Rubisco Function, Evolution, and Engineering.

Carbon fixation is the process by which CO2 is converted from a gas into biomass. The Calvin-Benson-Bassham cycle (CBB) is the dominant carbon-consuming pathway on Earth, driving >99.5% of the ∼120 billion tons of carbon that are converted to sugar by plants, algae, and cyanobacteria. The carboxylase enzyme in the CBB, ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco), fixes one CO2 molecule per turn of the cycle into bioavailable sugars. Despite being critical to the assimilation of carbon, rubisco's kinetic rate is not very fast, limiting flux through the pathway. This bottleneck presents a paradox: Why has rubisco not evolved to be a better catalyst? Many hypothesize that the catalytic mechanism of rubisco is subject to one or more trade-offs and that rubisco variants have been optimized for their native physiological environment. Here, we review the evolution and biochemistry of rubisco through the lens of structure and mechanism in order to understand what trade-offs limit its improvement. We also review the many attempts to improve rubisco itself and thereby promote plant growth.

Conversion of Escherichia coli to Generate All Biomass Carbon from CO2.

The living world is largely divided into autotrophs that convert CO2 into biomass and heterotrophs that consume organic compounds. In spite of widespread interest in renewable energy storage and more sustainable food production, the engineering of industrially relevant heterotrophic model organisms to use CO2 as their sole carbon source has so far remained an outstanding challenge. Here, we report the achievement of this transformation on laboratory timescales. We constructed and evolved Escherichia coli to produce all its biomass carbon from CO2. Reducing power and energy, but not carbon, are supplied via the one-carbon molecule formate, which can be produced electrochemically. Rubisco and phosphoribulokinase were co-expressed with formate dehydrogenase to enable CO2 fixation and reduction via the Calvin-Benson-Bassham cycle. Autotrophic growth was achieved following several months of continuous laboratory evolution in a chemostat under intensifying organic carbon limitation and confirmed via isotopic labeling.

A Spatial Interactome Reveals the Protein Organization of the Algal CO2-Concentrating Mechanism.

Approximately one-third of global CO2 fixation is performed by eukaryotic algae. Nearly all algae enhance their carbon assimilation by operating a CO2-concentrating mechanism (CCM) built around an organelle called the pyrenoid, whose protein composition is largely unknown. Here, we developed tools in the model alga Chlamydomonas reinhardtii to determine the localizations of 135 candidate CCM proteins and physical interactors of 38 of these proteins. Our data reveal the identity of 89 pyrenoid proteins, including Rubisco-interacting proteins, photosystem I assembly factor candidates, and inorganic carbon flux components. We identify three previously undescribed protein layers of the pyrenoid: a plate-like layer, a mesh layer, and a punctate layer. We find that the carbonic anhydrase CAH6 is in the flagella, not in the stroma that surrounds the pyrenoid as in current models. These results provide an overview of proteins operating in the eukaryotic algal CCM, a key process that drives global carbon fixation.

The Eukaryotic CO2-Concentrating Organelle Is Liquid-like and Exhibits Dynamic Reorganization.

Approximately 30%-40% of global CO2 fixation occurs inside a non-membrane-bound organelle called the pyrenoid, which is found within the chloroplasts of most eukaryotic algae. The pyrenoid matrix is densely packed with the CO2-fixing enzyme Rubisco and is thought to be a crystalline or amorphous solid. Here, we show that the pyrenoid matrix of the unicellular alga Chlamydomonas reinhardtii is not crystalline but behaves as a liquid that dissolves and condenses during cell division. Furthermore, we show that new pyrenoids are formed both by fission and de novo assembly. Our modeling predicts the existence of a "magic number" effect associated with special, highly stable heterocomplexes that influences phase separation in liquid-like organelles. This view of the pyrenoid matrix as a phase-separated compartment provides a paradigm for understanding its structure, biogenesis, and regulation. More broadly, our findings expand our understanding of the principles that govern the architecture and inheritance of liquid-like organelles.

