A Modular Cloning Toolkit for the production of recombinant proteins in Leishmania tarentolae.

Modular Cloning (MoClo) is based on libraries of standardized genetic parts that can be directionally assembled via Golden Gate cloning in one-pot reactions into transcription units and multigene constructs. Here, a team of bachelor students established a MoClo toolkit for the protist Leishmania tarentolae in the frame of the international Genetically Engineered Machine (iGEM) competition. Our modular toolkit is based on a domesticated version of a commercial LEXSY expression vector and comprises 34 genetic parts encoding various affinity tags, targeting signals as well as fluorescent and luminescent proteins. We demonstrated the utility of our kit by the successful production of 16 different tagged versions of the receptor binding domain (RBD) of the SARS-CoV-2 spike protein in L. tarentolae liquid cultures. While highest yields of secreted recombinant RBD were obtained for GST-tagged fusion proteins 48 h post induction, C-terminal peptide tags were often degraded and resulted in lower yields of secreted RBD. Fusing secreted RBD to a synthetic O-glycosylation SP20 module resulted in an apparent molecular mass shift around 10 kDa. No disadvantage regarding the production of RBD was detected when the three antibiotics of the LEXSY system were omitted during the 48-h induction phase. Furthermore, the successful purification of secreted RBD from the supernatant of L. tarentolae liquid cultures was demonstrated in pilot experiments. In summary, we established a MoClo toolkit and exemplified its application for the production of recombinant proteins in L. tarentolae.

TurboID reveals the proxiomes of Chlamydomonas proteins involved in thylakoid biogenesis and stress response.

In Chlamydomonas (Chlamydomonas reinhardtii), the VESICLE-INDUCING PROTEIN IN PLASTIDS 1 and 2 (VIPP1 and VIPP2) play roles in the sensing and coping with membrane stress and in thylakoid membrane biogenesis. To gain more insight into these processes, we aimed to identify proteins interacting with VIPP1/2 in the chloroplast and chose proximity labeling (PL) for this purpose. We used the transient interaction between the nucleotide exchange factor CHLOROPLAST GRPE HOMOLOG 1 (CGE1) and the stromal HEAT SHOCK PROTEIN 70B (HSP70B) as test system. While PL with APEX2 and BioID proved to be inefficient, TurboID resulted in substantial biotinylation in vivo. TurboID-mediated PL with VIPP1/2 as baits under ambient and H2O2 stress conditions confirmed known interactions of VIPP1 with VIPP2, HSP70B, and the CHLOROPLAST DNAJ HOMOLOG 2 (CDJ2). Proteins identified in the VIPP1/2 proxiomes can be grouped into proteins involved in the biogenesis of thylakoid membrane complexes and the regulation of photosynthetic electron transport, including PROTON GRADIENT REGULATION 5-LIKE 1 (PGRL1). A third group comprises 11 proteins of unknown function whose genes are upregulated under chloroplast stress conditions. We named them VIPP PROXIMITY LABELING (VPL). In reciprocal experiments, we confirmed VIPP1 in the proxiomes of VPL2 and PGRL1. Our results demonstrate the robustness of TurboID-mediated PL for studying protein interaction networks in the chloroplast of Chlamydomonas and pave the way for analyzing functions of VIPPs in thylakoid biogenesis and stress responses.

Analysis of Viral Promoters for Transgene Expression and of the Effect of 5'-UTRs on Alternative Translational Start Sites in Chlamydomonas.

Microalgae biotechnology has the potential to produce high quality bioproducts in a sustainable manner. Here, Chlamydomonas reinhardtii has shown great potential as a host for biotechnological exploitation. However, low expression of nuclear transgenes is still a problem and needs to be optimized. In many model organisms, viral promoters are used to drive transgene expression at high levels. However, no viruses are known to infect Chlamydomonas, and known viral promoters are not functional. Recently, two different lineages of giant viruses were identified in the genomes of Chlamydomonas reinhardtii field isolates. In this work, we tested six potentially strong promoters from these viral genomes for their ability to drive transgene expression in Chlamydomonas. We used ble, NanoLUC, and mCherry as reporter genes, and three native benchmark promoters as controls. None of the viral promoters drove expression of any reporter gene beyond background. During our study, we found that mCherry variants are produced by alternative in-frame translational start sites in Chlamydomonas. We show that this problem can be overcome by mutating the responsible methionine codons to codons for leucine and by using the 5'-UTR of ßTUB2 instead of the 5'-UTRs of PSAD or RBCS2. Apparently, the ßTUB2 5'-UTR promotes the use of the first start codon. This could be mediated by the formation of a stem-loop between sequences of the ßTUB2 5'-UTR and sequences downstream of the first AUG in the mCherry reporter, potentially increasing the dwell time of the scanning 40S subunit on the first AUG and thus decreasing the probability of leaky scanning.

CLPB3 is required for the removal of chloroplast protein aggregates and thermotolerance in Chlamydomonas.

