Photoreaction Dynamics of a Full-Length Protein YtvA and Intermolecular Interaction with RsbRA.

YtvA from Bacillus subtilis is a sensor protein that responds to blue light stress and regulates the activity of transcription factor σB. It is composed of the N-terminal LOV (light-oxygen-voltage) domain, the C-terminal STAS (sulfate transporter and anti-sigma factor antagonist) domain, and a linker region connecting them. In this study, the photoreaction and kinetics of full-length YtvA and the intermolecular interaction with a downstream protein, RsbRA, were revealed by the transient grating method. Although N-YLOV-linker, which is composed of the LOV domain of YtvA with helices A'α and Jα, exhibits a diffusion change due to the rotational motion of the helices, the YtvA dimer does not show the diffusion change. This result suggests that the STAS domain inhibits the rotational movement of helices A'α and Jα. We found that the YtvA dimer formed a heterotetramer with the RsbRA dimer probably via the interaction between the STAS domains, and we showed the diffusion change upon blue light illumination with a time constant faster than 70 µs. This result suggests a conformational change of the STAS domains; i.e., the interface between the STAS domains of the proteins changes to enhance the friction with water by the rotation structural change of helices A'α and Jα of YtvA.

Blue light induces major changes in the gene expression profile of the cyanobacterium Synechocystis sp. PCC 6803.

Although cyanobacteria absorb blue light, they use it less efficiently for photosynthesis than other colors absorbed by their photosynthetic pigments. A plausible explanation for this enigmatic phenomenon is that blue light is not absorbed by phycobilisomes and, hence, causes an excitation shortage at photosystem II (PSII). This hypothesis is supported by recent physiological studies, but a comprehensive understanding of the underlying changes in gene expression is still lacking. In this study, we investigate how a switch from artificial white light to blue, orange or red light affects the transcriptome of the cyanobacterium Synechocystis sp. PCC 6803. In total, 145 genes were significantly regulated in response to blue light, whereas only a few genes responded to orange and red light. In particular, genes encoding the D1 and D2 proteins of PSII, the PSII chlorophyll-binding protein CP47 and genes involved in PSII repair were upregulated in blue light, whereas none of the photosystem I (PSI) genes responded to blue light. These changes were accompanied by a decreasing PSI:PSII ratio. Furthermore, many genes involved in gene transcription and translation and several ATP synthase genes were transiently downregulated, concurrent with a temporarily decreased growth rate in blue light. After 6-7 days, when cell densities had strongly declined, the growth rate recovered and the expression of these growth-related genes returned to initial levels. Hence, blue light induces major changes in the transcriptome of cyanobacteria, in an attempt to increase the photosynthetic activity of PSII and cope with the adverse growth conditions imposed by blue light.

Combining retinal-based and chlorophyll-based (oxygenic) photosynthesis: Proteorhodopsin expression increases growth rate and fitness of a ∆PSI strain of Synechocystis sp. PCC6803.

To fill the "green absorption gap", a green absorbing proteorhodopsin was expressed in a PSI-deletion strain (ΔPSI) of Synechocystis sp. PCC6803. Growth-rate measurements, competition experiments and physiological characterization of the proteorhodopsin-expressing strains, relative to the ΔPSI control strain, allow us to conclude that proteorhodopsin can enhance the rate of photoheterotrophic growth of ΔPSI Synechocystis strain. The physiological characterization included measurement of the amount of residual glucose in the spent medium and analysis of oxygen uptake- and production rates. To explore the use of solar radiation beyond the PAR region, a red-shifted variant Proteorhodopsin-D212N/F234S was expressed in a retinal-deficient PSI-deletion strain (ΔPSI/ΔSynACO). Via exogenous addition of retinal analogue an infrared absorbing pigment (maximally at 740 nm) was reconstituted in vivo. However, upon illumination with 746 nm light, it did not significantly stimulate the growth (rate) of this mutant. The inability of the proteorhodopsin-expressing ΔPSI strain to grow photoautotrophically is most likely due to a kinetic rather than a thermodynamic limitation of its NADPH-dehydrogenase in NADP+-reduction.

Exploring the low photosynthetic efficiency of cyanobacteria in blue light using a mutant lacking phycobilisomes.

