A promiscuous mechanism to phase separate eukaryotic carbon fixation in the green lineage.

CO2 fixation is commonly limited by inefficiency of the CO2-fixing enzyme Rubisco. Eukaryotic algae concentrate and fix CO2 in phase-separated condensates called pyrenoids, which complete up to one-third of global CO2 fixation. Condensation of Rubisco in pyrenoids is dependent on interaction with disordered linker proteins that show little conservation between species. We developed a sequence-independent bioinformatic pipeline to identify linker proteins in green algae. We report the linker from Chlorella and demonstrate that it binds a conserved site on the Rubisco large subunit. We show that the Chlorella linker phase separates Chlamydomonas Rubisco and that despite their separation by ~800 million years of evolution, the Chlorella linker can support the formation of a functional pyrenoid in Chlamydomonas. This cross-species reactivity extends to plants, with the Chlorella linker able to drive condensation of some native plant Rubiscos in vitro and in planta. Our results represent an exciting frontier for pyrenoid engineering in plants, which is modelled to increase crop yields.

Bestrophin-like protein 4 is involved in photosynthetic acclimation to light fluctuations in Chlamydomonas.

In many eukaryotic algae, CO2 fixation by Rubisco is enhanced by a CO2-concentrating mechanism, which utilizes a Rubisco-rich organelle called the pyrenoid. The pyrenoid is traversed by a network of thylakoid membranes called pyrenoid tubules, which are proposed to deliver CO2. In the model alga Chlamydomonas (Chlamydomonas reinhardtii), the pyrenoid tubules have been proposed to be tethered to the Rubisco matrix by a bestrophin-like transmembrane protein, BST4. Here, we show that BST4 forms a complex that localizes to the pyrenoid tubules. A Chlamydomonas mutant impaired in the accumulation of BST4 (bst4) formed normal pyrenoid tubules, and heterologous expression of BST4 in Arabidopsis (Arabidopsis thaliana) did not lead to the incorporation of thylakoids into a reconstituted Rubisco condensate. Chlamydomonas bst4 mutants did not show impaired growth under continuous light at air level CO2 but were impaired in their growth under fluctuating light. By quantifying the non-photochemical quenching (NPQ) of chlorophyll fluorescence, we propose that bst4 has a transiently lower thylakoid lumenal pH during dark-to-light transition compared to control strains. We conclude that BST4 is not a tethering protein but is most likely a pyrenoid tubule ion channel involved in the ion homeostasis of the lumen with particular importance during light fluctuations.

Biogenesis, engineering and function of membranes in the CO 2 -fixing pyrenoid.

Approximately one-third of global CO 2 assimilation is performed by the pyrenoid 1 , a liquid-like organelle found in most algae and some plants 2 . Specialized membranes are hypothesized to drive CO 2 assimilation in the pyrenoid by delivering concentrated CO 2 3,4 , but their biogenesis and function have not been experimentally characterized. Here, we show that homologous proteins SAGA1 and MITH1 mediate the biogenesis of the pyrenoid membrane tubules in the model alga Chlamydomonas reinhardtii and are sufficient to reconstitute pyrenoid-traversing membranes in a heterologous system, the plant Arabidopsis thaliana . SAGA1 localizes to the regions where thylakoid membranes transition into tubules and is necessary to initiate tubule formation. MITH1 localizes to the tubules and is necessary for their extension through the pyrenoid. Tubule-deficient mutants exhibit growth defects under CO 2 -limiting conditions, providing evidence for the function of membrane tubules in CO 2 delivery to the pyrenoid. Furthermore, these mutants form multiple aberrant condensates of pyrenoid matrix, indicating that a normal tubule network promotes the coalescence of a single pyrenoid. The reconstitution of pyrenoid-traversing membranes in a plant represents a key milestone toward engineering a functional pyrenoid into crops for improving crop yields. More broadly, our study demonstrates the functional importance of pyrenoid membranes, identifies key biogenesis factors, and paves the way for the molecular characterization of pyrenoid membranes across the tree of life.

Engineering highly productive cyanobacteria towards carbon negative emissions technologies.

