Machine Learning-Supported Enzyme Engineering toward Improved CO2-Fixation of Glycolyl-CoA Carboxylase
Daniel G. Marchal, Luca Schulz,† Ingmar Schuster, Jelena Ivanovska, Nicole Paczia, Simone Prinz, Jan Zarzycki, Tobias J. Erb. ACS Synthetic Biology. 12(12): 3521–3530, November 2023. doi: https://doi.org/10.1021%2Facssynbio.3c00403
Glycolyl-CoA carboxylase (GCC) is a new-to-nature enzyme that catalyzes the key reaction in the tartronyl-CoA (TaCo) pathway, a synthetic photorespiration bypass that was recently designed to improve photosynthetic CO2 fixation. GCC was created from propionyl-CoA carboxylase (PCC) through five mutations. However, despite reaching activities of naturally evolved biotin-dependent carboxylases, the quintuple substitution variant GCC M5 still lags behind 4-fold in catalytic efficiency compared to its template PCC and suffers from futile ATP hydrolysis during CO2 fixation. To further improve upon GCC M5, we developed a machine learning-supported workflow that reduces screening efforts for identifying improved enzymes. Using this workflow, we present two novel GCC variants with 2-fold increased carboxylation rate and 60% reduced energy demand, respectively, which are able to address kinetic and thermodynamic limitations of the TaCo pathway. Our work highlights the potential of combining machine learning and directed evolution strategies to reduce screening efforts in enzyme engineering.
Brassicaceae display variation in efficiency of photorespiratory carbon-recapturing mechanisms
Urte Schlüter, Jacques W Bouvier, Ricardo Guerreiro, Milena Malisic, Carina Kontny, Philipp Westhoff, Benjamin Stich, Andreas P M Weber. Journal of Experimental Botany. 74(21):6631-6649, November 2023. doi: https://doi.org/10.1093/jxb/erad250
Carbon-concentrating mechanisms enhance the carboxylase efficiency of Rubisco by providing supra-atmospheric concentrations of CO2 in its surroundings. Beside the C4 photosynthesis pathway, carbon concentration can also be achieved by the photorespiratory glycine shuttle which requires fewer and less complex modifications. Plants displaying CO2 compensation points between 10 ppm and 40 ppm are often considered to utilize such a photorespiratory shuttle and are termed ‘C3-C4 intermediates’. In the present study, we perform a physiological, biochemical, and anatomical survey of a large number of Brassicaceae species to better understand the C3-C4 intermediate phenotype, including its basic components and its plasticity. Our phylogenetic analysis suggested that C3-C4 metabolism evolved up to five times independently in the Brassicaceae. The efficiency of the pathway showed considerable variation. Centripetal accumulation of organelles in the bundle sheath was consistently observed in all C3-C4-classified taxa, indicating a crucial role for anatomical features in CO2-concentrating pathways. Leaf metabolite patterns were strongly influenced by the individual species, but accumulation of photorespiratory shuttle metabolites glycine and serine was generally observed. Analysis of phosphoenolpyruvate carboxylase activities suggested that C4-like shuttles have not evolved in the investigated Brassicaceae. Convergent evolution of the photorespiratory shuttle indicates that it represents a distinct photosynthesis type that is beneficial in some environments.
Understanding regulation in complex environments: a route to enhance photosynthetic light-reactions in microalgae photobioreactors
Giorgio Perin, Tomas Morosinotto. Frontiers in Photobiology. Volume 1, November 2023. doi: https://doi.org/10.3389/fphbi.2023.1274525
Microalgae are recognized as a sustainable source of biomass to produce a wide range of bioproducts. To maximize the positive environmental impact and achieve economic competitiveness of microalgae-based products, it is however still essential to improve the biomass productivity during large-scale cultivation. Microalgae large-scale cultures are generally limited by light availability and thus the efficiency in conversion of radiation energy into biomass is a major factor impacting productivity. Natural light is a highly variable environmental parameter, and it constantly changes following seasons, time of day, and weather conditions. The artificial environment of large-scale microalgae cultures generates a further layer of complexity added to these natural light dynamics. In fact, because of biomass density and cell self-shading, light is unevenly distributed in the mass culture. Moreover, because of mixing, cells move between different parts of the volume, generating abrupt fluctuations in light exposure. Although microalgae evolved various regulatory mechanisms to cope with dynamic light conditions, these are not adapted to respond to the complex mixture of natural and artificial fluctuations commonly encountered in large-scale cultures, often causing reduction in photosynthetic efficiency. In the past years, genetic approaches to improve the light reactions of photosynthesis have been explored to optimise the composition and regulation of the photosynthetic machinery to large-scale cultivation. These approaches have shown promising results at the laboratory scale but have yet to be fully proven at the industrial scale. This can be explained by the fact that the complexity of the cultivation environment on microalgae photosynthesis and its impact on productivity is underestimated. This work aims for a systematic discussion on the complex role played by the growth environment in determining microalgae photosynthetic performances upon cultivation at industrial scale, with the objective of maximizing the impact of genetic modifications and ultimately fully realize the potential of microalgae for biomass productivity.
