The synthetic future of algal genomes.

Algae are diverse organisms with significant biotechnological potential for resource circularity. Taking inspiration from fermentative microbes, engineering algal genomes holds promise to broadly expand their application ranges. Advances in genome sequencing with improvements in DNA synthesis and delivery techniques are enabling customized molecular tool development to confer advanced traits to algae. Efforts to redesign and rebuild entire genomes to create fit-for-purpose organisms currently being explored in heterotrophic prokaryotes and eukaryotic microbes could also be applied to photosynthetic algae. Future algal genome engineering will enhance yields of native products and permit the expression of complex biochemical pathways to produce novel metabolites from sustainable inputs. We present a historical perspective on advances in engineering algae, discuss the requisite genetic traits to enable algal genome optimization, take inspiration from whole-genome engineering efforts in other microbes for algal systems, and present candidate algal species in the context of these engineering goals.

Bicistronic expression of nuclear transgenes in Chlamydomonas reinhardtii.

In eukaryotic organisms, proteins are typically translated from monocistronic messenger RNAs containing a single coding sequence (CDS). However, recent long transcript sequencing identified 87 nuclear polycistronic mRNAs in Chlamydomonas reinhardtii natively carrying multiple co-expressed CDSs. In this study, we investigated the dynamics of 22 short intergenic sequences derived from these native polycistronic loci by their application in genetic constructs for synthetic transgene expression. A promising candidate sequence was identified based on the quantification of transformation efficiency and expression strength of a fluorescence reporter protein. Subsequently, the expression of independent proteins from one mRNA was verified by cDNA amplification and protein molecular mass characterization. We demonstrated engineered bicistronic expression in vivo to drive successful co-expression of several terpene synthases with the selection marker aphVIII. Bicistronic transgene design resulted in significantly increased (E)-α-bisabolene production of 7.95 mg L-1 from a single open reading frame, 18.1× fold higher than previous reports. Use of this strategy simplifies screening procedures for identification of high-level expressing transformants, does not require the application of additional fluorescence reporters, and reduces the nucleotide footprint compared to classical monocistronic expression cassettes. Although clear advantages for bicistronic transgene expression were observed, this strategy was found to be limited to the aphVIII marker, and further studies are necessary to gain insights into the underlying mechanism that uniquely permits this co-expression from the algal nuclear genome.

Engineered ketocarotenoid biosynthesis in the polyextremophilic red microalga Cyanidioschyzon merolae 10D.

The polyextremophilic Cyanidiophyceae are eukaryotic red microalgae with promising biotechnological properties arising from their low pH and elevated temperature requirements which can minimize culture contamination at scale. Cyanidioschyzon merolae 10D is a cell wall deficient species with a fully sequenced genome that is amenable to nuclear transgene integration by targeted homologous recombination. C. merolae maintains a minimal carotenoid profile and here, we sought to determine its capacity for ketocarotenoid accumulation mediated by heterologous expression of a green algal ß-carotene ketolase (BKT) and hydroxylase (CHYB). To achieve this, a synthetic transgene expression cassette system was built to integrate and express Chlamydomonas reinhardtii (Cr) sourced enzymes by fusing native C. merolae transcription, translation and chloroplast targeting signals to codon-optimized coding sequences. Chloramphenicol resistance was used to select for the integration of synthetic linear DNAs into a neutral site within the host genome. CrBKT expression caused accumulation of canthaxanthin and adonirubin as major carotenoids while co-expression of CrBKT with CrCHYB generated astaxanthin as the major carotenoid in C. merolae. Unlike green algae and plants, ketocarotenoid accumulation in C. merolae did not reduce total carotenoid contents, but chlorophyll a reduction was observed. Light intensity affected global ratios of all pigments but not individual pigment compositions and phycocyanin contents were not markedly different between parental strain and transformants. Continuous illumination was found to encourage biomass accumulation and all strains could be cultivated in simulated summer conditions from two different extreme desert environments. Our findings present the first example of carotenoid metabolic engineering in a red eukaryotic microalga and open the possibility for use of C. merolae 10D for simultaneous production of phycocyanin and ketocarotenoid pigments.

Cultivation of the polyextremophile Cyanidioschyzon merolae 10D during summer conditions on the coast of the Red Sea and its adaptation to hypersaline sea water.