A systematic exploration of bacterial form I rubisco maximal carboxylation rates.

Autotrophy is the basis for complex life on Earth. Central to this process is rubisco-the enzyme that catalyzes almost all carbon fixation on the planet. Yet, with only a small fraction of rubisco diversity kinetically characterized so far, the underlying biological factors driving the evolution of fast rubiscos in nature remain unclear. We conducted a high-throughput kinetic characterization of over 100 bacterial form I rubiscos, the most ubiquitous group of rubisco sequences in nature, to uncover the determinants of rubisco's carboxylation velocity. We show that the presence of a carboxysome CO2 concentrating mechanism correlates with faster rubiscos with a median fivefold higher rate. In contrast to prior studies, we find that rubiscos originating from α-cyanobacteria exhibit the highest carboxylation rates among form I enzymes (≈10 s-1 median versus <7 s-1 in other groups). Our study systematically reveals biological and environmental properties associated with kinetic variation across rubiscos from nature.

Illuminating the coevolution of photosynthesis and Bacteria.

Life harnessing light energy transformed the relationship between biology and Earth-bringing a massive flux of organic carbon and oxidants to Earth's surface that gave way to today's organotrophy- and respiration-dominated biosphere. However, our understanding of how life drove this transition has largely relied on the geological record; much remains unresolved due to the complexity and paucity of the genetic record tied to photosynthesis. Here, through holistic phylogenetic comparison of the bacterial domain and all photosynthetic machinery (totally spanning >10,000 genomes), we identify evolutionary congruence between three independent biological systems-bacteria, (bacterio)chlorophyll-mediated light metabolism (chlorophototrophy), and carbon fixation-and uncover their intertwined history. Our analyses uniformly mapped progenitors of extant light-metabolizing machinery (reaction centers, [bacterio]chlorophyll synthases, and magnesium-chelatases) and enzymes facilitating the Calvin-Benson-Bassham cycle (form I RuBisCO and phosphoribulokinase) to the same ancient Terrabacteria organism near the base of the bacterial domain. These phylogenies consistently showed that extant phototrophs ultimately derived light metabolism from this bacterium, the last phototroph common ancestor (LPCA). LPCA was a non-oxygen-generating (anoxygenic) phototroph that already possessed carbon fixation and two reaction centers, a type I analogous to extant forms and a primitive type II. Analyses also indicate chlorophototrophy originated before LPCA. We further reconstructed evolution of chlorophototrophs/chlorophototrophy post-LPCA, including vertical inheritance in Terrabacteria, the rise of oxygen-generating chlorophototrophy in one descendant branch near the Great Oxidation Event, and subsequent emergence of Cyanobacteria. These collectively unveil a detailed view of the coevolution of light metabolism and Bacteria having clear congruence with the geological record.

An alcove at the acetyl-CoA synthase nickel active site is required for productive substrate CO binding and anaerobic carbon fixation.

One of the seven natural CO2 fixation pathways, the anaerobic Wood-Ljungdahl pathway (WLP) is unique in generating CO as a metabolic intermediate, operating through organometallic intermediates, and in conserving (versus utilizing) net ATP. The key enzyme in the WLP is acetyl-CoA synthase (ACS), which uses an active site [2Ni-4Fe-4S] cluster (A-cluster), a CO tunnel, and an organometallic (Ni-CO, Ni-methyl, and Ni-acetyl) reaction sequence to generate acetyl-CoA. Here, we reveal that an alcove, which interfaces the tunnel and the A-cluster, is essential for CO2 fixation and autotrophic growth by the WLP. In vitro spectroscopy, kinetics, binding, and in vivo growth experiments reveal that a Phe229A substitution at one wall of the alcove decreases CO affinity thirty-fold and abolishes autotrophic growth; however, a F229W substitution enhances CO binding 80-fold. Our results indicate that the structure of the alcove is exquisitely tuned to concentrate CO near the A-cluster; protect ACS from CO loss during catalysis, provide a haven for inhibitory CO, and stabilize the tetrahedral coordination at the Nip site where CO binds. The directing, concentrating, and protective effects of the alcove explain the inability of F209A to grow autotrophically. The alcove also could help explain current controversies over whether ACS binds CO and methyl through a random or ordered mechanism. Our work redefines what we historically refer to as the metallocenter "active site". The alcove is so crucial for enzymatic function that we propose it is part of the active site. The community should now look for such alcoves in all "gas handling" metalloenzymes.