Cyanobacteria are a diverse and ecologically important group of photosynthetic prokaryotes that contribute significantly to the global carbon cycle through the capture of CO2 as biomass. Cyanobacterial biotechnology could play a key role in a sustainable bioeconomy through negative emissions technologies (NETs), such as carbon sequestration or bioproduction. However, the primary issues of low productivities and high infrastructure costs currently limit the commercialisation of such applications. The isolation of several fast-growing strains and recent advancements in molecular biology tools now offer promising new avenues for improving yields, including metabolic engineering approaches guided by high-throughput screening and metabolic models. Furthermore, emerging research on engineering coculture communities could help to develop more robust culturing systems to support broader NET applications.

A toolbox to engineer the highly productive cyanobacterium Synechococcus sp. PCC 11901.

Synechococcus sp. PCC 11901 (PCC 11901) is a fast-growing marine cyanobacterial strain that has a capacity for sustained biomass accumulation to very high cell densities, comparable to that achieved by commercially relevant heterotrophic organisms. However, genetic tools to engineer PCC 11901 for biotechnology applications are limited. Here we describe a suite of tools based on the CyanoGate MoClo system to unlock the engineering potential of PCC 11901. First, we characterized neutral sites suitable for stable genomic integration that do not affect growth even at high cell densities. Second, we tested a suite of constitutive promoters, terminators, and inducible promoters including a 2,4-diacetylphloroglucinol (DAPG)-inducible PhlF repressor system, which has not previously been demonstrated in cyanobacteria and showed tight regulation and a 228-fold dynamic range of induction. Lastly, we developed a DAPG-inducible dCas9-based CRISPR interference (CRISPRi) system and a modular method to generate markerless mutants using CRISPR-Cas12a. Based on our findings, PCC 11901 is highly responsive to CRISPRi-based repression and showed high efficiencies for single insertion (31% to 81%) and multiplex double insertion (25%) genome editing with Cas12a. We envision that these tools will lay the foundations for the adoption of PCC 11901 as a robust model strain for engineering biology and green biotechnology.

Perspectives on improving photosynthesis to increase crop yield.

Improving photosynthesis, the fundamental process by which plants convert light energy into chemical energy, is a key area of research with great potential for enhancing sustainable agricultural productivity and addressing global food security challenges. This perspective delves into the latest advancements and approaches aimed at optimizing photosynthetic efficiency. Our discussion encompasses the entire process, beginning with light harvesting and its regulation and progressing through the bottleneck of electron transfer. We then delve into the carbon reactions of photosynthesis, focusing on strategies targeting the enzymes of the Calvin-Benson-Bassham (CBB) cycle. Additionally, we explore methods to increase carbon dioxide (CO2) concentration near the Rubisco, the enzyme responsible for the first step of CBB cycle, drawing inspiration from various photosynthetic organisms, and conclude this section by examining ways to enhance CO2 delivery into leaves. Moving beyond individual processes, we discuss two approaches to identifying key targets for photosynthesis improvement: systems modeling and the study of natural variation. Finally, we revisit some of the strategies mentioned above to provide a holistic view of the improvements, analyzing their impact on nitrogen use efficiency and on canopy photosynthesis.

SAGA1 and SAGA2 promote starch formation around proto-pyrenoids in Arabidopsis chloroplasts.

The pyrenoid is a chloroplastic microcompartment in which most algae and some terrestrial plants condense the primary carboxylase, Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase) as part of a CO2-concentrating mechanism that improves the efficiency of CO2 capture. Engineering a pyrenoid-based CO2-concentrating mechanism (pCCM) into C3 crop plants is a promising strategy to enhance yield capacities and resilience to the changing climate. Many pyrenoids are characterized by a sheath of starch plates that is proposed to act as a barrier to limit CO2 diffusion. Recently, we have reconstituted a phase-separated "proto-pyrenoid" Rubisco matrix in the model C3 plant Arabidopsis thaliana using proteins from the alga with the most well-studied pyrenoid, Chlamydomonas reinhardtii [N. Atkinson, Y. Mao, K. X. Chan, A. J. McCormick, Nat. Commun. 11, 6303 (2020)]. Here, we describe the impact of introducing the Chlamydomonas proteins StArch Granules Abnormal 1 (SAGA1) and SAGA2, which are associated with the regulation of pyrenoid starch biogenesis and morphology. We show that SAGA1 localizes to the proto-pyrenoid in engineered Arabidopsis plants, which results in the formation of atypical spherical starch granules enclosed within the proto-pyrenoid condensate and adjacent plate-like granules that partially cover the condensate, but without modifying the total amount of chloroplastic starch accrued. Additional expression of SAGA2 further increases the proportion of starch synthesized as adjacent plate-like granules that fully encircle the proto-pyrenoid. Our findings pave the way to assembling a diffusion barrier as part of a functional pCCM in vascular plants, while also advancing our understanding of the roles of SAGA1 and SAGA2 in starch sheath formation and broadening the avenues for engineering starch morphology.

