151 results found
Murray JW, Rutherford AW, Nixon PJ, 2020, Photosystem II in a State of Disassembly, Joule, ISSN: 2542-4351
Feng Y, Morgan M, Fraser PD, et al., 2020, Crystal structure of geranylgeranyl pyrophosphate synthase (CrtE) involved in cyanobacterial terpenoid biosynthesis, Frontiers in Plant Science, Vol: 11, ISSN: 1664-462X
Cyanobacteria are photosynthetic prokaryotes that perform oxygenic photosynthesis. Due to their ability to use the photon energy of sunlight to fix carbon dioxide into biomass, cyanobacteria are promising hosts for the sustainable production of terpenoids, also known as isoprenoids, a diverse class of natural products with potential as advanced biofuels and high-value chemicals. However, the cyanobacterial enzymes involved in the biosynthesis of the terpene precursors needed to make more complicated terpenoids are poorly characterized. Here we show that the predicted type II prenyltransferase CrtE encoded by the model cyanobacterium Synechococcus sp. PCC 7002 is homodimeric and able to synthesize C20-geranylgeranyl pyrophosphate (GGPP) from C5-isopentenyl pyrophosphate (IPP) and C5-dimethylallyl pyrophosphate (DMAPP). The crystal structure of CrtE solved to a resolution of 2.7 Å revealed a strong structural similarity to the large subunit of the heterodimeric geranylgeranyl pyrophosphate synthase 1 from Arabidopsis thaliana with each subunit containing 14 helices. Using mutagenesis, we confirmed that the fourth and fifth amino acids (Met-87 and Ser-88) before the first conserved aspartate-rich motif (FARM) play important roles in controlling chain elongation. While the WT enzyme specifically produced GGPP, variants M87F and S88Y could only generate C15-farnesyl pyrophosphate (FPP), indicating that residues with large side chains obstruct product elongation. In contrast, replacement of M87 with the smaller Ala residue allowed the formation of the longer C25-geranylfarnesyl pyrophosphate (GFPP) product. Overall, our results provide new structural and functional information on the cyanobacterial CrtE enzyme that could lead to the development of improved cyanobacterial platforms for terpenoid production.
Włodarczyk A, Selão TT, Norling B, et al., 2020, Newly discovered Synechococcus sp. PCC 11901 is a robust cyanobacterial strain for high biomass production, Communications Biology, Vol: 3, ISSN: 2399-3642
Cyanobacteria, which use solar energy to convert carbon dioxide into biomass, are potential solar biorefineries for the sustainable production of chemicals and biofuels. However, yields obtained with current strains are still uncompetitive compared to existing heterotrophic production systems. Here we report the discovery and characterization of a new cyanobacterial strain, Synechococcus sp. PCC 11901, with promising features for green biotechnology. It is naturally transformable, has a short doubling time of ≈2 hours, grows at high light intensities and in a wide range of salinities and accumulates up to ≈33 g dry cell weight per litre when cultured in a shake-flask system using a modified growth medium − 1.7 to 3 times more than other strains tested under similar conditions. As a proof of principle, PCC 11901 engineered to produce free fatty acids yielded over 6 mM (1.5 g L−1), an amount comparable to that achieved by similarly engineered heterotrophic organisms.
Ahmad N, Khan MO, Islam E, et al., 2020, Contrasting responses to stress displayed by tobacco overexpressing an algal plastid terminal oxidase in the chloroplast, Frontiers in Plant Science, Vol: 11, ISSN: 1664-462X
The plastid terminal oxidase (PTOX) – an interfacial diiron carboxylate protein found in the thylakoid membranes of chloroplasts – oxidizes plastoquinol and reduces molecular oxygen to water. It is believed to play a physiologically important role in the response of some plant species to light and salt (NaCl) stress by diverting excess electrons to oxygen thereby protecting photosystem II (PSII) from photodamage. PTOX is therefore a candidate for engineering stress tolerance in crop plants. Previously, we used chloroplast transformation technology to over express PTOX1 from the green alga Chlamydomonas reinhardtii in tobacco (generating line Nt-PTOX-OE). Contrary to expectation, growth of Nt-PTOX-OE plants was more sensitive to light stress. Here we have examined in detail the effects of PTOX1 on photosynthesis in Nt-PTOX-OE tobacco plants grown at two different light intensities. Under ‘low light’ (50 μmol photons m–2 s–1) conditions, Nt-PTOX-OE and WT plants showed similar photosynthetic activities. In contrast, under ‘high light’ (125 μmol photons m–2 s–1) conditions, Nt-PTOX-OE showed less PSII activity than WT while photosystem I (PSI) activity was unaffected. Nt-PTOX-OE grown under high light also failed to increase the chlorophyll a/b ratio and the maximum rate of CO2 assimilation compared to low-light grown plants, suggesting a defect in acclimation. In contrast, Nt-PTOX-OE plants showed much better germination, root length, and shoot biomass accumulation than WT when exposed to high levels of NaCl and showed better recovery and less chlorophyll bleaching after NaCl stress when grown hydroponically. Overall, our results strengthen the link between PTOX and the resistance of plants to salt stress.
