In vitro demonstration and in planta characterization of a condensed, reverse TCA (crTCA) cycle

Abstract

Introduction: Plants employ the Calvin-Benson cycle (CBC) to fix atmospheric CO₂ for the production of biomass. The flux of carbon through the CBC is limited by the activity and selectivity of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase (RuBisCO). Alternative CO₂ fixation pathways that do not use RuBisCO to fix CO₂ have evolved in some anaerobic, autotrophic microorganisms.

Methods: Rather than modifying existing routes of carbon metabolism in plants, we have developed a synthetic carbon fixation cycle that does not exist in nature but is inspired by metabolisms of bacterial autotrophs. In this work, we build and characterize a condensed, reverse tricarboxylic acid (crTCA) cycle in vitro and in planta.

Results: We demonstrate that a simple, synthetic cycle can be used to fix carbon in vitro under aerobic and mesophilic conditions and that these enzymes retain activity whenexpressed transiently in planta. We then evaluate stable transgenic lines of Camelina sativa that have both phenotypic and physiologic changes. Transgenic C. sativa are shorter than controls with increased rates of photosynthetic CO₂ assimilation and changes in photorespiratory metabolism.

Discussion: This first iteration of a build-test-learn phase of the crTCA cycle provides promising evidence that this pathway can be used to increase photosynthetic capacity in plants.

Keywords: CO2 fixation; Camelina sativa; carbon capture; photosynthesis; reverse TCA cycle; synthetic biology.

Figure 1

Linear representation of vectors used to express the crTCA enzymes and coenzymes. The constitutive promoters used in this study are CaMV35S (35S_P), Actin 2 (Act2_P) and EntCUP4. The chloroplast transit peptides used are RbcS from A. thaliana (RbcS_AT), RbcS from N. tabacum (RbcS_NT) and Biotin carboxyl carrier protein from A. thaliana (BCCP_AT). Terminators used are CaMV35S (35S_T), nopaline synthase (NOS_T) and octopine synthase (OCS_T). LB: Left Border, RB: Right Border. Figure made using Biorender.

Figure 2

The crTCA cycle. Theoretical representation of a condensed, reverse TCA (crTCA) cycle and how it could contribute to plant metabolism. The design of the cycle (red arrows) is to take succinate through four or five steps to yield glyoxylate and fixing two carbon molecules. The product of the crTCA cycle, glyoxylate, may have multiple fates in endogenous plant metabolism (black arrows). Reactions listed by number are 1) succinyl-CoA synthetase (SCS); 2) 2-oxoglutarate:ferredoxin oxidoreductase (KOR); 3) 2-oxoglutarate carboxylase (OGC); 4) oxalosuccinate reductase (OSR); 5) isocitrate lyase (ICL); 6) isocitrate dehydrogenase (ICDH); 7) plastidial glyoxylate reductase 2 (GLYR2) or plastidial hydroxypyruvate reductase 3 (HPR3); 8) mitochondrial GABA transaminase (GABA-T) or peroxisomal glutamate:glyoxylate aminotransferase (GGAT1/2).

Figure 3

Demonstration of cyclic in vitro activity under anaerobic and aerobic conditions. Enzyme reactions were provided with NaH¹³CO₃ as substrate and samples were taken over the course of two hours. Isotope incorporation into the metabolites were quantified using standard curves analyzed at the same time as the reaction samples. The mean is shown ± one standard deviation, n = 3.

Figure 4

Phenotype of transgenic C. sativa grown in the greenhouse. Seven-week old plants expressing the crTCA cycle genes are noticeably shorter than non-segregant WT or Empty Vector controls. Construct 1 expression led to a slightly shorter plant height but also a noticeable increase in axillary branches.

Figure 5

Light-dependent phenotype and physiology of transgenic C. sativa grown at elevated CO₂ concentrations. (A) At 1200 ppm CO₂ and 200 PPFD (top row), plants expressing the crTCA cycle (middle column) appear shorter than both WT (left column) and the partial crTCA line, Construct 1 (right column). At the same CO₂ concentration and 1200 PPFD (bottom row), plants expressing the crTCA cycle display no phenotype whereas Construct 1 plants are slightly smaller. (B) Physiologic measurements of photosynthetic gas exchange parameters. n = 4 plants/line; letters indicate statistically significant groups determined via Tukey HSD, p< 0.05. Means are displayed as bar graphs ± the standard error of the mean (SEM).

Figure 6

Transient ¹³C labeling differences in WT and crTCA lines. (A) The mass isotopomer distribution (MID) of glycine and serine is shown where the points represent averages and error bars represent ± one standard deviation. Glycine is the top row, serine is the bottom. WT is the left column and crTCA is the right column. For each time point, n = 3 biological replicates. Each color represents a distinct isotopomer with various degrees of ¹³C incorporation: m0 is unlabeled, m1 is one ¹³C label; m2 is two ¹³C labels. (B) ¹³C enrichment of aspartate, glutamate and sucrose. WT samples are the black circles while crTCA samples are the red circles. The mean is presented with error bars representing one standard deviation (n = 3).