Tapping the Unused Potential of Photosynthesis with a Heterologous Electron Sink

Abstract

Photosynthesis is the pivotal biochemical reaction on the planet, providing energy for the 30 global ecosystem. The evolution of oxygenic photosynthesis 2.7-3.2 billion years ago led to 31 the oxygenation of the planet. 1 This subsequently hampered the efficiency of 32 photosynthesis as oxygen competed for binding to the active site of the carbon-fixing 33 catalyst ribulose-1, 5-bisphosphate carboxylase/oxygenase (Rubisco). As a result, the 34 potential of photosynthesis is not achieved as the capacity for light capture and electron 35 transport is often greater than the capacity for carbon-fixation. Photosynthetic cells therefore 36 lose excess energy as heat and fluorescence, or through a number of alternative electron 37 dissipation pathways. 2 Improving photosynthetic efficiency is central to efforts to increase 38 food and/or biofuel yield, and also to realize the biotechnological potential of photosynthetic 39 species. 3 These efforts typically focus on modification of the pigments and proteins involved 40 in light capture, improving the efficiency/specificity of Rubisco or metabolic engineering of 41 product formation downstream of carbon fixation. 4 However, rewiring photosynthesis such 42 that excess reducing potential from light capture is diverted to catalyse the formation of high 43 value products has received little attention. 5 Such a strategy can, in theory, increase the 44 overall efficiency of photosynthetic electron usage by enabling the utilisation of electrons that 45 would otherwise be wasted. 46To increase overall photosynthetic efficiency, we installed the P450 CYP1A1 from 47Rattus norvegicus (brown rat) into Synechococcus PCC 7002 (henceforth Synechococcus) 48 as a new electron-sink in the photosynthetic electron transport chain (Figure 1a). 49Cytochrome P450s are a large and diverse class of monooxygenases that split molecular 50 oxygen (O2), inserting one atom into the substrate and reducing the other to water (Figure 51 1a). CYP1A1 plays a key role in the biotransformation of drugs and other chemical 52 compounds in mammals and has been widely studied. The catalytic activity of CYP1A1 is 53 well-defined and easily assayed, 6 its structure has been resolved at 2.6 Å 7 and it has been 54 expressed in other microbial hosts, allowing comparison of expression and activity levels 55 among species. 8 We designed plasmid pSy21 to express CYP1A1 in Synechococcus. The 56 expression cassette consisted of the cyp1a1 gene, a constitutive phycocyanin promoter from 57 4 Synechocystis PCC 6803, a kanamycin resistance cassette, the rrnB terminator from 58Escherichia coli, and targeting flanks to guide integration to the glpK genomic neutral site 59 (Figure 1b). The cyp1a1 gene was codon-optimized for expression in Synechococcus and 60 the FLAG peptide sequence was added in-frame, along with a 4 x glycine-alanine peptide 61 linker, to the 3' of cyp1a1 to simplify detection, quantification and purification; no other 62 modifications were made to the cyp1a1 sequence. This multipart construct was assemb...

Responses of Synechocystis sp. PCC 6803 to heterologous biosynthetic pathways

“…These numbers are small and appear to be well within the buffering capacity of the photosynthetic regulatory mechanisms. In support of this view, previous work [ 11 , 32 , 33 ], together with results presented here, indicate that the expression of cytochrome P450s does not drain a significant number of electrons out of the photosynthetic electron transfer chain. Dhurrin and manoyl oxide, being the product of long and complex metabolic routes, require a large amount of fixed carbons and subsequently harvested photons, as compared to simpler products, such as ethanol and succinic acid.…”

An inducible expression system in the alga Nannochloropsis gaditana controlled by the nitrate reductase promoter

“…Blotting was performed as described previously (Berepiki et al 2016). Membranes were incubated first with an anti-GFP antibody (N-terminal antibody produced in rabbit; Sigma-Aldrich) diluted to 1:1000 in blocking solution and then with goat anti-rabbit IgG (Agrisera) diluted to 1:1000 in blocking solution.…”

Photosynthetic fuel for heterologous enzymes: the role of electron carrier proteins

“…In another example, 40 µg g DW −1 of the antimicrobial diterpenoid isopimaric acid resulted from transiently expressing CYP720B4 from Sitka spruce in tobacco (Gnanasekaran et al 2015). While the physiological consequences of coupling cytochrome P450 enzymes to photosynthetic reducing power are not yet fully explored, introduction of the mammalian CYP1A1 into Synechococcus elongatus PCC 7002 caused a 31% increase in photosynthetic electron transfer rates (Berepiki et al 2016), which suggests that the photosynthetic machinery has the capacity to tolerate increased sink activity.…”