The west coast of the Arabian Peninsula borders the Red Sea, a water body which maintains high average temperatures and increased salinity compared to other seas or oceans. This geography has many resources which could be used to support algal biotechnology efforts in bio-resource circularity. However, summer conditions in this region may exceed the temperature tolerance of most currently cultivated microalgae. The Cyanidiophyceae are a class of polyextremophilic red algae that natively inhabit acidic hot springs. C. merolae 10D has recently emerged as an interesting model organism capable of high-cell density cultivation on pure CO2 with optimal growth at elevated temperatures and acidic pH. C. merolae biomass has an interesting macromolecular composition, is protein rich, and contains valuable bio-products like heat-stable phycocyanin, carotenoids, ß-glucan, and starch. Here, photobioreactors were used to model C. merolae 10D growth performance in simulated environmental conditions of the mid-Red Sea coast across four seasons, it was then grown at various scales outdoors in Thuwal, Saudi Arabia during the Summer of 2022. We show that C. merolae 10D is amenable to cultivation with industrial-grade nutrient and CO2 inputs outdoors in this location and that its biomass is relatively constant in biochemical composition across culture conditions. We also show the adaptation of C. merolae 10D to high salinity levels of those found in Red Sea waters and conducted further modeled cultivations in nutrient enriched local sea water. It was determined that salt-water adapted C. merolae 10D could be cultivated with reduced nutrient inputs in local conditions. The results presented here indicate this may be a promising alternative species for algal bioprocesses in outdoor conditions in extreme coastal desert summer environments.

Engineered production of isoprene from the model green microalga Chlamydomonas reinhardtii.

Isoprene is a clear, colorless, volatile 5-carbon hydrocarbon that is one monomer of all cellular isoprenoids and a platform chemical with multiple applications in industry. Many plants have evolved isoprene synthases (IspSs) with the capacity to liberate isoprene from dimethylallyl diphosphate (DMADP) as part of cellular thermotolerance mechanisms. Isoprene is hydrophobic and volatile, rapidly leaves plant tissues and is one of the main carbon emission sources from vegetation globally. The universality of isoprenoid metabolism allows volatile isoprene production from microbes expressing heterologous IspSs. Here, we compared heterologous overexpression from the nuclear genome and localization into the plastid of four plant terpene synthases (TPs) in the green microalga Chlamydomonas reinhardtii. Using sealed vial mixotrophic cultivation, direct quantification of isoprene production was achieved from the headspace of living cultures, with the highest isoprene production observed in algae expressing the Ipomoea batatas IspS. Perturbations of the downstream carotenoid pathway through keto carotenoid biosynthesis enhanced isoprene titers, which could be further enhanced by increasing flux towards DMADP through heterologous co-expression of a yeast isopentenyl-DP delta isomerase. Multiplexed controlled-environment testing revealed that cultivation temperature, rather than illumination intensity, was the main factor affecting isoprene yield from the engineered alga. This is the first report of heterologous isoprene production from a eukaryotic alga and sets a foundation for further exploration of carbon conversion to this commodity chemical.

Metabolic Engineering for Efficient Ketocarotenoid Accumulation in the Green Microalga Chlamydomonas reinhardtii.

Astaxanthin is a valuable ketocarotenoid with various pharmaceutical and nutraceutical applications. Green microalgae harbor natural capacities for pigment accumulation due to their 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway. Recently, a redesigned ß-carotene ketolase (BKT) was found to enable ketocarotenoid accumulation in the model microalga Chlamydomonas reinhardtii, and transformants exhibited reduced photoinhibition under high-light. Here, a systematic screening by synthetic transgene design of carotenoid pathway enzymes and overexpression from the nuclear genome identified phytoene synthase (PSY/crtB) as a bottleneck for carotenoid accumulation in C. reinhardtii. Increased ß-carotene hydroxylase (CHYB) activity was found to be essential for engineered astaxanthin accumulation. A combined BKT, crtB, and CHYB expression strategy resulted in a volumetric astaxanthin production of 9.5 ± 0.3 mg L-1 (4.5 ± 0.1 mg g-1 CDW) in mixotrophic and 23.5 mg L-1 (1.09 mg L-1 h-1) in high cell density conditions, a 4-fold increase compared to previous reports in C. reinhardtii. This work presents a systematic investigation of bottlenecks in astaxanthin accumulation in C. reinhardtii and the phototrophic green cell factory design for competitive use in industrial biotechnology.