Improving photosynthetic efficiency toward food security
Edward N. Smith, Marvin van Aalst, Tiina Tosens, Ülo Niinemets, Benjamin Stich, Tomas Morosinotto, Alessandro Alboresi, Tobias J. Erb, Paul A. Gómez-Coronado, Dimitri Tolleter, Giovanni Finazzi, Gilles Curien, Matthias Heinemann, Oliver Ebenhöh, Julian M. Hibberd, Andreas P.M. Weber. Molecular Plant. Volume 16, Issue 10, 1547–1563, October 2023. doi: https://doi.org/10.1016/j.molp.2023.08.017
Photosynthesis in crops and natural vegetation allows light energy to be converted into chemical energy and thus forms the foundation for almost all terrestrial trophic networks on Earth. The efficiency of photosynthetic energy conversion plays a crucial role in determining the portion of incident solar radiation that can be used to generate plant biomass throughout a growth season. Consequently, alongside the factors such as resource availability, crop management, crop selection, maintenance costs, and intrinsic yield potential, photosynthetic energy use efficiency significantly influences crop yield. Photosynthetic efficiency is relevant to sustainability and food security because it affects water use efficiency, nutrient use efficiency, and land use efficiency. This review focuses specifically on the potential for improvements in photosynthetic efficiency to drive a sustainable increase in crop yields. We discuss bypassing photorespiration, enhancing light use efficiency, harnessing natural variation in photosynthetic parameters for breeding purposes, and adopting new-to-nature approaches that show promise for achieving unprecedented gains in photosynthetic efficiency.
Impact of the carbon flux regulator protein pirC on ethanol production in engineered cyanobacteria
Böhm Julien, Kauss Karsten, Michl Klaudia, Engelhardt Lisa, Brouwer Eva-Maria, Hagemann Martin. Frontiers in Microbiology. Volume 14, August 2023, 104968. doi: https://doi.org/10.3389/fmicb.2023.1238737
Future sustainable energy production can be achieved using mass cultures of photoautotrophic microorganisms such as cyanobacteria, which are engineered to synthesize valuable products directly from CO2 and sunlight. For example, strains of the model organism Synechocystis sp. PCC 6803 have been generated to produce ethanol. Here, we performed a study to prove the hypothesis that carbon flux in the direction of pyruvate is one bottleneck to achieve high ethanol titers in cyanobacteria. Ethanol-producing strains of the cyanobacterium Synechocystis sp. PCC 6803 were generated that bear mutation in the gene pirC aiming to increase carbon flux towards pyruvate. The strains were cultivated at different nitrogen or carbon conditions and the ethanol production was analysed. Generally, a clear correlation between growth rate and ethanol production was found. The mutation of pirC, however, had only a positive impact on ethanol titers under nitrogen depletion. The increase in ethanol was accompanied by elevated pyruvate and lowered glycogen levels indicating that the absence of pirC indeed increased carbon partitioning towards lower glycolysis. Metabolome analysis revealed that this change in carbon flow had also a marked impact on the overall primary metabolism in Synechocystis sp. PCC 6803. Deletion of pirC improved ethanol production under specific conditions supporting the notion that a better understanding of regulatory mechanisms involved in cyanobacterial carbon partitioning is needed to engineer more productive cyanobacterial strains.
What controls carbon sequestration in plants under which conditions?