The ubiquitous chlorophyll a (Chl a) pigment absorbs both blue and red light. Yet, in contrast to green algae and higher plants, most cyanobacteria have much lower photosynthetic rates in blue than in red light. A plausible but not yet well-supported hypothesis is that blue light results in limited energy transfer to photosystem II (PSII), because cyanobacteria invest most Chl a in photosystem I (PSI), whereas their phycobilisomes (PBS) are mostly associated with PSII but do not absorb blue photons. In this paper, we compare the photosynthetic performance in blue and orange-red light of wildtype Synechocystis sp. PCC 6803 and a PBS-deficient mutant. Our results show that the wildtype had much lower biomass, Chl a content, PSI:PSII ratio and O2 production rate per PSII in blue light than in orange-red light, whereas the PBS-deficient mutant had a low biomass, Chl a content, PSI:PSII ratio, and O2 production rate per PSII in both light colors. More specifically, the wildtype displayed a similar low photosynthetic efficiency in blue light as the PBS-deficient mutant in both light colors. Our results demonstrate that the absorption of light energy by PBS and subsequent transfer to PSII are crucial for efficient photosynthesis in cyanobacteria, which may explain both the low photosynthetic efficiency of PBS-containing cyanobacteria and the evolutionary success of chlorophyll-based light-harvesting antennae in environments dominated by blue light.

On the use of oxygenic photosynthesis for the sustainable production of commodity chemicals.

A sustainable society will have to largely refrain from the use of fossil carbon deposits. In such a regime, renewable electricity can be harvested as a primary source of energy. However, as for the synthesis of carbon-based materials from bulk chemicals, an alternative is required. A sustainable approach towards this is the synthesis of commodity chemicals from CO2 , water and sunlight. Multiple paths to achieve this have been designed and tested in the domains of chemistry and biology. In the latter, the use of both chemotrophic and phototrophic organisms has been advocated. 'Direct conversion' of CO2 and H2 O, catalyzed by an oxyphototroph, has excellent prospects to become the most economically competitive of these transformations, because of the relative ease of scale-up of this process. Significantly, for a wide range of energy and commodity products, a proof of principle via engineering of the corresponding production organism has been provided. In the optimization of a cyanobacterial production organism, a wide range of aspects has to be addressed. Of these, here we will put our focus on: (1) optimizing the (carbon) flux to the desired product; (2) increasing the genetic stability of the producing organism and (3) maximizing its energy conversion efficiency. Significant advances have been made on all these three aspects during the past 2 years and these will be discussed: (1) increasing the carbon partitioning to >50%; (2) aligning product formation with the growth of the cells and (3) expanding the photosynthetically active radiation region for oxygenic photosynthesis.

Spectrally decomposed dark-to-light transitions in a PSI-deficient mutant of Synechocystis sp. PCC 6803.

Cyanobacterial thylakoid membranes are known to host photosynthetic and respiratory complexes. This hampers a straight forward interpretation of the highly dynamic fluorescence originating from photosynthetic units. The present study focuses on dark-to-light transitions in whole cells of a PSI-deficient mutant of the cyanobacterium Synechocystis sp. PCC 6803. The time-dependent cellular fluorescence spectrum has been measured, while having previously exposed the cells to different conditions that affect respiratory activity. The analysis method used allows the detected signal to be decomposed in a few components that are then assigned to functional emitting species. Additionally, we have worked out a minimal mathematical model consisting of sensible postulated species to interpret the recorded data. We conclude that the following two functional complexes play a major role: a phycobilisome antenna complex coupled to a PSII dimer with either two or no closed reaction centers. Crucially, we present evidence for an additional species capable of strongly quenching fluorescence, whose formation requires the presence of oxygen.

Deletion of sll1541 in Synechocystis sp. Strain PCC 6803 Allows Formation of a Far-Red-Shifted holo-Proteorhodopsin In Vivo.