Pope M, Hodge J, Nixon PJ, 2020, An improved natural transformation protocol for the cyanobacterium Synechocystis sp. PCC 6803, Frontiers in Plant Science, Vol: 11, ISSN: 1664-462X
The naturally transformable cyanobacterium Synechocystis sp. PCC 6803 is a widely used chassis strain for the photosynthetic production of chemicals. However, Synechocystis possesses multiple genome copies per cell which means that segregating mutations across all genome copies can be time-consuming. Here we use flow cytometry in combination with DNA staining to investigate the effect of phosphate deprivation on the genome copy number of the glucose-tolerant GT-P sub-strain of Synechocystis 6803. Like the PCC 6803 wild type strain, the ploidy of GT-P cells grown in BG-11 medium is growth phase dependent with an average genome copy number of 6.05 ± 0.27 in early growth (OD740 = 0.1) decreasing to 2.49 ± 0.11 in late stationary phase (OD740 = 7). We show that a 10-fold reduction in the initial phosphate concentration of the BG-11 growth medium reduces the average genome copy number of GT-P cells from 4.51 ± 0.20 to 2.94 ± 0.13 and increases the proportion of monoploid cells from 0 to 6% after 7 days of growth. In addition, we also show that the DnaA protein, which unusually for bacteria is not required for DNA replication in Synechocystis, plays a role in restoring polyploidy upon subsequent phosphate supplementation. Based on these observations, we have developed an alternative natural transformation protocol involving phosphate depletion that decreases the time required to obtain fully segregated mutants.
Trinugroho J, Bečková M, Shao S, et al., 2020, Chlorophyll f synthesis by a super-rogue photosystem II complex, Nature Plants, Vol: 6, Pages: 238-244, ISSN: 2055-026X
Certain cyanobacteria synthesize chlorophyll molecules (Chl d and Chl f) that absorb in the far-red region of the solar spectrum, thereby extending the spectral range of photosynthetically active radiation1,2. The synthesis and introduction of these far-red chlorophylls into the photosynthetic apparatus of plants might improve the efficiency of oxygenic photosynthesis, especially in far-red enriched environments, such as in the lower regions of the canopy3. Production of Chl f requires the ChlF subunit, also known as PsbA4 (ref. 4) or super-rogue D1 (ref. 5), a paralogue of the D1 subunit of photosystem II (PSII) which, together with D2, bind cofactors involved in the light-driven oxidation of water. Current ideas suggest that ChlF oxidizes Chl a to Chl f in a homodimeric ChlF reaction centre (RC) complex and represents a missing link in the evolution of the heterodimeric D1/D2 RC of PSII (refs. 4,6). However, unambiguous biochemical support for this proposal is lacking. Here, we show that ChlF can substitute for D1 to form modified PSII complexes capable of producing Chl f. Remarkably, mutation of just two residues in D1 converts oxygen-evolving PSII into a Chl f synthase. Overall, we have identified a new class of PSII complex, which we term ‘super-rogue’ PSII, with an unexpected role in pigment biosynthesis rather than water oxidation.
Selão TT, Jebarani J, Ismail NA, et al., 2020, Enhanced production of D-lactate in cyanobacteria by re-routing photosynthetic cyclic and pseudo-cyclic electron flow, Frontiers in Plant Science, Vol: 10, ISSN: 1664-462X
Cyanobacteria are promising chassis strains for the photosynthetic production of platform and specialty chemicals from carbon dioxide. Their efficient light harvesting and metabolic flexibility has allowed a wide range of biomolecules, such as the bioplastic polylactate precursor D lactate, to be produced, though usually at relatively low yields. In order to increase photosynthetic electron flow towards the production of D-lactate, we have generated several strains of the marine cyanobacterium Synechococcus sp. PCC 7002 (Syn7002) with deletions in genes involved in cyclic or pseudo-cyclic electron flow around photosystem I. Using a variant of the Chlamydomonas reinhardtii D-lactate dehydrogenase (LDHSRT 25 , engineered to efficiently utilize NADPH in vivo), we show that deletion of either of the two flavodiiron flv homologues (involved in pseudo-cyclic electron transport) or the Syn7002 pgr5 homologue (proposed to be a vital part of the cyclic electron transport pathway) is able to increase D-lactate production in Syn7002 strains expressing LDHSRT 29 and the Escherichia coli LldP (lactate permease), especially at low temperature (25 °C) and 0.04% (v/v) CO2, though at elevated temperatures (38 °C) and/or high (1%) CO2 concentrations the effect was less obvious. The Δpgr5 background seemed to be particularly beneficial at 25 °C and 0.04% (v/v) CO2, with a nearly 7-fold increase in D lactate accumulation in comparison to the wild-type background (≈1000 vs ≈150 mg/L) and decreased side effects in comparison to the flv deletion strains. Overall, our results show that manipulation of photosynthetic electron flow is a viable strategy to increase production of platform chemicals in cyanobacteria under ambient conditions.