Tim Nies, Marvin van Aalst, Nima Saadat, Josha Ebeling, Oliver Ebenhöh. Biosystems. Volume 231, September 2023, 104968. doi: https://doi.org/10.1016/j.biosystems.2023.104968
Photosynthetic organisms use photosynthesis to harvest sunlight and convert the solar energy into chemical energy, which is then used to reduce atmospheric carbon dioxide into organic molecules. This process forms the basis of all life on Earth, and stands at the beginning of the food chain which feeds the world population. Not surprisingly, many research efforts are currently ongoing aiming at improving growth and product yield of photosynthetic organisms, and several of these activities directly target the photosynthetic pathways. Metabolic Control Analysis (MCA) shows that, in general, the control over a metabolic flux, such as carbon fixation, is distributed among several steps and highly dependent on the external conditions. Therefore, the concept of a single ‘rate-limiting’ step is hardly ever applicable, and as a consequence, any strategy relying on improving a single molecular process in a complex metabolic system is bound to fail to yield the expected results. In photosynthesis, reports on which processes exert the highest control over carbon fixation are contradictory. This refers to both the photosynthetic ‘light’ reactions harvesting photons and the ’dark’ reactions of the Calvin–Benson–Bassham Cycle (CBB cycle). Here, we employ a recently developed mathematical model, which describes photosynthesis as an interacting supply–demand system, to systematically study how external conditions affect the control over carbon fixation fluxes.
Tailoring confocal microscopy for real-time analysis of photosynthesis at single-cell resolution
Giorgio Perin, Alessandra Bellan, Tim Michelberger, Dagmar Lyska, Setsuko Wakao, Krishna K Niyogi, Tomas MorosinottoMattia Storti, Haythem Hsine, Clarisse Uwizeye, Olivier Bastien, Daniel P. Yee, Fabien Chevalier, Johan Decelle, Cécile Giustini, Daniel Béal, Gilles Curien, Giovanni Finazzi, Dimitri Tolleter. Cell Reports Methods. Volume 3, Issue 9, 25 September 2023, 100568. doi: https://doi.org/10.1016/j.crmeth.2023.100568
Photoautotrophs’ environmental responses have been extensively studied at the organism and ecosystem level. However, less is known about their photosynthesis at the single-cell level. This information is needed to understand photosynthetic acclimation processes, as light changes as it penetrates cells, layers of cells, or organs. Furthermore, cells within the same tissue may behave differently, being at different developmental/physiological stages. Here, we describe an approach for single-cell and subcellular photophysiology based on the customization of confocal microscopy to assess chlorophyll fluorescence quenching by the saturation pulse method. We exploit this setup to (1) reassess the specialization of photosynthetic activities in developing tissues of non-vascular plants; (2) identify a specific subpopulation of phytoplankton cells in marine photosymbiosis, which consolidate energetic connections with their hosts; and (3) examine the link between light penetration and photoprotection responses inside the different tissues that constitute a plant leaf anatomy.
Modulation of xanthophyll cycle impacts biomass productivity in the marine microalga Nannochloropsis
Giorgio Perin, Alessandra Bellan, Tim Michelberger, Dagmar Lyska, Setsuko Wakao, Krishna K Niyogi, Tomas Morosinotto. Plant Biology. 2023 Jun 20;120(25):e2214119120. doi: https://doi.org/10.1073/pnas.2214119120
Life on earth depends on photosynthetic primary producers that exploit sunlight to fix CO2 into biomass. Approximately half of global primary production is associated with microalgae living in aquatic environments. Microalgae also represent a promising source of biomass to complement crop cultivation, and they could contribute to the development of a more sustainable bioeconomy. Photosynthetic organisms evolved multiple mechanisms involved in the regulation of photosynthesis to respond to highly variable environmental conditions. While essential to avoid photodamage, regulation of photosynthesis results in dissipation of absorbed light energy, generating a complex trade-off between protection from stress and light-use efficiency. This work investigates the impact of the xanthophyll cycle, the light-induced reversible conversion of violaxanthin into zeaxanthin, on the protection from excess light and on biomass productivity in the marine microalgae of the genus Nannochloropsis. Zeaxanthin is shown to have an essential role in protection from excess light, contributing to the induction of nonphotochemical quenching and scavenging of reactive oxygen species. On the contrary, the overexpression of zeaxanthin epoxidase enables a faster reconversion of zeaxanthin to violaxanthin that is shown to be advantageous for biomass productivity in dense cultures in photobioreactors. These results demonstrate that zeaxanthin accumulation is critical to respond to strong illumination, but it may lead to unnecessary energy losses in light-limiting conditions and accelerating its reconversion to violaxanthin provides an advantage for biomass productivity in microalgae.