In many pro- and eukaryotes, a retinal-based proton pump equips the cell to drive ATP synthesis with (sun)light. Such pumps, therefore, have been proposed as a plug-in for cyanobacteria to artificially increase the efficiency of oxygenic photosynthesis. However, little information on the metabolism of retinal, their chromophore, is available for these organisms. We have studied the in vivo roles of five genes (sll1541, slr1648, slr0091, slr1192, and slr0574) potentially involved in retinal metabolism in Synechocystis sp. strain PCC 6803. With a gene deletion approach, we have shown that Synechocystis apo-carotenoid-15,15-oxygenase (SynACO), encoded by gene sll1541, is an indispensable enzyme for retinal synthesis in Synechocystis, presumably via asymmetric cleavage of ß-apo-carotenal. The second carotenoid oxygenase (SynDiox2), encoded by gene slr1648, competes with SynACO for substrate(s) but only measurably contributes to retinal biosynthesis in stationary phase via an as-yet-unknown mechanism. In vivo degradation of retinal may proceed through spontaneous chemical oxidation and via enzyme-catalyzed processes. Deletion of gene slr0574 (encoding CYP120A1), but not of slr0091 or of slr1192, causes an increase (relative to the level in wild-type Synechocystis) in the retinal content in both the linear and stationary growth phases. These results suggest that CYP120A1 does contribute to retinal degradation. Preliminary data obtained using 13C-labeled retinal suggest that conversion to retinol and retinoic acid and subsequent further oxidation also play a role. Deletion of sll1541 leads to deficiency in retinal synthesis and allows the in vivo reconstitution of far-red-absorbing holo-proteorhodopsin with exogenous retinal analogues, as demonstrated here for all-trans 3,4-dehydroretinal and 3-methylamino-16-nor-1,2,3,4-didehydroretinal.IMPORTANCE Retinal is formed by many cyanobacteria and has a critical role in most forms of life for processes such as photoreception, growth, and stress survival. However, the metabolic pathways in cyanobacteria for synthesis and degradation of retinal are poorly understood. In this paper we identify genes involved in its synthesis, characterize their role, and provide an initial characterization of the pathway of its degradation. This led to the identification of sll1541 (encoding SynACO) as the essential gene for retinal synthesis. Multiple pathways for retinal degradation presumably exist. These results have allowed us to construct a strain that expresses a light-dependent proton pump with an action spectrum extending beyond 700 nm. The availability of this strain will be important for further work aimed at increasing the overall efficiency of oxygenic photosynthesis.

Blue light reduces photosynthetic efficiency of cyanobacteria through an imbalance between photosystems I and II.

Several studies have described that cyanobacteria use blue light less efficiently for photosynthesis than most eukaryotic phototrophs, but comprehensive studies of this phenomenon are lacking. Here, we study the effect of blue (450 nm), orange (625 nm), and red (660 nm) light on growth of the model cyanobacterium Synechocystis sp. PCC 6803, the green alga Chlorella sorokiniana and other cyanobacteria containing phycocyanin or phycoerythrin. Our results demonstrate that specific growth rates of the cyanobacteria were similar in orange and red light, but much lower in blue light. Conversely, specific growth rates of the green alga C. sorokiniana were similar in blue and red light, but lower in orange light. Oxygen production rates of Synechocystis sp. PCC 6803 were five-fold lower in blue than in orange and red light at low light intensities but approached the same saturation level in all three colors at high light intensities. Measurements of 77 K fluorescence emission demonstrated a lower ratio of photosystem I to photosystem II (PSI:PSII ratio) and relatively more phycobilisomes associated with PSII (state 1) in blue light than in orange and red light. These results support the hypothesis that blue light, which is not absorbed by phycobilisomes, creates an imbalance between the two photosystems of cyanobacteria with an energy excess at PSI and a deficiency at the PSII-side of the photosynthetic electron transfer chain. Our results help to explain why phycobilisome-containing cyanobacteria use blue light less efficiently than species with chlorophyll-based light-harvesting antennae such as Prochlorococcus, green algae and terrestrial plants.

Spectrally decomposed dark-to-light transitions in Synechocystis sp. PCC 6803.