Rubisco kinetic adaptations to extreme environments.

Photosynthetic and chemosynthetic extremophiles have evolved adaptations to thrive in challenging environments by finely adjusting their metabolic pathways through evolutionary processes. A prime adaptation target to allow autotrophy in extreme conditions is the enzyme Rubisco, which plays a central role in the conversion of inorganic to organic carbon. Here, we present an extensive compilation of Rubisco kinetic traits from a wide range of species of bacteria, archaea, algae, and plants, sorted by phylogenetic group, Rubisco type, and extremophile type. Our results show that Rubisco kinetics for the few extremophile organisms reported up to date are placed at the margins of the enzyme's natural variability. Form ID Rubisco from thermoacidophile rhodophytes and form IB Rubisco from halophile terrestrial plants exhibit higher specificity and affinity for CO2 than their non-extremophilic counterparts, as well as higher carboxylation efficiency, whereas form ID Rubisco from psychrophile organisms possess lower affinity for O2. Additionally, form IB Rubisco from thermophile cyanobacteria shows enhanced CO2 specificity when compared to form IB non-extremophilic cyanobacteria. Overall, these findings highlight the unique characteristics of extremophile Rubisco enzymes and provide useful clues to guide next explorations aimed at finding more efficient Rubiscos.

Orchestrated translation specializes dinoflagellate metabolism three times per day.

Many cells specialize for different metabolic tasks at different times over their normal ZT cycle by changes in gene expression. However, in most cases, circadian gene expression has been assessed at the mRNA accumulation level, which may not faithfully reflect protein synthesis rates. Here, we use ribosome profiling in the dinoflagellate Lingulodinium polyedra to identify thousands of transcripts showing coordinated translation. All of the components in carbon fixation are concurrently regulated at ZT0, predicting the known rhythm of carbon fixation, and many enzymes involved in DNA replication are concurrently regulated at ZT12, also predicting the known rhythm in this process. Most of the enzymes in glycolysis and the TCA cycle are also regulated together, suggesting rhythms in these processes as well. Surprisingly, a third cluster of transcripts show peak translation at approximately ZT16, and these transcripts encode enzymes involved in transcription, translation, and amino acid biosynthesis. The latter has physiological consequences, as measured free amino acid levels increase at night and thus represent a previously undocumented rhythm in this model. Our results suggest that ribosome profiling may be a more accurate predictor of changed metabolic state than transcriptomics.

Riboflavin synthesis from gaseous nitrogen and carbon dioxide by a hybrid inorganic-biological system.

Microbes can provide a more sustainable and energy-efficient method of food and nutrient production compared to plant and animal sources, but energy-intensive carbon (e.g., sugars) and nitrogen (e.g., ammonia) inputs are required. Gas-fixing microorganisms that can grow on H2 from renewable water splitting and gaseous CO2 and N2 offer a renewable path to overcoming these limitations but confront challenges owing to the scarcity of genetic engineering in such organisms. Here, we demonstrate that the hydrogen-oxidizing carbon- and nitrogen-fixing microorganism Xanthobacter autotrophicus grown on a CO2/N2/H2 gas mixture can overproduce the vitamin riboflavin (vitamin B2). We identify plasmids and promoters for use in this bacterium and employ a constitutive promoter to overexpress riboflavin pathway enzymes. Riboflavin production is quantified at 15 times that of the wild-type organism. We demonstrate that riboflavin overproduction is maintained when the bacterium is grown under hybrid inorganic-biological conditions, in which H2 from water splitting, along with CO2 and N2, is fed to the bacterium, establishing the viability of the approach to sustainably produce food and nutrients.