The role of BST4 in the pyrenoid of Chlamydomonas reinhardtii.

In many eukaryotic algae, CO2 fixation by Rubisco is enhanced by a CO2-concentrating mechanism, which utilizes a Rubisco-rich organelle called the pyrenoid. The pyrenoid is traversed by a network of thylakoid-membranes called pyrenoid tubules, proposed to deliver CO2. In the model alga Chlamydomonas reinhardtii (Chlamydomonas), the pyrenoid tubules have been proposed to be tethered to the Rubisco matrix by a bestrophin-like transmembrane protein, BST4. Here, we show that BST4 forms a complex that localizes to the pyrenoid tubules. A Chlamydomonas mutant impaired in the accumulation of BST4 (bst4) formed normal pyrenoid tubules and heterologous expression of BST4 in Arabidopsis thaliana did not lead to the incorporation of thylakoids into a reconstituted Rubisco condensate. Chlamydomonas bst4 mutant did not show impaired growth at air level CO2. By quantifying the non-photochemical quenching (NPQ) of chlorophyll fluorescence, we show that bst4 displays a transiently lower thylakoid lumenal pH during dark to light transition compared to control strains. When acclimated to high light, bst4 had sustained higher NPQ and elevated levels of light-induced H2O2 production. We conclude that BST4 is not a tethering protein, but rather is an ion channel involved in lumenal pH regulation possibly by mediating bicarbonate transport across the pyrenoid tubules.

Interlaboratory Reproducibility in Growth and Reporter Expression in the Cyanobacterium Synechocystis sp. PCC 6803.

In recent years, a plethora of new synthetic biology tools for use in cyanobacteria have been published; however, their reported characterizations often cannot be reproduced, greatly limiting the comparability of results and hindering their applicability. In this interlaboratory study, the reproducibility of a standard microbiological experiment for the cyanobacterial model organism Synechocystis sp. PCC 6803 was assessed. Participants from eight different laboratories quantified the fluorescence intensity of mVENUS as a proxy for the transcription activity of the three promoters PJ23100, PrhaBAD, and PpetE over time. In addition, growth rates were measured to compare growth conditions between laboratories. By establishing strict and standardized laboratory protocols, reflecting frequently reported methods, we aimed to identify issues with state-of-the-art procedures and assess their effect on reproducibility. Significant differences in spectrophotometer measurements across laboratories from identical samples were found, suggesting that commonly used reporting practices of optical density values need to be supplemented by cell count or biomass measurements. Further, despite standardized light intensity in the incubators, significantly different growth rates between incubators used in this study were observed, highlighting the need for additional reporting requirements of growth conditions for phototrophic organisms beyond the light intensity and CO2 supply. Despite the use of a regulatory system orthogonal to Synechocystis sp. PCC 6803, PrhaBAD, and a high level of protocol standardization, ∼32% variation in promoter activity under induced conditions was found across laboratories, suggesting that the reproducibility of other data in the field of cyanobacteria might be affected similarly.

The small subunit of Rubisco and its potential as an engineering target.