Zhang L, Selão TT, Nixon PJ, et al., 2019, Photosynthetic conversion of CO2 to hyaluronic acid by engineered strains of the cyanobacterium Synechococcus sp. PCC 7002, Algal Research, Vol: 44, ISSN: 2211-9264
Hyaluronic acid (HA), consisting of alternating N-acetylglucosamine and glucuronic acid units, is a natural polymer with diverse cosmetic and medical applications. Currently, HA is produced by overexpressing HA synthases from gram-negative Pasteurella multocida (encoded by pmHAS) or gram-positive Streptococcus equisimilis (encoded by seHasA) in various heterotrophic microbial production platforms. Here we introduced these two different types of HA synthase into the fast-growing cyanobacterium Synechococcus sp. PCC 7002 (Syn7002) to explore the capacity for producing HA in a photosynthetic system. Our results show that both HA synthases enable Syn7002 to produce HA photoautotrophically, but that overexpression of the soluble HA synthase (PmHAS) is less deleterious to cell growth and results in higher production. Genetic disruption of the competing cellulose biosynthetic pathway increased the HA titer by over 5-fold (from 14 mg/L to 80 mg/L) and the relative proportion of HA with molecular mass greater than 2 MDa. Introduction of glmS and glmU, coding for enzymes involved in the biosynthesis of the precursor UDP-N-acetylglucosamine, in combination with partial glycogen depletion, allowed photosynthetic production of 112 mg/L of HA in 5 days, an 8-fold increase in comparison to the initial PmHAS expressing strain. Addition of tuaD and gtaB (coding for genes involved in UDP-glucuronic acid biosynthesis) also improved the HA yield, albeit to a lesser extent. Overall our results have shown that cyanobacteria hold promise for the sustainable production of pharmaceutically important polysaccharides from sunlight and CO2.
Viola S, Bailleul B, Yu J, et al., 2019, Probing the electric field across thylakoid membranes in cyanobacteria., Proc Natl Acad Sci U S A, Vol: 116, Pages: 21900-21906
In plants, algae, and some photosynthetic bacteria, the ElectroChromic Shift (ECS) of photosynthetic pigments, which senses the electric field across photosynthetic membranes, is widely used to quantify the activity of the photosynthetic chain. In cyanobacteria, ECS signals have never been used for physiological studies, although they can provide a unique tool to study the architecture and function of the respiratory and photosynthetic electron transfer chains, entangled in the thylakoid membranes. Here, we identified bona fide ECS signals, likely corresponding to carotenoid band shifts, in the model cyanobacteria Synechococcus elongatus PCC7942 and Synechocystis sp. PCC6803. These band shifts, most likely originating from pigments located in photosystem I, have highly similar spectra in the 2 species and can be best measured as the difference between the absorption changes at 500 to 505 nm and the ones at 480 to 485 nm. These signals respond linearly to the electric field and display the basic kinetic features of ECS as characterized in other organisms. We demonstrate that these probes are an ideal tool to study photosynthetic physiology in vivo, e.g., the fraction of PSI centers that are prebound by plastocyanin/cytochrome c 6 in darkness (about 60% in both cyanobacteria, in our experiments), the conductivity of the thylakoid membrane (largely reflecting the activity of the ATP synthase), or the steady-state rates of the photosynthetic electron transport pathways.
Kiss E, Knoppova J, Pascual Aznar G, et al., 2019, A photosynthesis-specific rubredoxin-like protein is required for efficient association of the D1 and D2 proteins during the initial steps of photosystem II assembly, The Plant Cell, Vol: 31, Pages: 2241-2258, ISSN: 1040-4651
Oxygenic photosynthesis relies on accessory factors to promote the assembly and maintenance ofthe photosynthetic apparatus in the thylakoid membranes. The highly conserved membrane-boundrubredoxin-like protein RubA has previously been implicated in the accumulation of bothphotosystem I (PSI) and photosystem II (PSII) but its mode of action remains unclear. Here weshow that RubA in the cyanobacterium Synechocystis sp. PCC 6803 is required forphotoautotrophic growth in fluctuating light and acts early in PSII biogenesis by promoting theformation of the heterodimeric D1/D2 reaction center complex, the site of primary photochemistry.We find that RubA, like the accessory factor Ycf48, is a component of the initial D1 assemblymodule as well as larger PSII assembly intermediates and that the redox-responsive rubredoxinlike domain is located on the cytoplasmic surface of PSII complexes. Fusion of RubA to Ycf48still permits normal PSII assembly suggesting a spatiotemporal proximity of both proteins duringtheir action. RubA is also important for the accumulation of PSI but this is an indirect effectstemming from the downregulation of light-dependent chlorophyll biosynthesis induced by PSII deficiency. Overall our data support the involvement of RubA in the redox control of PSIIbiogenesis.