Photosynthetic activity and respiration share the thylakoid membrane in cyanobacteria. We present a series of spectrally resolved fluorescence experiments where whole cells of the cyanobacterium Synechocystis sp. PCC 6803 and mutants thereof underwent a dark-to-light transition after different dark-adaptation (DA) periods. Two mutants were used: (i) a PSI-lacking mutant (ΔPSI) and (ii) M55, a mutant without NAD(P)H dehydrogenase type-1 (NDH-1). For comparison, measurements of the wild-type were also carried out. We recorded spectrally resolved fluorescence traces over several minutes with 100 ms time resolution. The excitation light was at 590 nm so as to specifically excite the phycobilisomes. In ΔPSI, DA time has no influence, and in dichlorophenyl-dimethylurea (DCMU)-treated samples we identify three main fluorescent components: PB-PSII complexes with closed (saturated) RCs, a quenched or open PB-PSII complex, and a PB-PSII 'not fully closed.' For the PSI-containing organisms without DCMU, we conclude that mainly three species contribute to the signal: a PB-PSII-PSI megacomplex with closed PSII RCs and (i) slow PB → PSI energy transfer, or (ii) fast PB → PSI energy transfer and (iii) complexes with open (photochemically quenched) PSII RCs. Furthermore, their time profiles reveal an adaptive response that we identify as a state transition. Our results suggest that deceleration of the PB → PSI energy transfer rate is the molecular mechanism underlying a state 2 to state 1 transition.

Challenges in the Application of Synthetic Biology Toward Synthesis of Commodity Products by Cyanobacteria via "Direct Conversion".

Cyanobacterial direct conversion of CO2 to several commodity chemicals has been recognized as a potential contributor to support the much-needed sustainable development of human societies. However, the feasibility of this "green conversion" hinders on our ability to overcome the hurdles presented by the natural evolvability of microbes. The latter may result in the genetic instability of engineered cyanobacterial strains leading to impaired productivity. This challenge is general to any "cell factory" approach in which the cells grow for multiple generations, and based on several studies carried out in different microbial hosts, we could identify that three distinct strategies have been proposed to tackle it. These are (1) to reduce microbial evolvability by decreasing the native mutation rate, (2) to align product formation with cell growth/fitness, and, paradoxically, (3) to efficiently reallocate cellular resources to product formation by uncoupling it from growth. The implementation of either of these strategies requires an advanced synthetic biology toolkit. Here, we review the existing methods available for cyanobacteria and identify areas of focus in which specific developments are still needed. Furthermore, we discuss how potentially stabilizing strategies may be used in combination leading to further increases of productivity while ensuring the stability of the cyanobacterial-based direct conversion process.

Retinal-Based Proton Pumping in the Near Infrared.

Proteorhodopsin (PR) and Gloeobacter rhodopsin (GR) are retinal-based light-driven proton pumps that absorb visible light (maxima at 520-540 nm). Shifting the action spectra of these proton pumps beyond 700 nm would generate new prospects in optogenetics, membrane sensor technology, and complementation of oxygenic phototrophy. We therefore investigated the effect of red-shifting analogues of retinal, combined with red-shifting mutations, on the spectral properties and pump activity of the resulting pigments. We investigated a variety of analogues, including many novel ones. One of the novel analogues we tested, 3-methylamino-16-nor-1,2,3,4-didehydroretinal (MMAR), produced exciting results. This analogue red-shifted all of the rhodopsin variants tested, accompanied by a strong broadening of the absorbance band, tailing out to 850-950 nm. In particular, MMAR showed a strong synergistic effect with the PR-D212N,F234S double mutant, inducing an astonishing 200 nm red shift in the absorbance maximum. To our knowledge, this is by far the largest red shift reported for any retinal protein. Very importantly, all MMAR-containing holoproteins are the first rhodopsins retaining significant pump activity under near-infrared illumination (730 nm light-emitting diode). Such MMAR-based rhodopsin variants present very promising opportunities for further synthetic biology modification and for a variety of biotechnological and biophysical applications.

Ethylene production with engineered Synechocystis sp PCC 6803 strains.