PlantACT! - how to tackle the climate crisis.

Greenhouse gas (GHG) emissions have created a global climate crisis which requires immediate interventions to mitigate the negative effects on all aspects of life on this planet. As current agriculture and land use contributes up to 25% of total GHG emissions, plant scientists take center stage in finding possible solutions for a transition to sustainable agriculture and land use. In this article, the PlantACT! (Plants for climate ACTion!) initiative of plant scientists lays out a road map of how and in which areas plant scientists can contribute to finding immediate, mid-term, and long-term solutions, and what changes are necessary to implement these solutions at the personal, institutional, and funding levels.

Natural carbon fixation and advances in synthetic engineering for redesigning and creating new fixation pathways.

BACKGROUND: Autotrophic carbon fixation is the primary route through which organic carbon enters the biosphere, and it is a key step in the biogeochemical carbon cycle. The Calvin-Benson-Bassham pathway, which is predominantly found in plants, algae, and some bacteria (mainly cyanobacteria), was previously considered to be the sole carbon-fixation pathway. However, the discovery of a new carbon-fixation pathway in sulfurous green bacteria almost two decades ago encouraged further research on previously overlooked ancient carbon-fixation pathways in taxonomically and phylogenetically distinct microorganisms. AIM OF REVIEW: In this review, we summarize the six known natural carbon-fixation pathways and outline the newly proposed additions to this list. We also discuss the recent achievements in synthetic carbon fixation and the importance of the metabolism of thermophilic microorganisms in this field. KEY SCIENTIFIC CONCEPTS OF REVIEW: Currently, at least six carbon-fixation routes have been confirmed in Bacteria and Archaea. Other possible candidate routes have also been suggested on the basis of emerging "omics" data analyses, expanding our knowledge and stimulating discussions on the importance of these pathways in the way organisms acquire carbon. Notably, the currently known natural fixation routes cannot balance the excessive anthropogenic carbon emissions in a highly unbalanced global carbon cycle. Therefore, significant efforts have also been made to improve the existing carbon-fixation pathways and/or design new efficient in vitro and in vivo synthetic pathways.

Biomass generation and heterologous isoprenoid milking from engineered microalgae grown in anaerobic membrane bioreactor effluent.

Wastewater (WW) treatment in anaerobic membrane bioreactors (AnMBR) is considered more sustainable than in aerobic reactors. However, outputs from AnMBR are a mixed methane and carbon dioxide gas stream as well as ammonium- (N) and phosphate- (P) containing waters. Using AnMBR outputs as inputs for photoautotrophic algal cultivation can strip the CO2 while removing N and P from effluent which feed algal biomass generation. Recent advances in algal engineering have generated strains that produce high-value side products concomitant with biomass, although only shown in heavily domesticated, lab-adapted strains. Here, it was investigated whether engineered Chlamydomonas reinhardtii could be grown directly in AnMBR effluent with CO2 concentrations found in AnMBR off-gas. The strain was found to proliferate over bacteria in the non-sterile effluent, consume N and P to levels that meet general discharge or reuse limits, and tolerate cultivation in modelled (extreme) outdoor environmental conditions prevalent along the central Red Sea coast. In addition to ∼2.4 g CDW L-1 biomass production in 96 h, a high-value heterologous sesquiterpene co-product could be obtained from 'milking' up to 837 µg L-1 culture in 96 h. This is the first demonstration of a combined bio-process that employs a heavily engineered algal strain to enhance the product generation potentials from AnMBR effluent treatment. This study shows it is possible to convert waste into value through use of engineered algae while also improving wastewater treatment economics through co-product generation.

Teaching an old 'doc' new tricks for algal biotechnology: Strategic filter use enables multi-scale fluorescent protein signal detection.

