Introduction of a phenylalanine sink in fast growing cyanobacterium Synechococcus elongatus PCC 11801 leads to improved PSII efficiency, linear electron transport, and carbon fixation

Abstract

Cyanobacteria are investigated for fundamental photosynthesis research and sustainable production of valuable biochemicals. However, low product titer and biomass productivities are major bottlenecks to the economical scale-up. Recent studies have shown that the introduction of a metabolic sink, such as sucrose, 2,3-butanediol, and 2-phenyl ethanol, in cyanobacteria improves carbon fixation by relieving the "sink" limitation of photosynthesis. However, the impact of light intensity on the behavior of this sink-derived enhancement in carbon fixation is not well understood and is necessary for translation to outdoor cultivation. Here, using random mutagenesis, we engineered Synechococcus elongatus PCC 11801 to overproduce 1.24 g L^-1 phenylalanine (Phe) in 3 days, identified L531W in the TolC protein as an important driver of Phe efflux, and investigated the effect of light intensity on total carbon fixation. We found that low light results in competition between biomass and Phe, whereas under excess light, a higher flux of fixed carbon is directed to the Phe sink. The introduction of the Phe sink improves the quantum yields of photosystem I and II with a concomitant increase in the total electron flow leading to nearly 70% increase in carbon fixation at high light in the mutant strain. Additionally, the cyclic electron flow decreased, which has implications for the ATP/NADPH production ratio. Our data highlight how light intensity affects the sink-derived enhancement in carbon fixation, the role of CEF to balance the source-sink demand for ATP and NADPH, and the enhancement of inorganic carbon fixation in cyanobacteria with an engineered sink.

Keywords: Synechococcus elongatus; carbon fixation; cyclic electron transport; linear electron transport; phenylalanine; photosynthetic efficiency; source‐sink relationship.

Figure 1

Electron transport pathways in the light harvesting system of cyanobacteria. Schematic representation of cyanobacterial photosynthetic machinery showing electron transfer pathways, nonphotochemical quenching pathway, action of electron transport inhibitors, Calvin cycle, Biomass and Phe sinks, and ATP/NADPH synthesis and consumption. ^†ATP:NADPH ratio can vary depending on N source, carbon uptake, amino acid reuptake, carbon recycling, etc. The red arrows indicate loss of excitons/electrons to photoprotective mechanisms. CBB, Calvin‐Benson‐Bassham; CEF, cyclic electron flow; Cyt b ₆ f, cytochrome b ₆ f; DBMIB, dibromothymoquinone; DCMU, 3‐(3,4‐dichlorophenyl)‐1,1‐dimethylurea; E4P, erythrose 4‐phosphate; Fd, ferridoxin; FNR, ferridoxin NADP⁺ oxidoreductase; HA, hydroxylamine; LEF, linear electron flow; NPQ, nonphotochemical quenching; PC, plastocyanin; PEP, phosphoenolpyruvate; PQ, plastoquinone; PSI, photosystem I; PSII, photosystem II; Phe, phenylalanine.

Figure 2

Growth and Phe production of wild type and mutant strains. (a) Phe titer of wild type and mutants when cultured for 3 days at 240 μmol m⁻² sec⁻¹ under 3% CO₂. (b) Total capacity of biomass and Phe sinks on a mass basis calculated as biomass (g L⁻¹) + Phe (g L⁻¹). * indicates P < 0.05 using a two‐tailed two‐sample t‐test.

Figure 3

Carbon distribution between biomass and Phe. Distribution of fixed carbon in biomass, Phe, and the total sink (biomass + Phe) in WT, M14, and M14.2 under LL (40 μmol photons m⁻² sec⁻¹) (a–c), ML (240 μmol photons m⁻² sec⁻¹) (d–f), and HL (1000 μmol photons m⁻² sec⁻¹) (g, i) conditions. Data represent the mean and standard deviation of three biological replicates inoculated at the same density and cultured at 38°C and ambient CO₂. The x‐axis represents time in hours and the y‐axis, carbon content in mg L⁻¹.

Figure 4

Phe‐overproducing strains assimilate more carbon. (a) Ratio of total carbon fixed by M14 and M14.2 to WT at the end of 2 days. Statistical comparison was performed with a one‐tailed t‐test with the null hypothesis mean ≤1. (b, c) Percentage of carbon that is diverted to the Phe sink under different light conditions in M14 and M14.2. Data represent mean and standard deviation of three biological replicates inoculated at the same density and cultured at 38°C and ambient CO₂.

Figure 5

Characterization of PSII efficiency and linear electron transport rate. M14.2 shows higher PSII operating efficiency and linear electron transport (a) Effective quantum yield of PSII and (b) relative electron transfer rate through PSII (linear electron flow) under different light intensities in WT and M14.2. Data are the mean and standard deviations of three biological replicates.

Figure 6

PSI redox kinetics of WT and M14.2. (a–c) Light‐induced oxidation and dark re‐reduction kinetics of P700 in LL, ML, and HL‐acclimated strains. For each growth light, a corresponding actinic light of comparable intensity was used for determination of the intermediate P700 oxidation state P. Traces for untreated, DCMU and HA, and DBMIB‐treated samples are given. (d–f) The dark re‐reduction kinetics of P700 are shown after normalization to $P_m^{\prime }$ as described previously (Holland et al., 2016). Traces are representative of three biological replicates.

Figure 7

Partitioning of electrons into linear and cyclic pathways in WT and M14.2. (a) Estimation of the fraction of CEF in WT and M14.2 under different light intensities. (b) rETR1 and the contribution of linear and cyclic flow toward it. (c) ФPSI is plotted against ФPSII to determine the extent of cyclic and linear electron flow. Data represent mean and standard deviation from three biological replicates. Statistical significance is calculated using a two‐tailed t‐test. The P‐values are located above the * in the Figure.

Figure 8

Effect of Phe sink on PSI photochemical efficiency. (a) Photochemical efficiency of PSI (ФPSI) at LL, ML, and HL in WT and M14.2. The nonphotochemical energy dissipation due to (b) donor side limitation Y(ND) and (c) acceptor side limitation Y(NA). Data represent mean and standard deviation of three biological replicates. The P‐values are located above the * in the Figure.