In the cytosol of plant cells, heat-induced protein aggregates are resolved by the CASEIN LYTIC PROTEINASE/HEAT SHOCK PROTEIN 100 (CLP/HSP100) chaperone family member HSP101, which is essential for thermotolerance. For the chloroplast family member CLPB3 this is less clear, with controversial reports on its role in conferring thermotolerance. To shed light on this issue, we have characterized two clpb3 mutants in Chlamydomonas reinhardtii. We show that chloroplast CLPB3 is required for resolving heat-induced protein aggregates containing stromal TRIGGER FACTOR (TIG1) and the small heat shock proteins 22E/F (HSP22E/F) in vivo, and for conferring thermotolerance under heat stress. Although CLPB3 accumulation is similar to that of stromal HSP70B under ambient conditions, we observed no prominent constitutive phenotypes. However, we found decreased accumulation of the PLASTID RIBOSOMAL PROTEIN L1 (PRPL1) and increased accumulation of the stromal protease DEG1C in the clpb3 mutants, suggesting that a reduction in chloroplast protein synthesis capacity and an increase in proteolytic capacity may compensate for loss of CLPB3 function. Under ambient conditions, CLPB3 was distributed throughout the chloroplast, but reorganized into stromal foci upon heat stress, which mostly disappeared during recovery. CLPB3 foci were localized next to HSP22E/F, which accumulated largely near the thylakoid membranes. This suggests a possible role for CLPB3 in disentangling protein aggregates from the thylakoid membrane system.

Production and secretion of functional SARS-CoV-2 spike protein in Chlamydomonas reinhardtii.

The spike protein is the major protein on the surface of coronaviruses. It is therefore the prominent target of neutralizing antibodies and consequently the antigen of all currently admitted vaccines against SARS-CoV-2. Since it is a 1,273-amino acids glycoprotein with 22 N-linked glycans, the production of functional, full-length spike protein was limited to higher eukaryotes. Here we report the production of full-length SARS-CoV-2 spike protein - lacking the C-terminal membrane anchor - as a secreted protein in the prefusion-stabilized conformation in the unicellular green alga Chlamydomonas reinhardtii. We show that the spike protein is efficiently cleaved at the furin cleavage site during synthesis in the alga and that cleavage is abolished upon mutation of the multi-basic cleavage site. We could enrich the spike protein from culture medium by ammonium sulfate precipitation and demonstrate its functionality based on its interaction with recombinant ACE2 and ACE2 expressed on human 293T cells. Chlamydomonas reinhardtii is a GRAS organism that can be cultivated at low cost in simple media at a large scale, making it an attractive production platform for recombinant spike protein and other biopharmaceuticals in low-income countries.

New destination vectors facilitate Modular Cloning for Chlamydomonas.

Synthetic Biology is revolutionizing biological research by introducing principles of mechanical engineering, including the standardization of genetic parts and standardized part assembly routes. Both are realized in the Modular Cloning (MoClo) strategy. MoClo allows for the rapid and robust assembly of individual genes and multigene clusters, enabling iterative cycles of gene design, construction, testing, and learning in short time. This is particularly true if generation times of target organisms are short, as is the case for the unicellular green alga Chlamydomonas reinhardtii. Testing a gene of interest in Chlamydomonas with MoClo requires two assembly steps, one for the gene of interest itself and another to combine it with a selection marker. To reduce this to a single assembly step, we constructed five new destination vectors. They contain genes conferring resistance to commonly used antibiotics in Chlamydomonas and a site for the direct assembly of basic genetic parts. The vectors employ red/white color selection and, therefore, do not require costly compounds like X-gal and IPTG. mCherry expression is used to demonstrate the functionality of these vectors.

Real-time monitoring of subcellular H2O2 distribution in Chlamydomonas reinhardtii.

Hydrogen peroxide (H2O2) is recognized as an important signaling molecule in plants. We sought to establish a genetically encoded, fluorescent H2O2 sensor that allows H2O2 monitoring in all major subcompartments of a Chlamydomonas cell. To this end, we used the Chlamydomonas Modular Cloning toolbox to target the hypersensitive H2O2 sensor reduction-oxidation sensitive green fluorescent protein2-Tsa2ΔCR to the cytosol, nucleus, mitochondrial matrix, chloroplast stroma, thylakoid lumen, and endoplasmic reticulum (ER). The sensor was functional in all compartments, except for the ER where it was fully oxidized. Employing our novel sensors, we show that H2O2 produced by photosynthetic linear electron transport (PET) in the stroma leaks into the cytosol but only reaches other subcellular compartments if produced under nonphysiological conditions. Furthermore, in heat-stressed cells, we show that cytosolic H2O2 levels closely mirror temperature up- and downshifts and are independent from PET. Heat stress led to similar up- and downshifts of H2O2 levels in the nucleus and, more mildly, in mitochondria but not in the chloroplast. Our results thus suggest the establishment of steep intracellular H2O2 gradients under normal physiological conditions with limited diffusion into other compartments. We anticipate that these sensors will greatly facilitate future investigations of H2O2 biology in plant cells.

Structural basis for VIPP1 oligomerization and maintenance of thylakoid membrane integrity.