Fluorescent proteins (FPs) are powerful reporters with a broad range of applications in gene expression and subcellular localization. High-throughput screening is often required to identify individual transformed cell lines in organisms that favor non-homologous-end-joining integration of transgenes into genomes, like in the model green microalga Chlamydomonas reinhardtii. Strategic transgene design, including genetic fusion of transgenes to FPs, and strain domestication have aided engineering efforts in this host but have not removed the need for screening large numbers of transformants to identify those with robust transgene expression levels. FPs facilitate transformant screening by providing a visual signal indicating transgene expression. However, limited combinations of FPs have been described in alga and inherent background fluorescence from cell pigments can hinder FP detection efforts depending on available infrastructure. Here, an updated set of algal nuclear genome-domesticated plasmid parts for seven FPs and six epitope tags were generated and tested in C. reinhardtii. Strategic filter selection was found to enable detection of up to five independent FPs signals from cyan to far-red separately from inherent chlorophyll fluorescence in live algae at the agar plate-level and also in protein electrophoresis gels. This work presents technical advances for algal engineering that can assist reporter detection efforts in other photosynthetic host cells or organisms with inherent background fluorescence.

Chlamydomonas reinhardtii Alternates Peroxisomal Contents in Response to Trophic Conditions.

Chlamydomonas reinhardtii is a model green microalga capable of heterotrophic growth on acetic acid but not fatty acids, despite containing a full complement of genes for ß-oxidation. Recent reports indicate that the alga preferentially sequesters, rather than breaks down, lipid acyl chains as a means to rebuild its membranes rapidly. Here, we assemble a list of potential Chlamydomonas peroxins (PEXs) required for peroxisomal biogenesis to suggest that C. reinhardtii has a complete set of peroxisome biogenesis factors. To determine involvements of the peroxisomes in the metabolism of exogenously added fatty acids, we examined transgenic C. reinhardtii expressing fluorescent proteins fused to N- or C-terminal peptide of peroxisomal proteins, concomitantly with fluorescently labeled palmitic acid under different trophic conditions. We used confocal microscopy to track the populations of the peroxisomes in illuminated and dark conditions, with and without acetic acid as a carbon source. In the cells, four major populations of compartments were identified, containing: (1) a glyoxylate cycle enzyme marker and a protein containing peroxisomal targeting signal 1 (PTS1) tripeptide but lacking the fatty acid marker, (2) the fatty acid marker alone, (3) the glyoxylate cycle enzyme marker alone, and (4) the PTS1 marker alone. Less than 5% of the compartments contained both fatty acid and peroxisomal markers. Statistical analysis on optically sectioned images found that C. reinhardtii simultaneously carries diverse populations of the peroxisomes in the cell and modulates peroxisomal contents based on light conditions. On the other hand, the ratio of the compartment containing both fatty acid and peroxisomal markers did not change significantly regardless of the culture conditions. The result indicates that ß-oxidation may be only a minor occurrence in the peroxisomal population in C. reinhardtii, which supports the idea that lipid biosynthesis and not ß-oxidation is the primary metabolic preference of fatty acids in the alga.

Farnesyl pyrophosphate compartmentalization in the green microalga Chlamydomonas reinhardtii during heterologous (E)-α-bisabolene production.

BACKGROUND: Eukaryotic algae have recently emerged as hosts for metabolic engineering efforts to generate heterologous isoprenoids. Isoprenoid metabolic architectures, flux, subcellular localization, and transport dynamics have not yet been fully elucidated in algal hosts. RESULTS: In this study, we investigated the accessibility of different isoprenoid precursor pools for C15 sesquiterpenoid generation in the cytoplasm and chloroplast of Chlamydomonas reinhardtii using the Abies grandis bisabolene synthase (AgBS) as a reporter. The abundance of the C15 sesquiterpene precursor farnesyl pyrophosphate (FPP) was not increased in the cytosol by co-expression and fusion of AgBS with different FPP synthases (FPPSs), indicating limited C5 precursor availability in the cytoplasm. However, FPP was shown to be available in the plastid stroma, where bisabolene titers could be improved several-fold by FPPSs. Sesquiterpene production was greatest when AgBS-FPPS fusions were directed to the plastid and could further be improved by increasing the gene dosage. During scale-up cultivation with different carbon sources and light regimes, specific sesquiterpene productivities from the plastid were highest with CO2 as the only carbon source and light:dark illumination cycles. Potential prenyl unit transporters are proposed based on bioinformatic analyses, which may be in part responsible for our observations. CONCLUSIONS: Our findings indicate that the algal chloroplast can be harnessed in addition to the cytosol to exploit the full potential of algae as green cell factories for non-native sesquiterpenoid generation. Identification of a prenyl transporter may be leveraged for further extending this capacity.