Rubisco catalyses the first rate-limiting step in CO2 fixation and is responsible for the vast majority of organic carbon present in the biosphere. The function and regulation of Rubisco remain an important research topic and a longstanding engineering target to enhance the efficiency of photosynthesis for agriculture and green biotechnology. The most abundant form of Rubisco (Form I) consists of eight large and eight small subunits, and is found in all plants, algae, cyanobacteria, and most phototrophic and chemolithoautotrophic proteobacteria. Although the active sites of Rubisco are located on the large subunits, expression of the small subunit regulates the size of the Rubisco pool in plants and can influence the overall catalytic efficiency of the Rubisco complex. The small subunit is now receiving increasing attention as a potential engineering target to improve the performance of Rubisco. Here we review our current understanding of the role of the small subunit and our growing capacity to explore its potential to modulate Rubisco catalysis using engineering biology approaches.

New horizons for building pyrenoid-based CO2-concentrating mechanisms in plants to improve yields.

Many photosynthetic species have evolved CO2-concentrating mechanisms (CCMs) to improve the efficiency of CO2 assimilation by Rubisco and reduce the negative impacts of photorespiration. However, the majority of plants (i.e. C3 plants) lack an active CCM. Thus, engineering a functional heterologous CCM into important C3 crops, such as rice (Oryza sativa) and wheat (Triticum aestivum), has become a key strategic ambition to enhance yield potential. Here, we review recent advances in our understanding of the pyrenoid-based CCM in the model green alga Chlamydomonas reinhardtii and engineering progress in C3 plants. We also discuss recent modeling work that has provided insights into the potential advantages of Rubisco condensation within the pyrenoid and the energetic costs of the Chlamydomonas CCM, which, together, will help to better guide future engineering approaches. Key findings include the potential benefits of Rubisco condensation for carboxylation efficiency and the need for a diffusional barrier around the pyrenoid matrix. We discuss a minimal set of components for the CCM to function and that active bicarbonate import into the chloroplast stroma may not be necessary for a functional pyrenoid-based CCM in planta. Thus, the roadmap for building a pyrenoid-based CCM into plant chloroplasts to enhance the efficiency of photosynthesis now appears clearer with new challenges and opportunities.

Development of a Biotechnology Platform for the Fast-Growing Cyanobacterium Synechococcus sp. PCC 11901.

Synechococcus sp. PCC 11901 reportedly demonstrates the highest, most sustained growth of any known cyanobacterium under optimized conditions. Due to its recent discovery, our knowledge of its biology, including the factors underlying sustained, fast growth, is limited. Furthermore, tools specific for genetic manipulation of PCC 11901 are not established. Here, we demonstrate that PCC 11901 shows faster growth than other model cyanobacteria, including the fast-growing species Synechococcuselongatus UTEX 2973, under optimal growth conditions for UTEX 2973. Comparative genomics between PCC 11901 and Synechocystis sp. PCC 6803 reveal conservation of most metabolic pathways but PCC 11901 has a simplified electron transport chain and reduced light harvesting complex. This may underlie its superior light use, reduced photoinhibition, and higher photosynthetic and respiratory rates. To aid biotechnology applications, we developed a vitamin B12 auxotrophic mutant but were unable to generate unmarked knockouts using two negative selectable markers, suggesting that recombinase- or CRISPR-based approaches may be required for repeated genetic manipulation. Overall, this study establishes PCC 11901 as one of the most promising species currently available for cyanobacterial biotechnology and provides a useful set of bioinformatics tools and strains for advancing this field, in addition to insights into the factors underlying its fast growth phenotype.

Pilot scale production, extraction and purification of a thermostable phycocyanin from Synechocystis sp. PCC 6803.

Phycocyanin (PC) is a soluble blue pigment-protein primarily harvested from the cyanobacterium Arthrospira platensis. PC is in high demand from several industries, but its narrow stability range limits potential applications. Here, a pilot scale (120 L total) batch production, extraction and purification process for thermostable PC (Te-PC) from a Synechocystis sp. PCC 6803 'Olive' strain expressing the PC operon cpcBACD from Thermosynechococcus elongatus BP-1 on a self-replicating vector is presented. Batch cultivation without antibiotics had no impact on growth or Te-PC production and optimisation of growth conditions resulted in Te-PC contents of 75.3 ± 1.7 mg g DW-1. Wet biomass was harvested following chitosan-based flocculation with a 97 ± 2% efficiency, and Te-PC was extracted by high pressure homogenisation. Subsequent purification by heat-treatment and two-step ammonium sulfate precipitation removed chlorophyll and allophycocyanin contamination, resulting in Te-PC purities of 2.9 ± 0.7 and a mean Te-PC recovery of 84 ± 12%.