Selão TT, Włodarczyk A, Nixon PJ, et al., 2019, Growth and selection of the cyanobacterium Synechococcus sp. PCC 7002 using alternative nitrogen and phosphorus sources, Metabolic Engineering, Vol: 54, Pages: 255-263, ISSN: 1096-7176
Cyanobacteria, such as Synechococcus sp. PCC 7002 (Syn7002), are promising chassis strains for “green” biotechnological applications as they can be grown in seawater using oxygenic photosynthesis to fix carbon dioxide into biomass. Their other major nutritional requirements for efficient growth are sources of nitrogen (N) and phosphorus (P). As these organisms are more economically cultivated in outdoor open systems, there is a need to develop cost-effective approaches to prevent the growth of contaminating organisms, especially as the use of antibiotic selection markers is neither economically feasible nor ecologically desirable due to the risk of horizontal gene transfer. Here we have introduced a synthetic melamine degradation pathway into Syn7002 and evolved the resulting strain to efficiently use the nitrogen-rich xenobiotic compound melamine as the sole N source. We also show that expression of phosphite dehydrogenase in the absence of its cognate phosphite transporter permits growth of Syn7002 on phosphite and can be used as a selectable marker in Syn7002. We combined these two strategies to generate a strain that can grow on melamine and phosphite as sole N and P sources, respectively. This strain is able to resist deliberate contamination in large excess and should be a useful chassis for metabolic engineering and biotechnological applications using cyanobacteria.
Zhang L, Selao T, Norling B, et al., 2019, Photosynthetic hyaluronic acid production by metabolically engineered cyanobacteria
Shao S, Yu J, Nixon PJ, 2019, Selective Replacement of the Damaged D1 Reaction Center Subunit During the Repair of the Oxygen-Evolving Photosystem II Complex, Oxygen Production and Reduction in Artificial and Natural Systems, Editors: Barber, Ruban, Nixon, Publisher: World Scientific, Pages: 319-338
The multi-subunit photosystem II (PSII) pigment-protein complex found in plants, algae and cyanobacteria is nature’s biological catalyst for producing oxygen from water. PSII needs sunlight to drive water oxidation but too much light can cause irreversible damage to pigments and proteins within PSII and loss of enzyme activity. Damaged PSII complexes can, however, be repaired in a highly selective process involving the replacement of damaged components by newly synthesized copies and the recycling of undamaged protein subunits and co-factors. Although substantial progress has been made to identify the enzymes and accessory factors involved in repair, many fundamental questions remain unanswered. In this chapter we discuss recent ideas on how the damaged D1 reaction center subunit, which is the subunit most prone to damage in PSII, is specifically recognized for replacement. Detachment of CP43 allowing access by FtsH proteases to the N-terminal tail of D1 seems to underpin selective degradation.
Barber J, Ruban AV, Nixon PJ, 2019, Oxygen Production and Reduction in Artificial and Natural Systems, Publisher: World Scientific Publishing
Selao TT, Norling B, Nixon PJ, 2018, Genetically engineered cyanobacteria for growth in unsterilized conditions using antibiotic-free selection
Yu J, Knoppova J, Michoux F, et al., 2018, Ycf48 involved in the biogenesis of the oxygen-evolving photosystem II complex is a seven-bladed beta-propeller protein, Proceedings of the National Academy of Sciences, Vol: 115, Pages: E7824-E7833, ISSN: 0027-8424
Robust photosynthesis in chloroplasts and cyanobacteria requires the participation of accessory proteins to facilitate the assembly and maintenance of the photosynthetic apparatus located within the thylakoid membranes. The highly conserved Ycf48 protein acts early in the biogenesis of the oxygen-evolving photosystem II (PSII) complex by binding to newly synthesized precursor D1 subunit and by promoting efficient association with the D2 protein to form a PSII reaction center (PSII RC) assembly intermediate. Ycf48 is also required for efficient replacement of damaged D1 during the repair of PSII. However, the structural features underpinning Ycf48 function remain unclear. Here we show that Ycf48 proteins encoded by the thermophilic cyanobacterium Thermosynechococcus elongatus and the red alga Cyanidioschyzon merolae form seven-bladed beta-propellers with the 19-aa insertion characteristic of eukaryotic Ycf48 located at the junction of blades 3 and 4. Knowledge of these structures has allowed us to identify a conserved “Arg patch” on the surface of Ycf48 that is important for binding of Ycf48 to PSII RCs but also to larger complexes, including trimeric photosystem I (PSI). Reduced accumulation of chlorophyll in the absence of Ycf48 and the association of Ycf48 with PSI provide evidence of a more wide-ranging role for Ycf48 in the biogenesis of the photosynthetic apparatus than previously thought. Copurification of Ycf48 with the cyanobacterial YidC protein insertase supports the involvement of Ycf48 during the cotranslational insertion of chlorophyll-binding apopolypeptides into the membrane.