Vesicle-inducing protein in plastids 1 (VIPP1) is essential for the biogenesis and maintenance of thylakoid membranes, which transform light into life. However, it is unknown how VIPP1 performs its vital membrane-remodeling functions. Here, we use cryo-electron microscopy to determine structures of cyanobacterial VIPP1 rings, revealing how VIPP1 monomers flex and interweave to form basket-like assemblies of different symmetries. Three VIPP1 monomers together coordinate a non-canonical nucleotide binding pocket on one end of the ring. Inside the ring's lumen, amphipathic helices from each monomer align to form large hydrophobic columns, enabling VIPP1 to bind and curve membranes. In vivo mutations in these hydrophobic surfaces cause extreme thylakoid swelling under high light, indicating an essential role of VIPP1 lipid binding in resisting stress-induced damage. Using cryo-correlative light and electron microscopy (cryo-CLEM), we observe oligomeric VIPP1 coats encapsulating membrane tubules within the Chlamydomonas chloroplast. Our work provides a structural foundation for understanding how VIPP1 directs thylakoid biogenesis and maintenance.

VIPP2 interacts with VIPP1 and HSP22E/F at chloroplast membranes and modulates a retrograde signal for HSP22E/F gene expression.

VIPP proteins aid thylakoid biogenesis and membrane maintenance in cyanobacteria, algae, and plants. Some members of the Chlorophyceae contain two VIPP paralogs termed VIPP1 and VIPP2, which originate from an early gene duplication event during the evolution of green algae. VIPP2 is barely expressed under nonstress conditions but accumulates in cells exposed to high light intensities or H2 O2 , during recovery from heat stress, and in mutants with defective integration (alb3.1) or translocation (secA) of thylakoid membrane proteins. Recombinant VIPP2 forms rod-like structures in vitro and shows a strong affinity for phosphatidylinositol phosphate. Under stress conditions, >70% of VIPP2 is present in membrane fractions and localizes to chloroplast membranes. A vipp2 knock-out mutant displays no growth phenotypes and no defects in the biogenesis or repair of photosystem II. However, after exposure to high light intensities, the vipp2 mutant accumulates less HSP22E/F and more LHCSR3 protein and transcript. This suggests that VIPP2 modulates a retrograde signal for the expression of nuclear genes HSP22E/F and LHCSR3. Immunoprecipitation of VIPP2 from solubilized cells and membrane-enriched fractions revealed major interactions with VIPP1 and minor interactions with HSP22E/F. Our data support a distinct role of VIPP2 in sensing and coping with chloroplast membrane stress.

The Chlamydomonas deg1c Mutant Accumulates Proteins Involved in High Light Acclimation.

Degradation of periplasmic proteins (Deg)/high temperature requirement A (HtrA) proteases are ATP-independent Ser endopeptidases that perform key aspects of protein quality control in all domains of life. Here, we characterized Chlamydomonas reinhardtii DEG1C, which together with DEG1A and DEG1B is orthologous to Arabidopsis (Arabidopsis thaliana) Deg1 in the thylakoid lumen. We show that DEG1C is localized to the stroma and the periphery of thylakoid membranes. Purified DEG1C exhibited high proteolytic activity against unfolded model substrates and its activity increased with temperature and pH. DEG1C forms monomers, trimers, and hexamers that are in dynamic equilibrium. DEG1C protein levels increased upon nitrogen, sulfur, and phosphorus starvation; under heat, oxidative, and high light stress; and when Sec-mediated protein translocation was impaired. DEG1C depletion was not associated with any obvious aberrant phenotypes under nonstress conditions, high light exposure, or heat stress. However, quantitative shotgun proteomics revealed differences in the abundance of 307 proteins between a deg1c knock-out mutant and the wild type under nonstress conditions. Among the 115 upregulated proteins are PSII biogenesis factors, FtsH proteases, and proteins normally involved in high light responses, including the carbon dioxide concentrating mechanism, photorespiration, antioxidant defense, and photoprotection. We propose that the lack of DEG1C activity leads to a physiological state of the cells resembling that induced by high light intensities and therefore triggers high light protection responses.

Birth of a Photosynthetic Chassis: A MoClo Toolkit Enabling Synthetic Biology in the Microalga Chlamydomonas reinhardtii.

Microalgae are regarded as promising organisms to develop innovative concepts based on their photosynthetic capacity that offers more sustainable production than heterotrophic hosts. However, to realize their potential as green cell factories, a major challenge is to make microalgae easier to engineer. A promising approach for rapid and predictable genetic manipulation is to use standardized synthetic biology tools and workflows. To this end we have developed a Modular Cloning toolkit for the green microalga Chlamydomonas reinhardtii. It is based on Golden Gate cloning with standard syntax, and comprises 119 openly distributed genetic parts, most of which have been functionally validated in several strains. It contains promoters, UTRs, terminators, tags, reporters, antibiotic resistance genes, and introns cloned in various positions to allow maximum modularity. The toolkit enables rapid building of engineered cells for both fundamental research and algal biotechnology. This work will make Chlamydomonas the next chassis for sustainable synthetic biology.