Production of thermostable phycocyanin in a mesophilic cyanobacterium.

Phycocyanin (PC) is a soluble phycobiliprotein found within the light-harvesting phycobilisome complex of cyanobacteria and red algae, and is considered a high-value product due to its brilliant blue colour and fluorescent properties. However, commercially available PC has a relatively low temperature stability. Thermophilic species produce more thermostable variants of PC, but are challenging and energetically expensive to cultivate. Here, we show that the PC operon from the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1 (cpcBACD) is functional in the mesophile Synechocystis sp. PCC 6803. Expression of cpcBACD in an 'Olive' mutant strain of Synechocystis lacking endogenous PC resulted in high yields of thermostable PC (112 ± 1 mg g-1 DW) comparable to that of endogenous PC in wild-type cells. Heterologous PC also improved the growth of the Olive mutant, which was further supported by evidence of a functional interaction with the endogenous allophycocyanin core of the phycobilisome complex. The thermostability properties of the heterologous PC were comparable to those of PC from T. elongatus, and could be purified from the Olive mutant using a low-cost heat treatment method. Finally, we developed a scalable model to calculate the energetic benefits of producing PC from T. elongatus in Synechocystis cultures. Our model showed that the higher yields and lower cultivation temperatures of Synechocystis resulted in a 3.5-fold increase in energy efficiency compared to T. elongatus, indicating that producing thermostable PC in non-native hosts is a cost-effective strategy for scaling to commercial production.

Demonstration of protein capture and separation using three-dimensional printed anion exchange monoliths fabricated in one-step.

Three-dimensional printing applications in separation science are currently limited by the lack of materials compatible with chromatographic operations and three-dimensional printing technologies. In this work, we propose a new material for Digital Light Processing printing to fabricate functional ion exchange monoliths in a single step. Through copolymerization of the bifunctional monomer [2-(acryloyloxy)ethyl] trimethylammonium chloride, monolithic structures with quaternary amine ligands were fabricated. The novel formulation was optimized in terms of protein binding and recovery, microporous structure, and its swelling susceptibility by increasing its cross-link density and employing cyclohexanol and dodecanol as pore forming agents. In static conditions, the material demonstrated a maximum binding capacity of 104.2 ± 10.6 mg/mL for bovine serum albumin, in line with commercially available materials. Its anion exchange behavior was validated by separating bovine serum albumin and myoglobin on a monolithic bed with Schoen gyroid morphology. The same column geometry was tested for the purification of C-phycocyanin from clarified as well as cell-laden Arthrospira platensis feedstocks. This represents the first demonstration of one-step printed stationary phases to capture proteins directly from solid-laden feedstocks. We believe that the material presented here represents a significant improvement towards implementation of three-dimensional printed chromatography media in the field of separation science.

Microcystinase - a review of the natural occurrence, heterologous expression, and biotechnological application of MlrA.

Microcystinase (MlrA) was first described in 1996. Since then MlrA peptidase activity has proven to be both the most efficient enzymatic process and the most specific catalyst of all known microcystins detoxification pathways. Furthermore, MlrA and the MlrABC degradation pathway are presently the only enzymatic processes with clear genetic and biochemical descriptions available for microcystins degradation, greatly facilitating modern applied genetics for any relevant technological development. Recently, there has been increasing interest in the potential of sustainable, biologically inspired alternatives to current industrial practice, with note that biological microcystins degradation is the primary detoxification process found in nature. While previous reviews have broadly discussed microbial biodegradation processes, here we present a review focused specifically on MlrA. Following a general overview, we briefly highlight the initial discovery and present understanding of the MlrABC degradation pathway, before discussing the genetic and biochemical aspects of MlrA. We then review the potential biotechnology applications of MlrA in the context of available literature with emphasis on the optimization of MlrA for in situ applications including (i) direct modulation of Mlr activity within naturally existing populations, (ii) bioaugmentation of systems with introduced biodegradative capacity via whole cell biocatalysts, and (iii) bioremediation via direct MlrA application.