Sawa M, Fantuzzi A, Nixon P, et al., 2018, Development of printed solar biobattery for use in bioelectronics, Arm Summit 2018, Publisher: Arm
There is an urgent need to develop a sustainable battery technology that is cheap, environmentally friendly, easy to fabricate and to dispose of, especially to tackle the world-wide increase in illegally dumped electronic wastes. Microbial biophotovoltaic (BPV) technology is a renewable bioenergy system currently being developed at the laboratory scale. It generates electricity from the photosynthetic metabolism of cyanobacteria and microalgae and exploits their ability to convert light energy into electrical current using water as the source of electrons. Innovative approaches are needed to solve scale-up issues such as cost, ease of fabrication (particularly the fabrication of the inorganic and biological (microbes) parts).In this talk, I will report the feasibility of using a simple commercial thermal-inkjet printer to fabricate a thin-film paper-based BPV cell consisting of a layer of cyanobacterial cells on top of a carbon nanotube conducting surface on plain copy paper. The digitally printed thin-film BPV system produced electricity both in the light and dark, with a maximum electrical power output of 0.38 mW m-2 in one system and the sustained electrical current production over 100 hours in another more fully printed system. I will address limitations and challenges as well possible applications in the area of printed bioelectronics.
Cardona T, Shao S, Nixon PJ, 2018, Enhancing photosynthesis in plants: the light reactions, Essays in Biochemistry, Vol: 62, Pages: 85-94, ISSN: 0071-1365
In this review, we highlight recent research and current ideas on how to improve the efficiency of the light reactions of photosynthesis in crops. We note that the efficiency of photosynthesis is a balance between how much energy is used for growth and the energy wasted or spent protecting the photosynthetic machinery from photodamage. There are reasons to be optimistic about enhancing photosynthetic efficiency, but many appealing ideas are still on the drawing board. It is envisioned that the crops of the future will be extensively genetically modified to tailor them to specific natural or artificial environmental conditions.
Shao S, Cardona T, Nixon PJ, 2018, Early emergence of the FtsH proteases involved in Photosystem II repair, Photosynthetica, Vol: 56, Pages: 163-177, ISSN: 0300-3604
Efficient degradation of damaged D1 during the repair of PSII is carried out by a set of dedicated FtsH proteases in the thylakoid membrane. Here we investigated whether the evolution of FtsH could hold clues to the origin of oxygenic photosynthesis. A phylogenetic analysis of over 6000 FtsH protease sequences revealed that there are three major groups of FtsH proteases originating from gene duplication events in the last common ancestor of bacteria, and that the FtsH proteases involved in PSII repair make a distinct clade branching out before the divergence of FtsH proteases found in all groups of anoxygenic phototrophic bacteria. Furthermore, we showed that the phylogenetic tree of FtsH proteases in phototrophic bacteria is similar to that for Type I and Type II reaction centre proteins. We conclude that the phylogeny of FtsH proteases is consistent with an early origin of water oxidation chemistry.
Sawa M, Fantuzzi A, Bombelli P, et al., 2017, Electricity generation from digitally printed cyanobacteria, Nature Communications, Vol: 8, ISSN: 2041-1723
Microbial biophotovoltaic cells exploit the ability of cyanobacteria and microalgae to convert light energy into electrical current using water as the source of electrons. Such bioelectrochemical systems have a clear advantage over more conventional microbial fuel cells which require the input of organic carbon for microbial growth. However, innovative approaches are needed to address scale-up issues associated with the fabrication of the inorganic (electrodes) and biological (microbe) parts of the biophotovoltaic device. Here we demonstrate the feasibility of using a simple commercial inkjet printer to fabricate a thin-film paper-based biophotovoltaic cell consisting of a layer of cyanobacterial cells on top of a carbon nanotube conducting surface. We show that these printed cyanobacteria are capable of generating a sustained electrical current both in the dark (as a ‘solar bio-battery’) and in response to light (as a ‘bio-solar-panel’) with potential applications in low-power devices.
