Dual-cycle CO₂ fixation enhances growth and lipid synthesis in Arabidopsis thaliana.

Carbon fixation through the Calvin-Benson-Bassham (CBB) cycle accounts for the majority of carbon dioxide (CO2) uptake from the atmosphere. The CBB cycle generates C3 carbohydrates but is inefficient at producing acetyl–coenzyme A (CoA) (C2), which is the universal precursor for synthesizing lipids. In this work, we introduced in Arabidopsis thaliana a new-to-nature CO2 fixing cycle, malyl-CoA-glycerate (McG) cycle, which together with the CBB cycle forms a dual-cycle CO2 fixation system. This cycle can fix one additional carbon by phosphoenolpyruvate carboxylase and convert the photorespiration product, glycolate, to acetyl-CoA. Plants with the McG cycle show enhanced protein abundance in their photosystems and enhanced photosystem II efficiency. McG plants had doubled CO2 fixation rates under atmospheric CO2, increased lipid production, pronounced growth enhancement, and tripled the seed yield. Editor's summary: Plants use the Calvin-Benson-Bassham cycle to convert carbon dioxide into organic carbon compounds, which they use as fuel and construction materials during growth. However, this productivity is limited by the relative inefficiency of the primary carbon fixation enzyme Rubisco. Lu et al. introduced an additional metabolic pathway, the malyl-CoA glycerate (McG) cycle, into Arabidopsis thaliana. These plants convert a secondary product of Rubisco activity into acetyl coenzyme A, which can then feed into endogenous lipid synthesis pathways. Plants with the McG cycle had increased lipids, seed yield, and overall biomass. This work provides a proof of concept for enhancing carbon fixation and plant growth without the need to directly alter Rubisco performance. —Madeleine Seale INTRODUCTION: The Calvin-Benson-Bassham (CBB) cycle accounts for the majority of carbon dioxide (CO2) uptake on Earth through ribulose 1,5 bisphosphate (RuBP) carboxylase/oxygenase (RuBisCO). However, for every three carbon atoms fixed, one is lost to CO2 when synthesizing acetyl–coenzyme A (CoA) to generate lipids, phytohormones, and other essential metabolites. Additionally, RuBisCO also oxygenates its precursor and generates glycolate, which is oxidized to CO2 through photorespiration. We aimed to solve these two CO2-losing problems by constructing a new-to-nature cycle that can convert glycolate or 3-phosphoglycerate (3PG) to acetyl-CoA. RATIONALE: We designed the malyl-CoA-glycerate (McG) cycle, which uses phosphoenolpyruvate carboxylase (PPC) as the key enzyme to accomplish the above tasks. In the McG cycle, one additional carbon is fixed when 3PG is the input, or no carbon is lost when glycolate is the input. In both cases, acetyl-CoA is produced more efficiently, which is expected to enhance the production of lipids and other important plant metabolites, including phytohormones. RESULTS: We constructed the McG cycle in Arabidopsis. The McG transgenic plants grew much larger, resulting in doubled or tripled dry weight compared with that of the wild type during their comparable life spans. The McG plants also have increased rosette leaf numbers, leaf areas, silique and seed numbers, and lipids in leaves and seeds. To support the increased growth and productivity, the McG plants approximately doubled their CO2 assimilation under atmospheric CO2, and their photosystem II showed greater efficiency than that of the wild type. The proteins in photosystems I and II, cytochrome b6/f and electron transfer chain, and F-type ATP synthase were significantly enriched in the McG plants. We also found that cytokinin levels were increased in McG plants, which may explain their increased shoot apical meristem (SAM) sizes and number of leaf primordia. Introducing partial McG cycles or omitting one key enzyme, glycolate dehydrogenase (GDH), greatly reduced the above growth benefits. Growth in high CO2 concentrations also diminished the McG effects, suggesting that glycolate conversion to acetyl-CoA played a major role. CONCLUSION: The McG cycle can use both the RuBisCO carboxylation and oxygenation products to produce acetyl-CoA. The conversion of glycolate to acetyl-CoA is dominant under ambient CO2 conditions and avoids photorespiration. When 3PG is used as an input, one additional carbon is fixed to produce two acetyl-CoA. Furthermore, because PPC uses bicarbonate, it does not compete with RuBisCO for dissolved CO2 in the chloroplast, which has a higher pH. The conversion of glycolate or 3PG to acetyl-CoA leads to increased production of lipids and cytokinin, the latter of which may explain the increased leaf number and SAM sizes. The increased acetyl-CoA also results in a proteomic shift that increases abundance of photosystem proteins. Therefore, the McG cycle creates a positive feedback loop through the production of acetyl-CoA, which is a precursor to cytokinin, and through an unknown mechanism that boosts photosystems protein abundance and efficiency. Together, these lead to increased carbon assimilation to support the beneficial growth phenotypes and high lipid production. The McG design enables efficient acetyl-CoA production without carbon loss at a reduced energy expenditure, suggesting the success of this "C2-centric" approach. A C2-centric dual-cycle carbon fixation system in Arabidopsis.: The McG (blue) cycle is introduced to Arabidopsis chloroplasts to work with the native CBB cycle (black) for more efficient acetyl-CoA synthesis and bypassing photorespiration. The McG plants grew much larger and produced more seeds and lipids through a positive feedback loop. [The figure was created with BioRender.com.] [ABSTRACT FROM AUTHOR]