Trajectories for the evolution of bacterial CO2-concentrating mechanisms.

Cyanobacteria rely on CO2-concentrating mechanisms (CCMs) to grow in today's atmosphere (0.04% CO2). These complex physiological adaptations require ≈15 genes to produce two types of protein complexes: inorganic carbon (Ci) transporters and 100+ nm carboxysome compartments that encapsulate rubisco with a carbonic anhydrase (CA) enzyme. Mutations disrupting any of these genes prohibit growth in ambient air. If any plausible ancestral form-i.e., lacking a single gene-cannot grow, how did the CCM evolve? Here, we test the hypothesis that evolution of the bacterial CCM was "catalyzed" by historically high CO2 levels that decreased over geologic time. Using an E. coli reconstitution of a bacterial CCM, we constructed strains lacking one or more CCM components and evaluated their growth across CO2 concentrations. We expected these experiments to demonstrate the importance of the carboxysome. Instead, we found that partial CCMs expressing CA or Ci uptake genes grew better than controls in intermediate CO2 levels (≈1%) and observed similar phenotypes in two autotrophic bacteria, Halothiobacillus neapolitanus and Cupriavidus necator. To understand how CA and Ci uptake improve growth, we model autotrophy as colimited by CO2 and HCO3-, as both are required to produce biomass. Our experiments and model delineated a viable trajectory for CCM evolution where decreasing atmospheric CO2 induces an HCO3- deficiency that is alleviated by acquisition of CA or Ci uptake, thereby enabling the emergence of a modern CCM. This work underscores the importance of considering physiology and environmental context when studying the evolution of biological complexity.

Engineering nonphotosynthetic carbon fixation for production of bioplastics by methanogenic archaea.

The conversion of CO2 to value-added products allows both capture and recycling of greenhouse gas emissions. While plants and other photosynthetic organisms play a key role in closing the global carbon cycle, their dependence on light to drive carbon fixation can be limiting for industrial chemical synthesis. Methanogenic archaea provide an alternative platform as an autotrophic microbial species capable of non-photosynthetic CO2 fixation, providing a potential route to engineered microbial fermentation to synthesize chemicals from CO2 without the need for light irradiation. One major challenge in this goal is to connect upstream carbon-fixation pathways with downstream biosynthetic pathways, given the distinct differences in metabolism between archaea and typical heterotrophs. We engineered the model methanogen, Methanococcus maripaludis, to divert acetyl-coenzyme A toward biosynthesis of value-added chemicals, including the bioplastic polyhydroxybutyrate (PHB). A number of studies implicated limitations in the redox pool, with NAD(P)(H) pools in M. maripaludis measured to be <15% of that of Escherichia coli, likely since methanogenic archaea utilize F420 and ferredoxins instead. Multiple engineering strategies were used to precisely target and increase the cofactor pool, including heterologous expression of a synthetic nicotinamide salvage pathway as well as an NAD+-dependent formate dehydrogenase from Candida boidinii. Engineered strains of M. maripaludis with improved NADH pools produced up to 171 ± 4 mg/L PHB and 24.0 ± 1.9% of dry cell weight. The metabolic engineering strategies presented in this study broaden the utility of M. maripaludis for sustainable chemical synthesis using CO2 and may be transferable to related archaeal species.

Extracellular carbonic anhydrase activity promotes a carbon concentration mechanism in metazoan calcifying cells.