Beckova M, Yu J, Krynicka V, et al., 2017, Structure of Psb29/Thf1 and its association with the FtsH protease complex involved in photosystem II repair in cyanobacteria, Philosophical Transactions of the Royal Society B: Biological Sciences, Vol: 372, ISSN: 1471-2970
One strategy for enhancing photosynthesis in crop plants is to improve the ability to repair photosystem II (PSII) in response to irreversible damage by light. Despite the pivotal role of thylakoid embedded FtsH protease complexes in the selective degradation of PSII subunits during repair, little is known about the factors involved in regulating FtsH expression. Here we show using the cyanobacterium Synechocystis sp. PCC 6803 that the Psb29 subunit, originally identified as a minor component of His tagged PSII preparations, physically interacts with FtsH complexes in vivo and is required for normal accumulation of the FtsH2/FtsH3 hetero oligomeric complex involved in PSII repair. We show using X ray crystallography that Psb29 from Thermosynechococcus elongatushas a unique fold consisting of a helical bundle and an extended C terminal helix and contains a highly conserved region that might be involved in binding to FtsH. A similar interaction is likely to occur in Arabidopsis chloroplasts between the Psb29 homologue, termed THF1, and the FTSH2/FTSH5 complex. The direct involvement of Psb29/THF1 in FtsH accumulation helps explain why THF1 is a target during the hypersensitive response in plants induced by pathogen infection. Downregulating FtsH function and the PSII repair cycle via THF1 would contribute to the production
Foyer CH, Ruban AV, Nixon PJ, 2017, Photosynthesis solutions to enhance productivity, Philosophical Transactions of the Royal Society of London: Biological Sciences, Vol: 372, ISSN: 0962-8436
The concept that photosynthesis is a highly inefficient process in terms ofconversion of light energy into biomass is embedded in the literature. It isonly in the past decade that the processes limiting photosynthetic efficiencyhave been understood to an extent that allows a step change in our ability tomanipulate light energy assimilation into carbon gain. We can thereforeenvisage that future increases in the grain yield potential of our majorcrops may depend largely on increasing the efficiency of photosynthesis.The papers in this issue provide new insights into the nature of current limitationson photosynthesis and identify new targets that can be used for cropimprovement, together with information on the impacts of a changingenvironment on the productivity of photosynthesis on land and in ouroceans.This article is part of the themed issue ‘Enhancing photosynthesis in cropplants: targets for improvement’.
Barretto S, Michoux F, Hellgardt K, et al., 2016, Pneumatic hydrodynamics influence transplastomic protein yields and biological responses during in vitro shoot regeneration of Nicotiana tabacum callus: Implications for bioprocess routes to plant-made biopharmaceuticals, Biochemical Engineering Journal, Vol: 11, Pages: 73-81, ISSN: 1369-703X
Transplastomic plants are capable of high-yield production of recombinant biopharmaceutical proteins. Planttissue culture combines advantages of agricultural cultivation with the bioprocess consistency associated withsuspension culture. Overexpression of recombinant proteins through regeneration of transplastomic Nicotianatabacum shoots from callus tissue in RITA® temporary immersion bioreactors has been previously demonstrated.In this study we investigated the hydrodynamics of periodic pneumatic suspension of liquid medium duringtemporary immersion culture (4 minutes aeration every 8 hours), and the impact on biological responses andtransplastomic expression of fragment C of tetanus toxin (TetC). Biomass was grown under a range of aerationrates for 3, 20 and 40-day durations. Growth, mitochondrial activity (a viability indicator) and TetC protein yieldswere correlated against the hydrodynamic parameters, shear rate and energy dissipation rate (per kg of medium).A critical aeration rate of 440 ml min-1 was identified, corresponding to a shear rate of 96.7 s-1, pneumatic powerinput of 8.8 mW kg-1and initial 20-day pneumatic energy dissipation of 127 J kg-1, at which significant reductionsin biomass accumulation and mitochondrial activity were observed. There was an exponential decline in TetCyields with increasing aeration rates at 40 days, across the entire range of conditions tested. These observationshave important implications for the optimisation and scale-up of transplastomic plant tissue culture bioprocessesfor biopharmaceutical production.
