting the disruption of each of these genes. Then, we illustrate the potential of the approach for multiplexing by generating double-gene knock-out strains, with 65% to 100% efficiency, using RNPs targeting one of these markers and PtAureo1a, a photoreceptor-encoding gene. Finally, we created triple knock-out strains in one step by delivering six RNP complexes into Phaeodactylum cells. This approach could readily be applied to other hard-to-transfect organisms of biotechnological interest.

The orange carotenoid protein (OCP) serves as a sensor of light intensity and an effector of phycobilisome (PB)–associated photoprotection in cyanobacteria. Structurally, the OCP is composed of two distinct domains spanned by a single carotenoid chromophore. Functionally, in response to high light, the OCP converts from a dark-stable orange form, OCP^O, to an active red form, OCP^R. The C-terminal domain of the OCP has been implicated in the dynamic response to light intensity and plays a role in switching off the OCP’s photoprotective response through its interaction with the fluorescence recovery protein. The function of the N-terminal domain, which is uniquely found in cyanobacteria, is unclear. To investigate its function, we isolated the N-terminal domain in vitro using limited proteolysis of native OCP. The N-terminal domain retains the carotenoid chromophore; this red carotenoid protein (RCP) has constitutive PB fluorescence quenching activity comparable in magnitude to that of active, full-length OCP^R. A comparison of the spectroscopic properties of the RCP with OCP^R indicates that critical protein–chromophore interactions within the C-terminal domain are weakened in the OCP^R form. These results suggest that the C-terminal domain dynamically regulates the photoprotective activity of an otherwise constitutively active carotenoid binding N-terminal domain.

Significance: Protection from overexcitation is one of the most important requirements of all photosynthetic organisms. Here we present a model based on coupled cross-linking/mass spectrometry and site-directed mutagenesis of the means by which the orange carotenoid protein (OCP) binds to the phycobilisome (PBS) antenna complex to avoid photodamage. The model shows that the protein must actively burrow into the complex, separating the PBS rings in the process. This penetration explains for the first time, to our knowledge, how the OCP carotenoid could approach the PBS chromophores at a distance that enables nonphotochemical quenching. However, the alteration in the core structure caused by OCP binding could also prevent energy transmission to the reaction centers.

Summary: Photoacclimation consists of short‐ and long‐term strategies used by photosynthetic organisms to adapt to dynamic light environments. Observable photophysiology changes resulting from these strategies have been used in coarse‐grained models to predict light‐dependent growth and photosynthetic rates. However, the contribution of the broader metabolic network, relevant to species‐specific strategies and fitness, is not accounted for in these simple models. We incorporated photophysiology experimental data with genome‐scale modeling to characterize organism‐level, light‐dependent metabolic changes in the model diatomPhaeodactylum tricornutum. Oxygen evolution and photon absorption rates were combined with condition‐specific biomass compositions to predict metabolic pathway usage for cells acclimated to four different light intensities. Photorespiration, an ornithine‐glutamine shunt, and branched‐chain amino acid metabolism were hypothesized as the primary intercompartment reductant shuttles for mediating excess light energy dissipation. Additionally, simulations suggested that carbon shunted through photorespiration is recycled back to the chloroplast as pyruvate, a mechanism distinct from known strategies in photosynthetic organisms. Our results suggest a flexible metabolic network inP. tricornutumthat tunes intercompartment metabolism to optimize energy transport between the organelles, consuming excess energy as needed. Characterization of these intercompartment reductant shuttles broadens our understanding of energy partitioning strategies in this clade of ecologically important primary producers.

Photosynthetic microbes respond to changing light environments to balance photosynthetic process with light induced damage and photoinhibition. There have been very few characterizations of photosynthetic physiology or biomass partitioning during the day in mass culture. Understanding the constraints on photosynthetic efficiency and biomass accumulation are necessary for engineering superior strains or cultivation methods. We observed the photosynthetic physiology of nutrient replete Phaeodactylum tricornutum growing in light environments that mimic those found in rapidly mixing, outdoor, low biomass photobioreactors. We found little evidence for photoinhibition or non-photochemical quenching in situ, suggesting photosynthesis remains highly efficient throughout the day. Cells doubled their organic carbon from dawn to dusk and a small percentage – around 20% – of this carbon was allocated to carbohydrates or triacylglycerol. We conclude that the self-shading provided by dense culturing of P. tricornutum inhibits the induction of photodamage, and energy dissipation processes that would otherwise lower productivity in an outdoor photobioreactor