Many calcifying organisms utilize metabolic CO2 to generate CaCO3 minerals to harden their shells and skeletons. Carbonic anhydrases are evolutionary ancient enzymes that have been proposed to play a key role in the calcification process, with the underlying mechanisms being little understood. Here, we used the calcifying primary mesenchyme cells (PMCs) of sea urchin larva to study the role of cytosolic (iCAs) and extracellular carbonic anhydrases (eCAs) in the cellular carbon concentration mechanism (CCM). Molecular analyses identified iCAs and eCAs in PMCs and highlight the prominent expression of a glycosylphosphatidylinositol-anchored membrane-bound CA (Cara7). Intracellular pH recordings in combination with CO2 pulse experiments demonstrated iCA activity in PMCs. iCA activity measurements, together with pharmacological approaches, revealed an opposing contribution of iCAs and eCAs on the CCM. H+-selective electrodes were used to demonstrate eCA-catalyzed CO2 hydration rates at the cell surface. Knockdown of Cara7 reduced extracellular CO2 hydration rates accompanied by impaired formation of specific skeletal segments. Finally, reduced pHi regulatory capacities during inhibition and knockdown of Cara7 underscore a role of this eCA in cellular HCO3- uptake. This work reveals the function of CAs in the cellular CCM of a marine calcifying animal. Extracellular hydration of metabolic CO2 by Cara7 coupled to HCO3- uptake mechanisms mitigates the loss of carbon and reduces the cellular proton load during the mineralization process. The findings of this work provide insights into the cellular mechanisms of an ancient biological process that is capable of utilizing CO2 to generate a versatile construction material.

Plant diversity decreases greenhouse gas emissions by increasing soil and plant carbon storage in terrestrial ecosystems.

The decline in global plant diversity has raised concerns about its implications for carbon fixation and global greenhouse gas emissions (GGE), including carbon dioxide (CO2), nitrous oxide (N2O) and methane (CH4). Therefore, we conducted a comprehensive meta-analysis of 2103 paired observations, examining GGE, soil organic carbon (SOC) and plant carbon in plant mixtures and monocultures. Our findings indicate that plant mixtures decrease soil N2O emissions by 21.4% compared to monocultures. No significant differences occurred between mixtures and monocultures for soil CO2 emissions, CH4 emissions or CH4 uptake. Plant mixtures exhibit higher SOC and plant carbon storage than monocultures. After 10 years of vegetation development, a 40% reduction in species richness decreases SOC content and plant carbon storage by 12.3% and 58.7% respectively. These findings offer insights into the intricate connections between plant diversity, soil and plant carbon storage and GGE-a critical but previously unexamined aspect of biodiversity-ecosystem functioning.

Highly active rubiscos discovered by systematic interrogation of natural sequence diversity.

CO2 is converted into biomass almost solely by the enzyme rubisco. The poor carboxylation properties of plant rubiscos have led to efforts that made it the most kinetically characterized enzyme, yet these studies focused on < 5% of its natural diversity. Here, we searched for fast-carboxylating variants by systematically mining genomic and metagenomic data. Approximately 33,000 unique rubisco sequences were identified and clustered into ≈ 1,000 similarity groups. We then synthesized, purified, and biochemically tested the carboxylation rates of 143 representatives, spanning all clusters of form-II and form-II/III rubiscos. Most variants (> 100) were active in vitro, with the fastest having a turnover number of 22 ± 1 s-1 -sixfold faster than the median plant rubisco and nearly twofold faster than the fastest measured rubisco to date. Unlike rubiscos from plants and cyanobacteria, the fastest variants discovered here are homodimers and exhibit a much simpler folding and activation kinetics. Our pipeline can be utilized to explore the kinetic space of other enzymes of interest, allowing us to get a better view of the biosynthetic potential of the biosphere.

Widespread dissolved inorganic carbon-modifying toolkits in genomes of autotrophic Bacteria and Archaea and how they are likely to bridge supply from the environment to demand by autotrophic pathways.

