Harry Oliver Jackson

The Nostocales are filamentous cyanobacteria that include bloom-forming, nitrogen-fixing species with major ecological and biotechnological relevance. Owing to their metabolic diversity, nitrogen-fixing capabilities and nutritional profile, they hold potential for various biotechnological applications including the production of nutraceuticals and biofertilizers. However, the capacity of certain Nostocales species to produce cyanotoxins also raises health concerns. This study provides a comparative bioinformatic analysis of 161 Nostocales genomes from the National Center for Biotechnology Information (NCBI) and three newly sequenced Aphanizomenon flos-aquae ( AFA ) strains, including Klamath AFA , with a focus on taxonomy, cyanotoxin-associated biosynthetic gene clusters (BGCs), natural competence, restriction-modification systems and vitamin B ₁₂ metabolism. Our analysis supports reassessment of several AFA -labelled genomes and refines the boundaries of the core AFA cluster. Cyanotoxin-associated BGCs, specifically for anatoxin-a, saxitoxin, cylindrospermopsin and microcystin/nodularin, were detected in only 7.3% of the genomes. Natural competence genes required for horizontal gene transfer were found in 89.6% of the genomes, although the presence of multiple restriction-modification system-associated proteins in Nostocales species, especially Aphanizomenon, Anabaena, Dolichospermum and Nostoc sp. may limit foreign gene acquisition. Additionally, ~30% of the genomes harbour a shared btu operon arrangement associated with B ₁₂ import, observed here across multiple Nostocales genera. B ₁₂ content in the AFA strains was measured at ~0.4–0.5 µg g ⁻¹ dry biomass and is likely linked to associated B ₁₂ -producing bacteria. Fluorescent B ₁₂ analogue uptake experiments in NIES 4052 and Klamath AFA support a model for the uptake of exogenous B ₁₂ in these strains. Together with the detection of cobalamin-producing commensal bacteria, this suggests that B ₁₂ in AFA biomass may be linked to cyanobacterium–bacteria interactions. Overall, this study provides a genomic and experimental framework for evaluating the taxonomy, safety, genetic tractability and nutritional potential of AFA and related Nostocales .

Genome scale engineering has enabled codon compression of the universal genetic code to eliminate seven codons in Escherichia coli , but to allow more radical schemes for codon compression and reassignment to be tested at genome scale, while avoiding significant technical challenges, smaller, simpler genetic systems are needed. Here, we report a recoding scheme for the 205 kb Chlamydomonas reinhardtii chloroplast genome, in which two stop codons and one or more of the codons for arginine, glycine, isoleucine, leucine, and serine, all of which have two cognate transfer RNAs (tRNAs), are absent, compressing the genetic code to 51 codons. Several recoding strategies were tested on the essential rpoA gene, encoding a subunit of the chloroplast RNA polymerase. A defined compression scheme, which relied on swapping the target codons with the permitted frequent codons, could replace the native sequence without affecting expression of a reporter protein or strain fitness under standard laboratory conditions. The same strategy was successfully used for codon compression of ycf1 , encoding a subunit of the chloroplast translocon, psaA and psbA , intron-containing highly expressed genes encoding reaction center subunits of both photosystems, and an 8.5 kb operon encoding essential and nonessential genes. Finally, we tested degeneracy of the 51-codon genetic code by exploring the combinatorial design for the large subunit of Rubisco, relying on restoration of photosynthesis in an rbcL mutant strain. More than 70 functional sequences with diverse codons were recovered. For all recoded genes, viable homoplasmic lines were obtained, showing the efficacy of our codon compression scheme.

Genome scale engineering has enabled codon compression of the universal genetic code of up to three codons in E. coli, providing the means for genetic code expansion. To go much beyond this number, smaller and simpler genetic systems are needed to avoid significant technical challenges. Chloroplast genomes offer multiple advantages for codon compression and reassignment. Here we report a recoding scheme for the Chlamydomonas reinhardtii chloroplast genome, in which two stop codons and one or more of the codons for arginine, glycine, isoleucine, leucine and serine, all of which have two cognate tRNAs, are absent, compressing the genetic code to 51 codons. Firstly, several recoding strategies were tested on the essential rpoA gene, encoding a subunit of the chloroplast RNA polymerase. A defined compression scheme, which relied on swapping the target codons with the permitted frequent codons, could replace the native protein coding sequence without affecting chloroplast protein expression levels or the strain fitness. The same strategy was successfully used for codon compression of ycf1 , encoding a subunit of the chloroplast translocon, psaA and psbA , intron-containing highly expressed genes encoding reaction centres subunits of both photosystems, and an 8.5 kb operon encoding essential and non-essential genes. Finally, we tested degeneracy of the 51-codon genetic code by exploring the combinatorial design for the large subunit of RuBisCO, relying on restoration of photosynthesis in an rbcL mutant strain. More than 70 functional sequences with diverse codon adaptation indices were recovered. In all codon-compressed genes there was no observable penalty on photosynthetic growth. Significance Statement: We demonstrate the use of a chloroplast genome as a synthetic biology platform to test modifications of the universal genetic code. Elimination of 13 of the 64 possible triplet codes (codons) was possible for several essential and/or highly expressed genes without affecting chloroplast function. The technology is mature, paving the way for codon compression of the entire genome and thus enabling further radical rearrangement or expansion of the genetic code.