BACKGROUND: Metabolic engineering and synthetic biology of cyanobacteria offer a promising sustainable alternative approach for fossil-based ethylene production, by using sunlight via oxygenic photosynthesis, to convert carbon dioxide directly into ethylene. Towards this, both well-studied cyanobacteria, i.e., Synechocystis sp PCC 6803 and Synechococcus elongatus PCC 7942, have been engineered to produce ethylene by introducing the ethylene-forming enzyme (Efe) from Pseudomonas syringae pv. phaseolicola PK2 (the Kudzu strain), which catalyzes the conversion of the ubiquitous tricarboxylic acid cycle intermediate 2-oxoglutarate into ethylene. RESULTS: This study focuses on Synechocystis sp PCC 6803 and shows stable ethylene production through the integration of a codon-optimized version of the efe gene under control of the Ptrc promoter and the core Shine-Dalgarno sequence (5'-AGGAGG-3') as the ribosome-binding site (RBS), at the slr0168 neutral site. We have increased ethylene production twofold by RBS screening and further investigated improving ethylene production from a single gene copy of efe, using multiple tandem promoters and by putting our best construct on an RSF1010-based broad-host-self-replicating plasmid, which has a higher copy number than the genome. Moreover, to raise the intracellular amounts of the key Efe substrate, 2-oxoglutarate, from which ethylene is formed, we constructed a glycogen-synthesis knockout mutant (ΔglgC) and introduced the ethylene biosynthetic pathway in it. Under nitrogen limiting conditions, the glycogen knockout strain has increased intracellular 2-oxoglutarate levels; however, surprisingly, ethylene production was lower in this strain than in the wild-type background. CONCLUSION: Making use of different RBS sequences, production of ethylene ranging over a 20-fold difference has been achieved. However, a further increase of production through multiple tandem promoters and a broad-host plasmid was not achieved speculating that the transcription strength and the gene copy number are not the limiting factors in our system.

Photochemical Reactions of the LOV and LOV-Linker Domains of the Blue Light Sensor Protein YtvA.

YtvA is a blue light sensor protein composed of an N-terminal LOV (light-oxygen-voltage) domain, a linker helix, and the C-terminal sulfate transporter and anti-σ factor antagonist domain. YtvA is believed to act as a positive regulator for light and salt stress responses by regulating the σB transcription factor. Although its biological function has been studied, the reaction dynamics and molecular mechanism underlying the function are not well understood. To improve our understanding of the signaling mechanism, we studied the reaction of the LOV domain (YLOV, amino acids 26-127), the LOV domain with its N-terminal extension (N-YLOV, amino acids 1-127), the LOV domain with its C-terminal linker helix (YLOV-linker, amino acids 26-147), and the YLOV domain with the N-terminal extension and the C-terminal linker helix (N-YLOV-linker, amino acids 1-147) using the transient grating method. The signals of all constructs showed adduct formation, thermal diffusion, and molecular diffusion. YLOV showed no change in the diffusion coefficient (D), while the other three constructs showed a significant decrease in D within ∼70 µs of photoexcitation. This indicates that conformational changes in both the N- and C-terminal helices of the YLOV domain indeed do occur. The time constant in the YtvA derivatives was much faster than the corresponding dynamics of phototropins. Interestingly, an additional reaction was observed as a volume expansion as well as a slight increase in D only when both helices were included. These findings suggest that although the rearrangement of the N- and C-terminal helices occurs independently on the fast time scale, this change induces an additional conformational change only when both helices are present.

Time-series analysis of the transcriptome and proteome of Escherichia coli upon glucose repression.

Time-series transcript- and protein-profiles were measured upon initiation of carbon catabolite repression in Escherichia coli, in order to investigate the extent of post-transcriptional control in this prototypical response. A glucose-limited chemostat culture was used as the CCR-free reference condition. Stopping the pump and simultaneously adding a pulse of glucose, that saturated the cells for at least 1h, was used to initiate the glucose response. Samples were collected and subjected to quantitative time-series analysis of both the transcriptome (using microarray analysis) and the proteome (through a combination of 15N-metabolic labeling and mass spectrometry). Changes in the transcriptome and corresponding proteome were analyzed using statistical procedures designed specifically for time-series data. By comparison of the two sets of data, a total of 96 genes were identified that are post-transcriptionally regulated. This gene list provides candidates for future in-depth investigation of the molecular mechanisms involved in post-transcriptional regulation during carbon catabolite repression in E. coli, like the involvement of small RNAs.

Expression of holo-proteorhodopsin in Synechocystis sp. PCC 6803.