Biocompatible fluorocarbon liquid underlays for in situ extraction of isoprenoids from microbial cultures.

Microbial production of heterologous metabolites is now a mature technology in many host organisms, opening new avenues for green production processes of specialty chemicals. At lab scale, petroleum-based hydrophobic bio-compatible solvents like dodecane can be used as a second phase on top of microbial cultures to act as a physical sink for heterologous hydrocarbon products like isoprenoids. However, this approach has significant drawbacks at scale due to the difficulty of handling solvents and their potential contamination with unwanted byproducts of their manufacture. We discovered that synthetic perfluorocarbon liquids (FCs), commonly used for heat transfer, can also act as physical sinks for microbially produced isoprenoid compounds. FCs are stable, inert, and are amenable to direct liquid-liquid extraction with alcohols for rapid product isolation. These liquids are more dense than water and form a lower phase to microbial cultures rather than an upper phase as with other solvents. Their ability to form an under-layer or 'underlay' also enables the cultivation of microbes directly at the FC-culture medium interface via gravity settling, which could open their application for filamentous or mat-forming organisms. We present comparisons of the isoprenoid extraction potential of three commercial FCs: FC-3283, FC-40, and FC-770 with engineered green microalga cultures producing patchoulol, taxadiene, casbene, or 13R(+) manoyl oxide. We demonstrate that FCs are promising alternatives to traditional solvents and open new avenues in bio-process design for microbial heterologous metabolite milking.

A New Approach to the Study of Plastidial Stress Granules: The Integrated Use of Arabidopsis thaliana and Chlamydomonas reinhardtii as Model Organisms.

The field of stress granules (SGs) has recently emerged in the study of the plant stress response, yet these structures, their dynamics and importance remain poorly characterized. There is currently a gap in our understanding of the physiological function of SGs during stress. Since there are only a few studies addressing SGs in planta, which are primarily focused on cytoplasmic SGs. The recent observation of SG-like foci in the chloroplast (cpSGs) of Arabidopsis thaliana opened even more questions regarding the role of these subcellular features. In this opinion article, we review the current knowledge of cpSGs and propose a workflow for the joint use of the long-established model organisms Chlamydomonas reinhardtii and A. thaliana to accelerate the evaluation of individual plant cpSGs components and their impact on stress responses. Finally, we present a short outlook and what we believe are the significant gaps that need to be addressed in the following years.

Combinatorial Engineering Enables Photoautotrophic Growth in High Cell Density Phosphite-Buffered Media to Support Engineered Chlamydomonas reinhardtii Bio-Production Concepts.

Chlamydomonas reinhardtii has emerged as a powerful green cell factory for metabolic engineering of sustainable products created from the photosynthetic lifestyle of this microalga. Advances in nuclear genome modification and transgene expression are allowing robust engineering strategies to be demonstrated in this host. However, commonly used lab strains are not equipped with features to enable their broader implementation in non-sterile conditions and high-cell density concepts. Here, we used combinatorial chloroplast and nuclear genome engineering to augment the metabolism of the C. reinhardtii strain UVM4 with publicly available genetic tools to enable the use of inorganic phosphite and nitrate as sole sources of phosphorous and nitrogen, respectively. We present recipes to create phosphite-buffered media solutions that enable high cell density algal cultivation. We then combined previously reported engineering strategies to produce the heterologous sesquiterpenoid patchoulol to high titers from our engineered green cell factories and show these products are possible to produce in non-sterile conditions. Our work presents a straightforward means to generate C. reinhardtii strains for broader application in bio-processes for the sustainable generation of products from green microalgae.

Subcellular Localizations of Catalase and Exogenously Added Fatty Acid in Chlamydomonas reinhardtii.

