UC Berkeley

Carbon fixation by photosynthetic organisms provides the bulk of energy that most other life depends on. Many photosynthetic organisms can shift metabolism in order to metabolize other sources of energy when necessary. The unicellular, Chlorophycean alga, Chromochloris zofingiensis, is one example of a photosynthetic organism with metabolic flexibility. When supplemented with glucose, C. zofingiensis will utilize both photosynthesis and glucose as an energy source. In specific culture conditions, supplemental glucose causes the total loss of photosynthetic capacity. Additionally, this phenomenon is reversible, and C. zofingiensis is able to rapidly regenerate the photosynthetic apparatus if glucose is removed. This observation, that glucose can trigger a reversible collapse of photosynthesis, provides the opportunity to investigate fundamental metabolic and signaling processes and the genes required to rebuild photosynthetic capacity. Chapter 1 introduces C. zofingiensis, its scientific history, and places it in the context of current research.

Chapter 2 presents results of a two-phase experiment, in which we driveC. zofingiensis into a state of photosynthetic repression using glucose in the first phase. In the second phase, we release that repression by removing glucose. We observe the severe impact on photosynthetic capacity, where oxygen evolution stops occurring and fluorescence signatures of a functional photosystem decrease to near zero. The proteins and lipids required for photosynthesis are greatly diminished. Cells grow larger in glucose, but thylakoid membranes in the chloroplast shrink, while storage molecules in the form of triacylglycerols and starch increase, as well as the keto-carotenoid, astaxanthin. In the second phase, when these cells are transferred into a glucose-free medium, photosynthetic repression is released, and the cells restart photosynthesizing within 12 hours and continue to regreen over the course of 48 hours. We analyze and observe gene expression during critical metabolic transitions and find a wholesale transcriptional reordering in glucose conditions that results in the differential expression of 41% of the genes in the genome. We find that glycolysis, lipid metabolism, chlorophyll biosynthesis, and the expression of photosynthetic proteins are tightly controlled by the presence or absence of glucose. This chapter presents C. zofingiensis as a model system to understand metabolic flexibility between photoautotrophic and heterotrophic modes, furthering our understanding of the regulation of algal metabolism. This work will facilitate engineering efforts to reroute metabolism towards beneficial bioproducts for energy, food, and potentially pharmaceuticals.

In Chapter 3, we seek to understand genetic determinants of trophic flexibility in C. zofingiensis. We utilize a genetic selection to identify mutants thatare unable to shift their trophic strategy when supplemented with glucose. UV- mutagenized cells were plated on medium containing the glucose analog, 2- deoxy-D-glucose, which induces photosynthetic repression, but is unable to be metabolized by C. zofingiensis. We identify potential causative mutations using whole-genome sequencing and find that all eight mutants generated and investigated have mutations in the critical glucose-phosphorylating enzyme, hexokinase (HXK1). We further characterize two of these independently generated mutants, which share the same hxk1 mutation. When treated with glucose, photosynthetic capacity, oxygen evolution, and proteins related to photosynthesis of hxk1 mutants are all much more similar to non-glucose-treated wild-type C. zofingiensis. The expression of genes representative of key pathways described in chapter 1, such as HXK1, a light-harvesting antenna protein (LHC16), A fatty acid de-esterase (FAD2), and the major lipid-droplet storage protein (MLDP1), is unresponsive to glucose. This provides the basis for our model of glucose-induced repression of photosynthesis, where the cellular response to glucose is mediated by HXK1.

Finally, in Chapter 4, we describe the potential of C. zofingiensis as an inducible model system of oxygenic photosynthesis to study thylakoid biogenesis. Utilizing a comparative genomics framework coupled with extant co- expression data for genes of the model plant Arabidopsis thaliana and data from a mutant library in model green alga Chlamydomonas reinhardtii, which contains many potential photosynthetic defects, we identify genes likely to be essential for thylakoid biosynthesis. By comparing sets of C. zofingiensis genes that are differentially expressed during the transition from glucose to non-glucose conditions to the A. thaliana and C. reinhardtii gene sets, we identify sets of genes, many of which are well-known components of thylakoid biogenesis, whereas others have roles that have yet to be described in thylakoid biogenesis. This work utilizes a recently developed bioinformatic tool called OrthoLang and presents a systematic approach to the integration of disparate data types, including RNA-seq data, a sequenced mutant library, and co-expression analysis.In summary, C. zofingiensis is emerging as a powerful model organism to study multiple aspects of photosynthetic metabolism. Signals that rewire metabolism in the presence of glucose pass through the HXK1 and result in global photosynthetic repression. Release of that repression allows C. zofingiensis to rapidly recover photosynthesis, providing an additional window into thylakoid biogenesis.