Photorespiration is the solution, not the problem
Broncano LS, Pukacz KR, Reichel-Deland V, Schlüter U, Triesch S, Weber AP. Journal of Plant Physiology. 2023 Mar 1;282:153928. doi: https://doi.org/10.1016/j.jplph.2023.153928
The entry of carbon dioxide from the atmosphere into the biosphere is mediated by the enzyme Rubisco, which catalyzes the carboxylation of ribulose 1,5-bisphosphate (RuBP) as the entry reaction of the Calvin Benson Bassham cycle (CBBC), leading to the formation of 2 molecules of 3-phosphoglyceric acid (3PGA) per CO2 fixed. 3PGA is reduced to triose phosphates at the expense of NADPH+ H⁺ and ATP that are provided by the photosynthetic light reactions. Triose phosphates are the principal products of the CBBC and the precursors for almost any compound in the biosphere.
PlantACT! : How to Tackle the Climate Crisis
Hirt, H., Al-Babili, S., Almeida-Trapp, M., Antoine, M., Aranda, M., Bartels, D., … Young, I. M. TRENDS IN PLANT SCIENCE, vol. 28, no. 5, 2023, pp. 537–43, doi: 10.1016/j.tplants.2023.01.005
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.
COBREXA. jl: constraint-based reconstruction and exascale analysis
Kratochvíl M, Heirendt L, Wilken SE, Pusa T, Arreckx S, Noronha A, van Aalst M, Satagopam VP, Ebenhöh O, Schneider R, Trefois C.
Bioinformatics, Feb 2022, 15;38(4):1171-2, doi: 10.1093/bioinformatics/btab782
COBREXA.jl is a Julia package for scalable, high-performance constraint-based reconstruction and analysis of very large-scale biological models. Its primary purpose is to facilitate the integration of modern high performance computing environments with the processing and analysis of large-scale metabolic models of challenging complexity. We report the architecture of the package, and demonstrate how the design promotes analysis scalability on several use-cases with multi-organism community models.
Computational Analysis of Alternative Photosynthetic
Electron Flows Linked With Oxidative Stress
Saadat N.P., Nies T, van Aalst M, Hank B, Demirtas B, Ebenhöh O, Matuszyńska A. Frontiers in Plant Science. 2021, 12, p.750580. doi: 10.3389/fpls.2021.750580
During photosynthesis, organisms respond to their energy demand and ensure the supply of energy and redox equivalents that sustain metabolism. Hence, the photosynthetic apparatus can, and in fact should, be treated as an integrated supply-demand system. Any imbalance in the energy produced and consumed can lead to adverse reactions, such as the production of reactive oxygen species (ROS). Reaction centres of both photosystems are known sites of ROS production. Here, we investigate in particular the central role of Photosystem I (PSI) in this tightly regulated system. Using a computational approach we have expanded a previously published mechanistic model of C3 photosynthesis by including ROS producing and scavenging reactions around PSI. These include two water to water reactions mediated by Plastid terminal oxidase (PTOX) and Mehler and the ascorbate-glutathione (ASC-GSH) cycle, as a main non-enzymatic antioxidant. We have used this model to predict flux distributions through alternative electron pathways under various environmental stress conditions by systematically varying light intensity and enzymatic activity of key reactions. In particular, we studied the link between ROS formation and activation of pathways around PSI as potential scavenging mechanisms. This work shines light on the role of alternative electron pathways in photosynthetic acclimation and investigates the effect of environmental perturbations on PSI activity in the context of metabolic productivity.
Implementation of the β-hydroxyaspartate cycle increases growth performance of Pseudomonas putida on the PET monomer ethylene glycol
von Borzyskowski, L.S., Schulz-Mirbach, H., Castellanos, M.T., Severi, F., Gómez-Coronado, P.A., Paczia, N., Glatter, T., Bar-Even, A., Lindner, S.N. and Erb, T.J., Metabolic Engineering, 76, pp.97-109. doi: 10.1016/j.ymben.2023.01.011
Ethylene glycol (EG) is a promising next generation feedstock for bioprocesses. It is a key component of the ubiquitous plastic polyethylene terephthalate (PET) and other polyester fibers and plastics, used in antifreeze formulations, and can also be generated by electrochemical conversion of syngas, which makes EG a key compound in a circular bioeconomy. The majority of biotechnologically relevant bacteria assimilate EG via the glycerate pathway, a wasteful metabolic route that releases CO2 and requires reducing equivalents as well as ATP. In contrast, the recently characterized β-hydroxyaspartate cycle (BHAC) provides a more efficient, carbon-conserving route for C2 assimilation. Here we aimed at overcoming the natural limitations of EG metabolism in the industrially relevant strain Pseudomonas putida KT2440 by replacing the native glycerate pathway with the BHAC. We first prototyped the core reaction sequence of the BHAC in Escherichia coli before establishing the complete four-enzyme BHAC in Pseudomonas putida. Directed evolution on EG resulted in an improved strain that exhibits 35% faster growth and 20% increased biomass yield compared to a recently reported P. putida strain that was evolved to grow on EG via the glycerate pathway. Genome sequencing and proteomics highlight plastic adaptations of the genetic and metabolic networks in response to the introduction of the BHAC into P. putida and identify key mutations for its further integration during evolution. Taken together, our study shows that the BHAC can be utilized as ‘plug-and-play’ module for the metabolic engineering of two important microbial platform organisms, paving the way for multiple applications for a more efficient and carbon-conserving upcycling of EG in the future.