Ahmad N, Michoux F, Lossl AG, et al., 2016, Challenges and perspectives in commercializing plastid transformation technology, Journal of Experimental Botany, Vol: 67, Pages: 5945-5960, ISSN: 1460-2431
Plastid transformation has emerged as an alternative platform to generate transgenic plants. Attractive features of this technology include specific integration of transgenes—either individually or as operons—into the plastid genome through homologous recombination, the potential for high-level protein expression, and transgene containment because of the maternal inheritance of plastids. Several issues associated with nuclear transformation such as gene silencing, variable gene expression due to the Mendelian laws of inheritance, and epigenetic regulation have not been observed in the plastid genome. Plastid transformation has been successfully used for the production of therapeutics, vaccines, antigens, and commercial enzymes, and for engineering various agronomic traits including resistance to biotic and abiotic stresses. However, these demonstrations have usually focused on model systems such as tobacco, and the technology per se has not yet reached the market. Technical factors limiting this technology include the lack of efficient protocols for the transformation of cereals, poor transgene expression in non-green plastids, a limited number of selection markers, and the lengthy procedures required to recover fully segregated plants. This article discusses the technology of transforming the plastid genome, the positive and negative features compared with nuclear transformation, and the current challenges that need to be addressed for successful commercialization.
Bečková M, Gardian Z, Yu J, et al., 2016, Association of Psb28 and Psb27 proteins with PSII-PSI supercomplexes upon exposure of Synechocystis sp. PCC 6803 to high light, Molecular Plant, Vol: 10, Pages: 62-72, ISSN: 1752-9867
Formation of the multi-subunit oxygen-evolving Photosystem II (PSII) complex involvesa number of auxiliary protein factors. In this study we compared the location and possiblefunction of two homologous PSII assembly factors, Psb28-1 and Psb28-2, from thecyanobacterium Synechocystis sp. PCC 6803. We show that FLAG-tagged Psb28-2 ispresent in both the monomeric PSII core complex and a PSII core complex lacking theinner antenna CP43 (RC47) whereas Psb28-1 preferentially binds to RC47. When cellsare exposed to increased irradiance, both tagged Psb28 proteins now associate witholigomeric forms of PSII and with PSII-PSI supercomplexes composed of trimericPhotosystem I (PSI) and two PSII monomers as deduced from negative stain electronmicroscopy. The presence of the Psb27 accessory protein in these complexes suggests theinvolvement of PSI in PSII biogenesis, possibly by photoprotecting PSII through energyspillover. Under standard cultivation conditions the distribution of PSII complexes issimilar in WT and each of the single psb28 null mutants except for loss of RC47 in theabsence of Psb28-1. In comparison with WT, growth of mutants lacking Psb28-1 andPsb27, but not Psb28-2, was retarded under high-light and, especially, intermittent highlight-darkconditions, emphasizing the physiological importance of PSII assembly factorsfor light acclimation.
Knoppova J, Yu J, Konik P, et al., 2016, CyanoP Is involved in the early steps of Photosystem two assembly in thecyanobacterium synechocystis sp. PCC 6803, Plant and Cell Physiology, Vol: 57, Pages: 1921-1931, ISSN: 1471-9053
Although the Photosystem II (PSII) complex is highly conserved in cyanobacteria and chloroplasts, the PsbU and PsbV subunits stabilizing the oxygen-evolving Mn4 CaO5 cluster in cyanobacteria are absent in chloroplasts and have been replaced by the PsbP and PsbQ subunits. There is, however, a distant cyanobacterial homologue of PsbP, termed CyanoP, of unknown function. Here we show that CyanoP plays a role in the early stages of PSII biogenesis in Synechocystis sp. PCC 6803. CyanoP is present in the PSII reaction centre assembly complex (RCII) lacking both the CP47 and CP43 modules and binds to the smaller D2 module. A small amount of larger PSII core complexes co-purifying with FLAG-tagged CyanoP indicates that CyanoP can accompany PSII on most of its assembly pathway. A role in biogenesis is supported by the accumulation of unassembled D1 precursor and impaired formation of RCII in a mutant lacking CyanoP. Interestingly, the pull-down preparations of CyanoP-FLAG from a strain lacking CP47 also contained PsbO indicating engagement of this protein with PSII at a much earlier stage in assembly than previously assumed.