Rab proteins constitute an important subfamily of molecular effectors whose function is related to the modulation of several important cellular and metabolic processes, including controlling intracellular endocytic vesicle trafficking, phagosome formation, endosome maturation, and innate immunity. In crustaceans, the study and characterization of these proteins have garnered great interest, mainly due to their pivotal role in the innate immune response mechanisms against infections. Among the Rab family, the Rab7 stands out as a prominent example of the potential of these proteins as therapeutic targets for the development of genetic therapies to control viral pathogens in crustacean aquaculture. In this review, a compendium of the information currently available in relation to Rab proteins in crustaceans is made, taking the Rab7 protein as a model, emphasizing not only its general aspects of functioning at the molecular level, but also describing in‐depth both mechanisms of action and potential interactions, as well as its implications in relation to the immune system, particularly in shrimp.

Aphanizomenon flos-aquae (AFA) is a Gram-negative nitrogen-fixing freshwater filamentous cyanobacterium with a rich nutritional profile that is approved for human consumption by both the Food and Drug Administration and the European Food and Safety Authorities. It has a high protein content (60-70%) and contains numerous vitamins, minerals and trace elements together with several high-value compounds with nutraceutical properties, such as C-phycocyanin and β-phenylethylamine. 500-1000 t of AFA dry biomass are currently wild harvested from natural blooms that occur seasonally in Klamath Lake, Oregon, USA, and distributed as a nutritional supplement worldwide. The requirements and unreliability of wild harvesting, owing to the dependence of AFA growth on environmental conditions and potential contamination by microcystin toxins, threaten the availability of biomass supply and restricts commercial expansion. In this review we propose AFA cultivation in open ponds or closed photobioreactors to obtain a reliable production of unialgal biomass to resolve the supply issue and enhance AFA as a feedstock for specific high-value by-products. Furthermore, we explore potential strategies for maximising overall yield and seasonal robustness by using a synergistic combination of cultivation and wild harvesting. Controlled AFA cultivation would also facilitate the use of genetic manipulation to generate bespoke strains with improved commercial applications, such as increasing the cyanobacterium’s nitrogen-fixation rate to enhance its value as a biofertiliser. Ultimately, realising the untapped biotechnological potential of AFA requires a better understanding of its fundamental biology, robust methodologies for laboratory and large-scale cultivation, and the development of AFA-specific genetic engineering techniques.

‘Marker-free’ strategies for creating transgenic microorganisms avoid the issue of potential transmission of antibiotic resistance genes to other microorganisms. An already-established strategy for engineering the chloroplast genome (=plastome) of the green microalga Chlamydomonas reinhardtii involves the restoration of photosynthetic function using a recipient strain carrying a plastome mutation in a key photosynthesis gene. Selection for transformant colonies is carried out on minimal media, such that only those cells in which the mutated gene has been replaced with a wild-type copy carried on the transgenic DNA are capable of phototrophic growth. However, this approach can suffer from issues of efficiency due to the slow growth of C. reinhardtii on minimal media and the slow die-back of the untransformed lawn of cells when using mutant strains with a limited photosensitivity phenotype. Furthermore, such phototrophic rescue has tended to rely on existing mutants that are not necessarily ideal for transformation and targeted transgene insertion: Mutants carrying point mutations can easily revert, and those with deletions that do not extend to the intended transgene insertion site can give rise to a sub-population of rescued lines that lack the transgene. In order to improve and accelerate the transformation pipeline for C. reinhardtii, we have created a novel recipient line, HNT6, carrying an engineered deletion in exon 3 of psaA, which encodes one of the core subunits of photosystem I (PSI). Such PSI mutants are highly light-sensitive allowing faster recovery of transformant colonies by selecting for light-tolerance on acetate-containing media, rather than phototrophic growth on minimal media. The deletion extends to a site upstream of psaA-3 that serves as a neutral locus for transgene insertion, thereby ensuring that all of the recovered colonies are transformants containing the transgene. We demonstrate the application of HNT6 using a luciferase reporter.

Microalgae are promising host organisms for the production of encapsulated recombinant proteins such as vaccines. However, bottlenecks in bioprocess development, such as the drying stage, need to be addressed to ensure feasibility at scale. In this study, we investigated the potential of spray drying to produce a recombinant vaccine in microalgae. A transformant line of Chlamydomonas reinhardtii carrying a subunit vaccine against salmonid alphavirus was created via chloroplast engineering. The integrity of the recombinant protein after spray drying and its stability after 27 months storage at –80 °C, +4 °C and room temperature were assessed by immunoblotting. The protein withstood spray drying without significant losses. Long-term storage at +4 °C and room temperature resulted in 50% and 92% degradation, respectively. Optimizing spray drying and storage conditions should minimize degradation and favour short-term storage at positive temperatures. Using data on yield and productivity, the economics of spray drying- and freeze drying-based bioprocesses were compared. The drying stage corresponded to 41% of the total production cost. Process optimization, genetic engineering and new market strategies were identified as potential targets for cost reduction. Overall, this study successfully demonstrates the suitability of spray drying as a process option for recombinant protein production in microalgae at the industrial scale.