Network Reconstruction and Modelling Made Reproducible with moped
Saadat NP, van Aalst M, Ebenhöh O. Metabolites. 2022; 12(4):275. doi: 10.3390/metabo12040275
Mathematical modeling of metabolic networks is a powerful approach to investigate the underlying principles of metabolism and growth. Such approaches include, among others, differential-equation-based modeling of metabolic systems, constraint-based modeling and metabolic network expansion of metabolic networks. Most of these methods are well established and are implemented in numerous software packages, but these are scattered between different programming languages, packages and syntaxes. This complicates establishing straight forward pipelines integrating model construction and simulation. We present a Python package moped that serves as an integrative hub for reproducible construction, modification, curation and analysis of metabolic models. moped supports draft reconstruction of models directly from genome/proteome sequences and pathway/genome databases utilizing GPR annotations, providing a completely reproducible model construction and curation process within executable Python scripts. Alternatively, existing models published in SBML format can be easily imported. Models are represented as Python objects, for which a wide spectrum of easy-to-use modification and analysis methods exist. The model structure can be manually altered by adding, removing or modifying reactions, and gap-filling reactions can be found and inspected. This greatly supports the development of draft models, as well as the curation and testing of models. Moreover, moped provides several analysis methods, in particular including the calculation of biosynthetic capacities using metabolic network expansion. The integration with other Python-based tools is facilitated through various model export options. For example, a model can be directly converted into a CobraPy object for constraint-based analyses. moped is a fully documented and expandable Python package. We demonstrate the capability to serve as a hub for integrating reproducible model construction and curation, database import, metabolic network expansion and export for constraint-based analyses.
Knowledge of Regulation of Photosynthesis in Outdoor Microalgae Cultures Is Essential for the Optimization of Biomass Productivity
Perin G, Gambaro F, Morosinotto T. Frontiers in Plant Science. 2022 Apr 4;13:751. doi: 10.3389/fpls.2022.846496
Microalgae represent a sustainable source of biomass that can be exploited for pharmaceutical, nutraceutical, cosmetic applications, as well as for food, feed, chemicals, and energy. To make microalgae applications economically competitive and maximize their positive environmental impact, it is however necessary to optimize productivity when cultivated at a large scale. Independently from the final product, this objective requires the optimization of biomass productivity and thus of microalgae ability to exploit light for CO2 fixation. Light is a highly variable environmental parameter, continuously changing depending on seasons, time of the day, and weather conditions. In microalgae large scale cultures, cell self-shading causes inhomogeneity in light distribution and, because of mixing, cells move between different parts of the culture, experiencing abrupt changes in light exposure. Microalgae evolved multiple regulatory mechanisms to deal with dynamic light conditions that, however, are not adapted to respond to the complex mixture of natural and artificial fluctuations found in large-scale cultures, which can thus drive to oversaturation of the photosynthetic machinery, leading to consequent oxidative stress. In this work, the present knowledge on the regulation of photosynthesis and its implications for the maximization of microalgae biomass productivity are discussed. Fast mechanisms of regulations, such as Non-Photochemical-Quenching and cyclic electron flow, are seminal to respond to sudden fluctuations of light intensity. However, they are less effective especially in the 1–100 s time range, where light fluctuations were shown to have the strongest negative impact on biomass productivity. On the longer term, microalgae modulate the composition and activity of the photosynthetic apparatus to environmental conditions, an acclimation response activated also in cultures outdoors. While regulation of photosynthesis has been investigated mainly in controlled lab-scale conditions so far, these mechanisms are highly impactful also in cultures outdoors, suggesting that the integration of detailed knowledge from microalgae large-scale cultivation is essential to drive more effective efforts to optimize biomass productivity.
Europe’s farm to fork strategy and its commitment to biotechnology and organic farming: conflicting or complementary goals?