Using dissolved inorganic carbon (DIC) as a major carbon source, as autotrophs do, is complicated by the bedeviling nature of this substance. Autotrophs using the Calvin-Benson-Bassham cycle (CBB) are known to make use of a toolkit comprised of DIC transporters and carbonic anhydrase enzymes (CA) to facilitate DIC fixation. This minireview provides a brief overview of the current understanding of how toolkit function facilitates DIC fixation in Cyanobacteria and some Proteobacteria using the CBB and continues with a survey of the DIC toolkit gene presence in organisms using different versions of the CBB and other autotrophic pathways (reductive citric acid cycle, Wood-Ljungdahl pathway, hydroxypropionate bicycle, hydroxypropionate-hydroxybutyrate cycle, and dicarboxylate-hydroxybutyrate cycle). The potential function of toolkit gene products in these organisms is discussed in terms of CO2 and HCO3- supply from the environment and demand by the autotrophic pathway. The presence of DIC toolkit genes in autotrophic organisms beyond those using the CBB suggests the relevance of DIC metabolism to these organisms and provides a basis for better engineering of these organisms for industrial and agricultural purposes.

Production of succinate with two CO2 fixation reactions from fatty acids in Cupriavidus necator H16.

BACKGROUND: Biotransformation of CO2 into high-value-added carbon-based products is a promising process for reducing greenhouse gas emissions. To realize the green transformation of CO2, we use fatty acids as carbon source to drive CO2 fixation to produce succinate through a portion of the 3-hydroxypropionate (3HP) cycle in Cupriavidus necator H16. RESULTS: This work can achieve the production of a single succinate molecule from one acetyl-CoA molecule and two CO2 molecules. It was verified using an isotope labeling experiment utilizing NaH13CO3. This implies that 50% of the carbon atoms present in succinate are derived from CO2, resulting in a twofold increase in efficiency compared to prior methods of succinate biosynthesis that relied on the carboxylation of phosphoenolpyruvate or pyruvate. Meanwhile, using fatty acid as a carbon source has a higher theoretical yield than other feedstocks and also avoids carbon loss during acetyl-CoA and succinate production. To further optimize succinate production, different approaches including the optimization of ATP and NADPH supply, optimization of metabolic burden, and optimization of carbon sources were used. The resulting strain was capable of producing succinate to a level of 3.6 g/L, an increase of 159% from the starting strain. CONCLUSIONS: This investigation established a new method for the production of succinate by the implementation of two CO2 fixation reactions and demonstrated the feasibility of ATP, NADPH, and metabolic burden regulation strategies in biological carbon fixation.

Metagenomic analysis revealed highly diverse carbon fixation microorganisms in a petroleum-hydrocarbon-contaminated aquifer.

As one of the ultimate products of hydrocarbon biodegradation, inorganic carbon always be used to evaluate hydrocarbon biodegradation rates in petroleum-hydrocarbon-contaminated (PHC) aquifers. The evaluation method was challenged because of the existence of carbon fixation microorganisms, which may uptake inorganic carbons and consequently cause the biodegradation rates to be underestimated. We wonder if there are carbon fixation microorganisms in PHC aquifers. Although an extremely limited number of carbon fixation microorganisms in PHC sites have been studied in previous studies, the vast majority of microorganisms that participate in carbon fixation have not been systematically identified. To systematically reveal carbon fixation microorganisms and their survival environmental conditions, high-throughput metagenomic sequencing technologies, which are characterized by culture-independent, unbiased, and comprehensive methods for the detection and taxonomic characterization of microorganisms, were introduced to analyze the groundwater samples collected from a PHC aquifer. Results showed that 1041 genera were annotated as carbon fixation microorganisms, which accounted for 49% of the total number of genera in the PHC aquifer. Carbon fixation genes involved in Calvin-Benson-Bassham (CBB), 3-hydroxy propionate (3HP), reductive tricarboxylic acid (rTCA), and Wood-Ljungdahl (WL) cycles accounted for 2%, 41%, 34%, and 23% of the total carbon fixation genes, respectively, and 3HP, rTCA, and WL can be deemed as the dominant carbon fixation pathways. Most of the identified carbon fixation microorganisms are potential hydrocarbon biodegraders, and the most abundant carbon fixation microorganisms, such as Microbacterium, Novosphingobium, and Reyranella, were just the most abundant microorganisms in the aquifer system. It's deduced that most of the microorganisms in the aquifer were facultative autotrophic, and undertaking the dual responsibilities of degrading hydrocarbons to inorganic carbon and uptaking inorganic carbon to biomass.