Retinal-based photosynthesis may contribute to the free energy conversion needed for growth of an organism carrying out oxygenic photosynthesis, like a cyanobacterium. After optimization, this may even enhance the overall efficiency of phototrophic growth of such organisms in sustainability applications. As a first step towards this, we here report on functional expression of the archetype proteorhodopsin in Synechocystis sp. PCC 6803. Upon use of the moderate-strength psbA2 promoter, holo-proteorhodopsin is expressed in this cyanobacterium, at a level of up to 10(5) molecules per cell, presumably in a hexameric quaternary structure, and with approximately equal distribution (on a protein-content basis) over the thylakoid and the cytoplasmic membrane fraction. These results also demonstrate that Synechocystis sp. PCC 6803 has the capacity to synthesize all-trans-retinal. Expressing a substantial amount of a heterologous opsin membrane protein causes a substantial growth retardation Synechocystis, as is clear from a strain expressing PROPS, a non-pumping mutant derivative of proteorhodopsin. Relative to this latter strain, proteorhodopsin expression, however, measurably stimulates its growth.

Chirality Matters: Synthesis and Consumption of the d-Enantiomer of Lactic Acid by Synechocystis sp. Strain PCC6803.

Both enantiomers of lactic acid, l-lactic acid and d-lactic acid, can be produced in a sustainable way by a photosynthetic microbial cell factory and thus from CO2, sunlight, and water. Several properties of polylactic acid (a polyester of polymerized lactic acid) depend on the controlled blend of these two enantiomers. Recently, cyanobacterium Synechocystis sp. strain PCC6803 was genetically modified to allow formation of either of these two enantiomers. This report elaborates on the d-lactic acid production achieved by the introduction of a d-specific lactate dehydrogenase from the lactic acid bacterium Leuconostoc mesenteroides into Synechocystis. A typical batch culture of this recombinant strain initially shows lactic acid production, followed by a phase of lactic acid consumption, until production "outcompetes" consumption at later growth stages. We show that Synechocystis is able to use d-lactic acid, but not l-lactic acid, as a carbon source for growth. Deletion of the organism's putative d-lactate dehydrogenase (encoded by slr1556), however, does not eliminate this ability with respect to d-lactic acid consumption. In contrast, d-lactic acid consumption does depend on the presence of glycolate dehydrogenase GlcD1 (encoded by sll0404). Accordingly, this report highlights the need to match a product of interest of a cyanobacterial cell factory with the metabolic network present in the host used for its synthesis and emphasizes the need to understand the physiology of the production host in detail.

Culturing Synechocystis sp. Strain PCC 6803 with N2 and CO2 in a Diel Regime Reveals Multiphase Glycogen Dynamics with Low Maintenance Costs.

UNLABELLED: Investigating the physiology of cyanobacteria cultured under a diel light regime is relevant for a better understanding of the resulting growth characteristics and for specific biotechnological applications that are foreseen for these photosynthetic organisms. Here, we present the results of a multiomics study of the model cyanobacterium Synechocystis sp. strain PCC 6803, cultured in a lab-scale photobioreactor in physiological conditions relevant for large-scale culturing. The culture was sparged with N2 and CO2, leading to an anoxic environment during the dark period. Growth followed the availability of light. Metabolite analysis performed with (1)H nuclear magnetic resonance analysis showed that amino acids involved in nitrogen and sulfur assimilation showed elevated levels in the light. Most protein levels, analyzed through mass spectrometry, remained rather stable. However, several high-light-response proteins and stress-response proteins showed distinct changes at the onset of the light period. Microarray-based transcript analysis found common patterns of ∼56% of the transcriptome following the diel regime. These oscillating transcripts could be grouped coarsely into genes that were upregulated and downregulated in the dark period. The accumulated glycogen was degraded in the anaerobic environment in the dark. A small part was degraded gradually, reflecting basic maintenance requirements of the cells in darkness. Surprisingly, the largest part was degraded rapidly in a short time span at the end of the dark period. This degradation could allow rapid formation of metabolic intermediates at the end of the dark period, preparing the cells for the resumption of growth at the start of the light period. IMPORTANCE: Industrial-scale biotechnological applications are anticipated for cyanobacteria. We simulated large-scale high-cell-density culturing of Synechocystis sp. PCC 6803 under a diel light regime in a lab-scale photobioreactor. In BG-11 medium, Synechocystis grew only in the light. Metabolite analysis grouped the collected samples according to the light and dark conditions. Proteome analysis suggested that the majority of enzyme-activity regulation was not hierarchical but rather occurred through enzyme activity regulation. An abrupt light-on condition induced high-light-stress proteins. Transcript analysis showed distinct patterns for the light and dark periods. Glycogen gradually accumulated in the light and was rapidly consumed in the last quarter of the dark period. This suggests that the circadian clock primed the cellular machinery for immediate resumption of growth in the light.