Purnhagen KP, Clemens S, Eriksson D, Fresco LO, Tosun J, Qaim M, Visser RG, Weber AP, Wesseler JH, Zilberman D. Trends in plant science. 2021 Jun 1;26(6):600-6. doi: 10.1016/j.tplants.2021.03.012
The European Commission’s Farm to Fork (F2F) strategy, under the European Green Deal, acknowledges that innovative techniques, including biotechnology, may play a role in increasing sustainability. At the same time, organic farming will be promoted, and at least 25% of the EU’s agricultural land shall be under organic farming by 2030. How can both biotechnology and organic farming be developed and promoted simultaneously to contribute to achieving the Sustainable Development Goals (SDGs)? We illustrate that achieving the SDGs benefits from the inclusion of recent innovations in biotechnology in organic farming. This requires a change in the law. Otherwise, the planned increase of organic production in the F2F strategy may result in less sustainable, not more sustainable, food systems.
Constructing and analysing dynamic models with modelbase v1. 2.3: a software update
van Aalst M, Ebenhöh O, Matuszyńska A. BMC bioinformatics. 2021 Dec;22(1):1-5. doi: 10.1186/s12859-021-04122-7
Computational mathematical models of biological and biomedical systems have been successfully applied to advance our understanding of various regulatory processes, metabolic fluxes, effects of drug therapies, and disease evolution and transmission. Unfortunately, despite community efforts leading to the development of SBML and the BioModels database, many published models have not been fully exploited, largely due to a lack of proper documentation or the dependence on proprietary software. To facilitate the reuse and further development of systems biology and systems medicine models, an open-source toolbox that makes the overall process of model construction more consistent, understandable, transparent, and reproducible is desired.
Results and discussion
We provide an update on the development of modelbase, a free, expandable Python package for constructing and analysing ordinary differential equation-based mathematical models of dynamic systems. It provides intuitive and unified methods to construct and solve these systems. Significantly expanded visualisation methods allow for convenient analysis of the structural and dynamic properties of models. After specifying reaction stoichiometries and rate equations modelbase can automatically assemble the associated system of differential equations. A newly provided library of common kinetic rate laws reduces the repetitiveness of the computer programming code. modelbase is also fully compatible with SBML. Previous versions provided functions for the automatic construction of networks for isotope labelling studies. Now, using user-provided label maps, modelbase v1.2.3 streamlines the expansion of classic models to their isotope-specific versions. Finally, the library of previously published models implemented in modelbase is growing continuously. Ranging from photosynthesis to tumour cell growth to viral infection evolution, all these models are now available in a transparent, reusable and unified format through modelbase.
With this new Python software package, which is written in currently one of the most popular programming languages, the user can develop new models and actively profit from the work of others. modelbase enables reproducing and replicating models in a consistent, tractable and expandable manner. Moreover, the expansion of models to their isotopic label-specific versions enables simulating label propagation, thus providing quantitative information regarding network topology and metabolic fluxes.
A synthetic C4 shuttle via the β-hydroxyaspartate cycle in C3 plants
Roell MS, Schada von Borzykowski L, Westhoff P, Plett A, Paczia N, Claus P, Urte S, Erb TJ, Weber APM. Proc Natl Acad Sci U S A. 2021 May 25;118(21):e2022307118. doi: 10.1073/pnas.2022307118.
Plants depend on the enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) for CO2 fixation. However, especially in C3 plants, photosynthetic yield is reduced by formation of 2-phosphoglycolate, a toxic oxygenation product of Rubisco, which needs to be recycled in a high-flux-demanding metabolic process called photorespiration. Canonical photorespiration dissipates energy and causes carbon and nitrogen losses. Reducing photorespiration through carbon-concentrating mechanisms, such as C4 photosynthesis, or bypassing photorespiration through metabolic engineering is expected to improve plant growth and yield. The β-hydroxyaspartate cycle (BHAC) is a recently described microbial pathway that converts glyoxylate, a metabolite of plant photorespiration, into oxaloacetate in a highly efficient carbon-, nitrogen-, and energy-conserving manner. Here, we engineered a functional BHAC in plant peroxisomes to create a photorespiratory bypass that is independent of 3-phosphoglycerate regeneration or decarboxylation of photorespiratory precursors. While efficient oxaloacetate conversion in Arabidopsis thaliana still masks the full potential of the BHAC, nitrogen conservation and accumulation of signature C4 metabolites demonstrate the proof of principle, opening the door to engineering a photorespiration-dependent synthetic carbon-concentrating mechanism in C3 plants.