Engineering Improved Photosynthesis in the Era of Synthetic Biology

Abstract

Much attention has been given to the enhancement of photosynthesis as a strategy for the optimization of crop productivity. As traditional plant breeding is most likely reaching a plateau, there is a timely need to accelerate improvements in photosynthetic efficiency by means of novel tools and biotechnological solutions. The emerging field of synthetic biology offers the potential for building completely novel pathways in predictable directions and, thus, addresses the global requirements for higher yields expected to occur in the 21st century. Here, we discuss recent advances and current challenges of engineering improved photosynthesis in the era of synthetic biology toward optimized utilization of solar energy and carbon sources to optimize the production of food, fiber, and fuel.

Keywords: genetic engineering; photosynthesis; synthetic biology; targeted manipulation; yield improvement.

Figure 1

Light Reactions and Potential Targets for Improvement Strategies. Overview of linear electron flow from water oxidation by OEC (gray rectangle) in the PSII LHCII supercomplex (light green), though cytochrome b₆f (orange), to PTOX (light red circle) and/or PC (circle red) or algal cytochrome c₆ (opened rectangle), then to PSI-LHCI (light green), followed by reduction of NADP⁺ by ferredoxin (brown) and ferredoxin:NADP⁺ reductase (oval purple circle), releasing protons into the lumen, which is then used to drive the ATP biosynthesis by ATP synthase (blue). In addition to linear electron flow, there are also cyclic electron transfer pathways mediated by PGR5/PGRL1 or NDH complex (purple). These pathways confer dynamic protection, preventing the production of ROS. Of note, ion channels, such as TPK3 and KEA3 (yellow), can be modulated by light fluctuations, regulating the proton motive force in the chloroplast and thus be important regulators of ATP biosynthesis by exchanging K⁺ and H⁺ between stroma and lumen. Moreover, recently in Nicotiana benthamiana a new strategy rerouting electron transferred by PSI to P450s monooxygenase was performed (Mellor et al., 2016, Urlacher and Girhard, 2019). CYP fused into Fd was expressed in the thylakoid membrane of chloroplasts, enabling direct coupling of photosynthetic electron transfer to the heme iron reduction. The reducing power (e⁻) needed for dhurrin formation in the chloroplast is thus ultimately derived by the water-splitting activity of PSII (pink, gray, and yellow intermembrane canes). PSI-LHCI, photosystem I light-harvesting complex I; PSII-LHCII, photosystem II light-harvesting complex II; PQ, plastoquinone; PQH, semi-plastoquinone; PTOX, plastoquinol terminal oxidase; PC, plastocyanin; Cyt C6, cytochrome c₆; Cytb6f, cytochrome b₆f; OEC, oxygen-evolving complex; Fd, ferredoxin; NDH, NAD(P)H dehydrogenase; PGR5, proton gradient regulation 5; PGRL1, PGR5-like protein 1; TPK3, two-pore K⁺ channel 3; KEA3, potassium cation efflux antiporter 3.

Figure 2

Photosynthetic Mechanisms and Strategies Used to Introduce Carbon Concentration Mechanisms into C3 Plants. (A) Converting C3 into C4 mechanism. The transition from C3 to C4 metabolism requires the differentiation of photosynthetically active vascular bundle sheath cells, modification in the biochemistry reactions of several enzymes, and modulation of metabolite transport in both inter- and intracellular compartments, as well as transferring GDC into bundle sheath cells (Schuler et al., 2016). (B) Converting C3 into crassulacean acid metabolism (CAM). The pathway of CAM in a mesophyll cell is temporally separated. The different background color indicates light at the top and dark at the bottom. The green boxes on both sides indicate the epidermis under these two different conditions (opened, night; closed, day). As alternative engineering target and less complicated process not involving changes in morphological structure is the introduction of CAM metabolism into C3 plants. Such engineering requires precise control of several key enzymes, such as PEP carboxylase, malic enzyme, and RuBisCO (Kubis and Bar-Even, 2019). (C) Transferring cyanobacteria and algal carbon concentration mechanism (CCM) components to C3 chloroplasts. Transfer of HCO₃⁻ transporter (red and yellow circles) on inner chloroplast membrane, expression of functional carboxysome (yellow icosahedron), and introducing an algal pyrenoid CCM (brown circle) in the chloroplast stroma. Long et al. (2018), using sophisticated approaches, took us a step closer to achieving a high stromal HCO₃⁻ pool in the presence of functional carboxysome, increasing CO₂ fixation and yield up to 60% in transformed tobacco, as previously predicted by McGrath and Long (2014). Asp, aspartate; CA, carbonic anhydrase; GDC, glycine decarboxylase; PEP, phosphoenolpyruvate; PEPcase, phosphoenolpyruvate carboxylase; PPDK, pyruvate phosphate dikinase; ME, malic enzyme.

Figure 3

Calvin–Benson Cycle Advances, RuBP Supply, and Photorespiratory Bypasses. RuBisCO catalyzes CO₂ and O₂ fixation. The product of CO₂ fixation is the 3PGA that enters in the CBC and can be directed to starch biosynthesis and/or sugars in the cytosol (black arrows). In addition, the oxygenation drives the production of 2PGA, which is metabolized in the C2 photorespiratory pathway (blue arrows). Recently, three new synthetic photorespiratory bypasses have been proposed to improve the carbon assimilation and reduce photorespiration losses in C3 plants. See details in pink circles: (1) glycolate is diverted into glycerate within the chloroplast, shifting the release of CO₂ from mitochondria to chloroplasts, and reducing ammonia release (dashed light-green arrows) (for details see Kebeish et al., 2007); (2) peroxisomal pathway, catalyzed by two Escherichia coli enzymes, converts glyoxylate into hydroxypyruvate and CO₂ in a two-step process (brown) (for details see Peterhänsel et al., 2013); (3) lastly, bypass 3 is considered a non-real bypass since the glycolate is completely oxidized into CO₂ inside chloroplasts by both newly introduced and native enzymes (dashed red arrows) (for details see Peterhänsel et al., 2013, Fonseca-Pereira et al., 2020). In parallel, overexpression of GDC (gray oval) in Arabidopsis increased net carbon assimilation. Besides this, RuBisCO activity is also limited by RuBP regeneration, which involves two main enzymes, SBPase and FBPA (yellow oval). The combination of SBPase and FBPA overexpression results in cumulative positive effects on leaf area and biomass accumulation (Driever et al., 2017). Recent studies have shown that BSD2 (red star) chaperone is crucial for cyanobacterial RuBisCO assembly into functional enzyme in tobacco (Conlan et al., 2019). Therefore, all these changes are considered important checkpoint targets to optimize and improve the photosynthetic efficiency. 3PGA, 3-phosphoglycerate; CBC, Calvin–Benson cycle; FBPA, fructose-1,6-bisphosphate aldolase; SBPase, sedoheptulose-1,7-bisphosphatase; BSD2, bundle sheath defective 2 protein.