Michoux F, Ahmad N, Wei Z-Y, et al., 2016, Testing the role of the N-terminal tail of D1 in the maintenance of photosystem II in tobacco chloroplasts, Frontiers in Plant Science, Vol: 7, ISSN: 1664-462X
A key step in the repair of photoinactivated oxygen-evolving photosystem II (PSII) complexes is the selective recognition and degradation of the damaged PSII subunit, usually the D1 reaction centre subunit. FtsH proteases play a major role in D1 degradation in both cyanobacteria and chloroplasts. In the case of the cyanobacterium Synechocystis sp. PCC 6803, analysis of an N-terminal truncation mutant of D1 lacking 20 amino-acid residues has provided evidence that FtsH complexes can remove damaged D1 in a processive reaction initiated at the exposed N-terminal tail. To test the importance of the N-terminal D1 tail in higher plants, we have constructed the equivalent truncation mutant in tobacco using chloroplast transformation techniques. The resulting mutant grew poorly and only accumulated about 25 % of wild-type levels of PSII in young leaves which declined as the leaves grew so that there was little PSII activity in mature leaves. Truncating D1 led to the loss of PSII supercomplexes and dimeric complexes in the membrane. Extensive and rapid non-photochemical quenching (NPQ) was still induced in the mutant, supporting the conclusion that PSII complexes are not required for NPQ. Analysis of leaves exposed to high light indicated that PSII repair in the truncation mutant was impaired at the level of synthesis and/or assembly of PSII but that D1 could still be degraded. These data support the idea that tobacco plants possess a number of back-up and compensatory pathways for removal of damaged D1 upon severe light stress.
Barretto S, Michoux F, Nixon PJ, 2016, Temporary Immersion Bioreactors for the Contained Production of Recombinant Proteins in Transplastomic Plants., Recombinant Proteins from Plants: Methods and Protocols, Publisher: Springer, Pages: 149-160, ISBN: 978-1-4939-3288-7
Despite the largely maternal inheritance of plastid genomes, the risk of transgene dissemination from transplastomic plants can limit the scope for field cultivation. There is a need for a cost-effective, scalable process to grow large quantities of transplastomic plant biomass for biosynthesis of biopharmaceuticals and other high-value heterologous proteins. Temporary immersion culture is a means of achieving this under fully contained conditions. This method describes the organogenesis of transplastomic Nicotiana tabacum callus in RITA(®) temporary immersion bioreactors to produce rootless leafy biomass, and subsequent total soluble protein extraction, SDS-PAGE, and Western immunoblot analysis of heterologous protein expression. This method can be used for propagation of plastid or nuclear transformants, though is especially suitable for transplastomic biomass, as organogenesis leads to greater expression and accumulation of transplastomic proteins due to increases in chloroplast number and size.
Burgess SJ, Hussein T, Yeoman JA, et al., 2015, Identification of the elusive pyruvate reductase of Chlamydomonas reinhardtii chloroplasts, Plant and Cell Physiology, Vol: 57, Pages: 82-94, ISSN: 1471-9053
Under anoxic conditions the green alga Chlamydomonas reinhardtii activates various 67 fermentation pathways leading to the creation of formate, acetate, ethanol and small 68 amounts of other metabolites including D-lactate and hydrogen. Progress has been 69 made in identifying the enzymes involved in these pathways and their sub-cellular 70 locations; however, the identity of the enzyme involved in reducing pyruvate to D-71 lactate has remained unclear. Based on sequence comparisons, enzyme activity 72 measurements, X-ray crystallography, biochemical fractionation and analysis of 73 knock-down mutants we conclude that pyruvate reduction in the chloroplast is 74 catalysed by a tetrameric NAD⁺-dependent D-lactate dehydrogenase encoded by 75 Cre07.g324550. Its expression during aerobic growth supports a possible function as a 76 ‘lactate valve’ for the export of lactate to the mitochondrion for oxidation by 77 cytochrome-dependent D-lactate dehydrogenases and by glycolate dehydrogenase. 78 We also present a revised spatial model of fermentation based on our 79 immunochemical detection of the likely pyruvate decarboxylase, PDC3, in the 80 cytoplasm.
Krynická V, Shao S, Nixon PJ, et al., 2015, Accessibility controls selective degradation ofphotosystem II subunits by FtsH protease, Nature Plants, Vol: 1, ISSN: 2055-0278
The oxygen-evolving photosystem II (PSII) complex located inchloroplasts and cyanobacteria is sensitive to light-induceddamage1 that unless repaired causes reduction in photosyntheticcapacity and growth. Although a potential target forcrop improvement, the mechanism of PSII repair remainsunclear. The D1 reaction center protein is the main target forphotodamage2, with repair involving the selective degradationof the damaged protein by FtsH protease3. How a singledamaged PSII subunit is recognized for replacement isunknown. Here, we have tested the dark stability of PSII subunitsin strains of the cyanobacterium Synechocystis PCC6803 blocked at specific stages of assembly. We have foundthat when D1, which is normally shielded by the CP43subunit, becomes exposed in a photochemically active PSIIcomplex lacking CP43, it is selectively degraded by FtsH evenin the dark. Removal of the CP47 subunit, which increasesaccessibility of FtsH to the D2 subunit, induced dark degradationof D2 at a faster rate than that of D1. In contrast,CP47 and CP43 are resistant to degradation in the dark. Ourresults indicate that protease accessibility induced by PSII disassemblyis an important determinant in the selection of the D1and D2 subunits to be degraded by FtsH.
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