Genetic engineering of Synechocystis PCC6803 for the photoautotrophic production of the sweetener erythritol.

BACKGROUND: Erythritol is a polyol that is used in the food and beverage industry. Due to its non-caloric and non-cariogenic properties, the popularity of this sweetener is increasing. Large scale production of erythritol is currently based on conversion of glucose by selected fungi. In this study, we describe a biotechnological process to produce erythritol from light and CO2, using engineered Synechocystis sp. PCC6803. METHODS: By functionally expressing codon-optimized genes encoding the erythrose-4-phosphate phosphatase TM1254 and the erythrose reductase Gcy1p, or GLD1, this cyanobacterium can directly convert the Calvin cycle intermediate erythrose-4-phosphate into erythritol via a two-step process and release the polyol sugar in the extracellular medium. Further modifications targeted enzyme expression and pathway intermediates. CONCLUSIONS: After several optimization steps, the best strain, SEP024, produced up to 2.1 mM (256 mg/l) erythritol, excreted in the medium.

Modulation of spectral properties and pump activity of proteorhodopsins by retinal analogues.

Proteorhodopsins are heptahelical membrane proteins which function as light-driven proton pumps. They use all-trans-retinal A1 as a ligand and chromophore and absorb visible light (520-540 nm). In the present paper, we describe modulation of the absorbance band of the proteorhodopsin from Monterey Bay SAR 86 gammaproteobacteria (PR), its red-shifted double mutant PR-D212N/F234S (PR-DNFS) and Gloeobacter rhodopsin (GR). This was approached using three analogues of all-trans-retinal A1, which differ in their electronic and conformational properties: all-trans-6,7-s-trans-locked retinal A1, all-trans-phenyl-retinal A1 and all-trans-retinal A2. We further probed the effect of these retinal analogues on the proton pump activity of the proteorhodopsins. Our results indicate that, whereas the constraints of the retinal-binding pocket differ for the proteorhodopsins, at least two of the retinal analogues are capable of shifting the absorbance bands of the pigments either bathochromically or hypsochromically, while maintaining their proton pump activity. Furthermore, the shifts implemented by the analogues add up to the shift induced by the double mutation in PR-DNFS. This type of chromophore substitution may present attractive applications in the field of optogenetics, towards increasing the flexibility of optogenetic tools or for membrane potential probes.

The redox potential of the plastoquinone pool of the cyanobacterium Synechocystis species strain PCC 6803 is under strict homeostatic control.

A method is presented for rapid extraction of the total plastoquinone (PQ) pool from Synechocystis sp. strain PCC 6803 cells that preserves the in vivo plastoquinol (PQH2) to -PQ ratio. Cells were rapidly transferred into ice-cold organic solvent for instantaneous extraction of the cellular PQ plus PQH2 content. After high-performance liquid chromatography fractionation of the organic phase extract, the PQH2 content was quantitatively determined via its fluorescence emission at 330 nm. The in-cell PQH2-PQ ratio then followed from comparison of the PQH2 signal in samples as collected and in an identical sample after complete reduction with sodium borohydride. Prior to PQH2 extraction, cells from steady-state chemostat cultures were exposed to a wide range of physiological conditions, including high/low availability of inorganic carbon, and various actinic illumination conditions. Well-characterized electron-transfer inhibitors were used to generate a reduced or an oxidized PQ pool for reference. The in vivo redox state of the PQ pool was correlated with the results of pulse-amplitude modulation-based chlorophyll a fluorescence emission measurements, oxygen exchange rates, and 77 K fluorescence emission spectra. Our results show that the redox state of the PQ pool of Synechocystis sp. strain PCC 6803 is subject to strict homeostatic control (i.e. regulated between narrow limits), in contrast to the more dynamic chlorophyll a fluorescence signal.