Pursuit of chlorovirus genetic transformation and CRISPR/Cas9-mediated gene editing

Genetic and molecular modifications of the large dsDNA chloroviruses, with genomes of 290 to 370 kb, would expedite studies to elucidate the functions of both identified and unidentified virus-encoded proteins. These plaque-forming viruses replicate in certain unicellular, eukaryotic chlorella-like green algae. However, to date, only a few of these algal species and virtually none of their viruses have been genetically manipulated due to lack of practical methods for genetic transformation and genome editing. Attempts at using Agrobacterium-mediated transfection of chlorovirus host Chlorella variabilis NC64A with a specially-designed binary vector resulted in successful transgenic cell selection based on expression of a hygromycin-resistance gene, initial expression of a green fluorescence gene and demonstration of integration of Agrobacterium T-DNA. However, expression of the integrated genes was soon lost. To develop gene editing tools for modifying specific chlorovirus CA-4B genes using preassembled Cas9 protein-sgRNA ribonucleoproteins (RNPs), we tested multiple methods for delivery of Cas9/sgRNA RNP complexes into infected cells including cell wall-degrading enzymes, electroporation, silicon carbide (SiC) whiskers, and cell-penetrating peptides (CPPs). In one experiment two independent virus mutants were isolated from macerozyme-treated NC64A cells incubated with Cas9/sgRNA RNPs targeting virus CA-4B-encoded gene 034r, which encodes a glycosyltransferase. Analysis of DNA sequences from the two mutant viruses showed highly targeted nucleotide sequence modifications in the 034r gene of each virus that were fully consistent with Cas9/RNP-directed gene editing. However, in ten subsequent experiments, we were unable to duplicate these results and therefore unable to achieve a reliable system to genetically edit chloroviruses. Nonetheless, these observations provide strong initial suggestions that Cas9/RNPs may function to promote editing of the chlorovirus genome, and that further experimentation is warranted and worthwhile.

Multi-omics Analysis of the Symbiotic Green Algae, Chlorella Variabilis, Revealing the Genetic Basis of the Obligate Endosymbiotic Lifestyle

Background: Photosynthetic eukaryotes have evolved through the acquisition of plastids by secondary endosymbiosis, a process that requires several steps. Immediately before plastid acquisition, the genome of the symbiont is known to be dramatically reduced, but few studies have focused on the genomic changes in the symbiont at the early stages of secondary endosymbiosis. Methods: To investigate the genetic basis of the transition from facultative to obligate endosymbiosis, we compared the genomes of Chlorella variabilis, a representative symbiotic alga, with that of Paramecium bursaria, to compare closely related free-living species and transcriptomes between organisms in symbiotic and non-symbiotic conditions. Results: We found that the non-reduced genome of C. variabilis and its genes play a crucial role in endosymbiosis, being involved in cell wall biogenesis and degradation, and metabolic exchanges with the host. Our results suggest that the genetic mechanism underlying the enhancement of photosynthesis under symbiosis is the increasing light absorption efficiency and carbon fixation capacity of the endosymbiont, resulting in an increase in the supply of maltose to P. bursaria.

Light-driven decarboxylative deuteration enabled by a divergently engineered photodecarboxylase

Despite the well-established chemical processes for C-D bond formation, the toolbox of enzymatic methodologies for deuterium incorporation has remained underdeveloped. Here we describe a photodecarboxylase from Chlorella variabilis NC64A (CvFAP)-catalyzed approach for the decarboxylative deuteration of various carboxylic acids by employing D2O as a cheap and readily available deuterium source. Divergent protein engineering of WT-CvFAP is implemented using Focused Rational Iterative Site-specific Mutagenesis (FRISM) as a strategy for expanding the substrate scope. Using specific mutants, several series of substrates including different chain length acids, racemic substrates as well as bulky cyclic acids are successfully converted into the deuterated products (>40 examples). In many cases WT-CvFAP fails completely. This approach also enables the enantiocomplementary kinetic resolution of racemic acids to afford chiral deuterated products, which can hardly be accomplished by existing methods. MD simulations explain the results of improved catalytic activity and stereoselectivity of WT CvFAP and mutants.

Distribution of the Water-Soluble Astaxanthin Binding Carotenoprotein (AstaP) in Scenedesmaceae

Photooxidative stress-inducible water-soluble astaxanthin-binding proteins, designated as AstaP, were identified in two Scenedesmaceae strains, Coelastrella astaxanthina Ki-4 and Scenedesmus obtusus Oki-4N; both strains were isolated under high light conditions. These AstaPs are classified as a novel family of carotenoprotein and are useful for providing valuable astaxanthin in water-soluble form; however, the distribution of AstaP orthologs in other microalgae remains unknown. Here, we examined the distribution of AstaP orthologs in the family Scenedesmaceae with two model microalgae, Chlamydomonas reinhardtii and Chlorella variabilis. The expression of AstaP orthologs under photooxidative stress conditions was detected in cell extracts of Scenedesmaceae strains, but not in model algal strains. Aqueous orange proteins produced by Scenedesmaceae strains were shown to bind astaxanthin. The protein from Scenedesmus costatus SAG 46.88 was purified. It was named ScosAstaP and found to bind astaxanthin. The deduced amino acid sequence from a gene encoding ScosAstaP showed 62% identity to Ki-4 AstaP. The expression of the genes encoding AstaP orthologs was shown to be inducible under photooxidative stress conditions; however, the production amounts of AstaP orthologs were estimated to be approximately 5 to 10 times lower than that of Ki-4 and Oki-4N.

Pursuit of chlorovirus genetic transformation and CRISPR/Cas9-mediated gene editing

The ability to carry out genetic and molecular modifications of the large dsDNA chloroviruses, with genomes of 290 to 370 kb, would expedite studies to elucidate the functions of both identified and unidentified virus-encoded proteins. These plaque-forming viruses replicate in certain unicellular, eukaryotic chlorella-like green algae and are present in freshwater environments throughout the world. However, to date, only a few of these algal species and virtually none of their viruses have been genetically manipulated due to lack of practical methods for genetic transformation and genome editing. In an effort to develop gene editing tools for modifying specific chlorovirus CA-4B genes using preassembled Cas9 protein-sgRNA ribonucleoproteins (RNPs), we first tested multiple methods for delivery of Cas9/sgRNA RNP complexes into infected cells including cell wall-degrading enzymes, electroporation, silicon carbide (SiC) whiskers, and cell-penetrating peptides (CPPs). Agrobacterium -mediated transfection of chlorovirus host Chlorella variabilis NC64A with a binary vector containing a chlorovirus-encoded glycosyltransferase mutant gene was also examined. Attempts at developing a reliable chlorovirus transformation system were unsuccessful. However, in one experiment two independent virus mutants were isolated from macerozyme-treated NC64A cells incubated with Cas9/sgRNA RNPs targeting CA-4B-encoded gene 034r , which encodes a putative glycosyltransferase. Selection of these mutants using antibodies was dependent on a specific change in the pattern of glycans attached to the virus’ major capsid protein (MCP). Analysis of DNA sequences from the two mutant viruses showed highly targeted nucleotide sequence modifications in the 034r gene of each virus that were fully consistent with Cas9/RNP-directed gene editing. However, we were unable to duplicate these results and therefore unable to achieve a reliable system to genetically edit chloroviruses. Nonetheless, these observations provide strong initial suggestions that Cas9/RNPs may function to promote editing of the chlorovirus genome, and that further experimentation is warranted and worthwhile.