Absence of carbonic anhydrase in chloroplasts affects C3 plant development but not photosynthesis.

The enzyme carbonic anhydrase (CA), which catalyzes the interconversion of bicarbonate with carbon dioxide (CO2) and water, has been hypothesized to play a role in C3 photosynthesis. We identified two tobacco stromal CAs, ß-CA1 and ß-CA5, and produced CRISPR/Cas9 mutants affecting their encoding genes. While single knockout lines Δß-ca1 and Δß-ca5 had no striking phenotypic differences compared to wild type (WT) plants, Δß-ca1ca5 leaves developed abnormally and exhibited large necrotic lesions even when supplied with sucrose. Leaf development of Δß-ca1ca5 plants normalized at 9,000 ppm CO2 Leaves of Δß-ca1ca5 mutants and WT that had matured in high CO2 had identical CO2 fixation rates and photosystem II efficiency. Fatty acids, which are formed through reactions with bicarbonate substrates, exhibited abnormal profiles in the chloroplast CA-less mutant. Emerging Δß-ca1ca5 leaves produce reactive oxygen species in chloroplasts, perhaps due to lower nonphotochemical quenching efficiency compared to WT. Δß-ca1ca5 seedling germination and development is negatively affected at ambient CO2 Transgenes expressing full-length ß-CA1 and ß-CA5 proteins complemented the Δß-ca1ca5 mutation but inactivated (ΔZn-ßCA1) and cytoplasm-localized (Δ62-ßCA1) forms of ß-CA1 did not reverse the growth phenotype. Nevertheless, expression of the inactivated ΔZn-ßCA1 protein was able to restore the hypersensitive response to tobacco mosaic virus, while Δß-ca1 and Δß-ca1ca5 plants failed to show a hypersensitive response. We conclude that stromal CA plays a role in plant development, likely through providing bicarbonate for biosynthetic reactions, but stromal CA is not needed for maximal rates of photosynthesis in the C3 plant tobacco.

Knocking Out Chloroplastic Aldolases/Rubisco Lysine Methyltransferase Enhances Biomass Accumulation in Nannochloropsis oceanica under High-Light Stress.

Rubisco large-subunit methyltransferase (LSMT), a SET-domain protein lysine methyltransferase, catalyzes the formation of trimethyl-lysine in the large subunit of Rubisco or in fructose-1,6-bisphosphate aldolases (FBAs). Rubisco and FBAs are both vital proteins involved in CO2 fixation in chloroplasts; however, the physiological effect of their trimethylation remains unknown. In Nannochloropsis oceanica, a homolog of LSMT (NoLSMT) is found. Phylogenetic analysis indicates that NoLSMT and other algae LSMTs are clustered in a basal position, suggesting that algal species are the origin of LSMT. As NoLSMT lacks the His-Ala/ProTrp triad, it is predicted to have FBAs as its substrate instead of Rubisco. The 18-20% reduced abundance of FBA methylation in NoLSMT-defective mutants further confirms this observation. Moreover, this gene (nolsmt) can be induced by low-CO2 conditions. Intriguingly, NoLSMT-knockout N. oceanica mutants exhibit a 9.7-13.8% increase in dry weight and enhanced growth, which is attributed to the alleviation of photoinhibition under high-light stress. This suggests that the elimination of FBA trimethylation facilitates carbon fixation under high-light stress conditions. These findings have implications in engineering carbon fixation to improve microalgae biomass production.