Fatty acids are important biological components, yet the metabolism of fatty acids in microalgae is not clearly understood. Previous studies found that Chlamydomonas reinhardtii, the model microalga, incorporates exogenously added fatty acids but metabolizes them differently from animals and yeast. Furthermore, a recent metabolic flux analysis found that the majority of lipid turnover in C. reinhardtii is the recycling of acyl chains from and to membranes, rather than ß -oxidation. This indicates that for the alga, the maintenance of existing acyl chains may be more valuable than their breakdown for energy. To gain cell-biological knowledge of fatty acid metabolism in C. reinhardtii, we conducted microscopy analysis with fluorescent probes. First, we found that CAT1 (catalase isoform 1) is in the peroxisomes while CAT2 (catalase isoform 2) is localized in the endoplasmic reticulum, indicating the alga is capable of detoxifying hydrogen peroxide that would be produced during ß-oxidation in the peroxisomes. Second, we compared the localization of exogenously added FL-C16 (fluorescently labelled palmitic acid) with fluorescently marked endosomes, mitochondria, peroxisomes, lysosomes, and lipid droplets. We found that exogenously added FL-C16 are incorporated and compartmentalized via a non-endocytic route within 10 min. However, the fluorescence signals from FL-C16 did not colocalize with any marked organelles, including peroxisomes. During triacylglycerol accumulation, the fluorescence signals from FL-C16 were localized in lipid droplets. These results support the idea that membrane turnover is favored over ß-oxidation in C. reinhardtii. The knowledge gained in these analyses would aid further studies of the fatty acid metabolism.

Alternative glycosylation controls endoplasmic reticulum dynamics and tubular extension in mammalian cells.

The endoplasmic reticulum (ER) is a central eukaryotic organelle with a tubular network made of hairpin proteins linked by hydrolysis of guanosine triphosphate nucleotides. Among posttranslational modifications initiated at the ER level, glycosylation is the most common reaction. However, our understanding of the impact of glycosylation on the ER structure remains unclear. Here, we show that exostosin-1 (EXT1) glycosyltransferase, an enzyme involved in N-glycosylation, is a key regulator of ER morphology and dynamics. We have integrated multiomics and superresolution imaging to characterize the broad effect of EXT1 inactivation, including the ER shape-dynamics-function relationships in mammalian cells. We have observed that inactivating EXT1 induces cell enlargement and enhances metabolic switches such as protein secretion. In particular, suppressing EXT1 in mouse thymocytes causes developmental dysfunctions associated with the ER network extension. Last, our data illuminate the physical and functional aspects of the ER proteome-glycome-lipidome structure axis, with implications in biotechnology and medicine.

Gene Delivery Technologies with Applications in Microalgal Genetic Engineering.

Microalgae and cyanobacteria are photosynthetic microbes that can be grown with the simple inputs of water, carbon dioxide, (sun)light, and trace elements. Their engineering holds the promise of tailored bio-molecule production using sustainable, environmentally friendly waste carbon inputs. Although algal engineering examples are beginning to show maturity, severe limitations remain in the transformation of multigene expression cassettes into model species and DNA delivery into non-model hosts. This review highlights common and emerging DNA delivery methods used for other organisms that may find future applications in algal engineering.

Efflux Transporters' Engineering and Their Application in Microbial Production of Heterologous Metabolites.

Metabolic engineering of microbial hosts for the production of heterologous metabolites and biochemicals is an enabling technology to generate meaningful quantities of desired products that may be otherwise difficult to produce by traditional means. Heterologous metabolite production can be restricted by the accumulation of toxic products within the cell. Efflux transport proteins (transporters) provide a potential solution to facilitate the export of these products, mitigate toxic effects, and enhance production. Recent investigations using knockout lines, heterologous expression, and expression profiling of transporters have revealed candidates that can enhance the export of heterologous metabolites from microbial cell systems. Transporter engineering efforts have revealed that some exhibit flexible substrate specificity and may have broader application potentials. In this Review, the major superfamilies of efflux transporters, their mechanistic modes of action, selection of appropriate efflux transporters for desired compounds, and potential transporter engineering strategies are described for potential applications in enhancing engineered microbial metabolite production. Future studies in substrate recognition, heterologous expression, and combinatorial engineering of efflux transporters will assist efforts to enhance heterologous metabolite production in microbial hosts.

Engineering Biocatalytic Solar Fuel Production: The PHOTOFUEL Consortium.

The EU Horizon2020 consortium PHOTOFUEL joined academic and industrial partners from biology, chemistry, engineering, engine design, and lifecycle assessment, making tremendous progress towards engine-ready fuels from CO2 via engineered photosynthetic microbes. Technical, environmental, economic, and societal opportunities and challenges were explored to frame future technology realization at scale.