Biosynthesis of platform chemical 3-hydroxypropionic acid (3-HP) directly from CO2 in cyanobacterium Synechocystis sp. PCC 6803

3-hydroxypropionic acid (3-HP) is an important platform chemical with a wide range of applications. So far large-scale production of 3-HP has been mainly through petroleum-based chemical processes, whose sustainability and environmental issues have attracted widespread attention. With the ability to fix CO₂ directly, cyanobacteria have been engineered as an autotrophic microbial cell factory to produce fuels and chemicals. In this study, we constructed the biosynthetic pathway of 3-HP in cyanobacterium Synechocystis sp. PCC 6803, and then optimized the system through the following approaches: i) increasing expression of malonyl-CoA reductase (MCR) gene using different promoters and cultivation conditions; ii) enhancing supply of the precursor malonyl-CoA by overexpressing acetyl-CoA carboxylase and biotinilase; iii) improving NADPH supply by overexpressing the NAD(P) transhydrogenase gene; iv) directing more carbon flux into 3-HP by inactivating the competing pathways of PHA and acetate biosynthesis. Together, the efforts led to a production of 837.18 mg L⁻¹ (348.8 mg/g dry cell weight) 3-HP directly from CO₂ in Synechocystis after 6 days cultivation, demonstrating the feasibility photosynthetic production of 3-HP directly from sunlight and CO₂ in cyanobacteria. In addition, the results showed that overexpression of the ribulose-1, 5-bisphosphate carboxylase/oxygenase (Rubisco) gene from Anabaena sp. PCC 7120 and Synechococcus sp. PCC 7942 led to no increase of 3-HP production, suggesting CO₂ fixation may not be a rate-limiting step for 3-HP biosynthesis in Synechocystis.     

Production of optically pure d-lactate from CO2 by blocking the PHB and acetate pathways and expressing d-lactate dehydrogenase in cyanobacterium Synechocystis sp. PCC 6803

Lactate is an important industrial material with numerous potential applications, and its production from carbon dioxide is very attractive. d-Lactate is an essential monomer for production of thermostable polylactide. The photoautotrophic prokaryote cyanobacterium Synechocystis sp. PCC 6803 represents a promising host for biosynthesis of d-lactate from CO₂ as it only contains d-lactate dehydrogenase. The production of d-lactate from CO₂ by an engineered strain of Synechocystis sp. PCC 6803 with overexpressing d-lactate dehydrogenase and a soluble transhydrogenase has been reported recently. Here, we report an alternative engineering strategy to produce d-lactate from CO₂. This strategy involves blocking two competitive pathways, the native poly-3-hydroxybutyrate and acetate pathways from the acetyl-CoA node, and introducing a more efficient d-lactate dehydrogenase into Synechocystis sp. PCC 6803. The engineered strain of Synechocystis sp. PCC 6803 was capable of producing 1.06 g/L of d-lactate from CO₂. This alternative strategy for the production of optically pure d-lactate could also be used to produce other acetyl-CoA-derived chemicals from CO₂ by using engineered cyanobacteria.     

Engineering Cupriavidus necator H16 for the autotrophic production of (R)-1,3-butanediol

Butanediols are widely used in the synthesis of polymers, specialty chemicals and important chemical intermediates. Optically pure R-form of 1,3-butanediol (1,3-BDO) is required for the synthesis of several industrial compounds and as a key intermediate of β-lactam antibiotic production. The (R)-1,3-BDO can only be produced by application of a biocatalytic process. Cupriavidus necator H16 is an established production host for biosynthesis of biodegradable polymer poly-3-hydroxybutryate (PHB) via acetyl-CoA intermediate. Therefore, the utilisation of acetyl-CoA or its upstream precursors offers a promising strategy for engineering biosynthesis of value-added products such as (R)-1,3-BDO in this bacterium. Notably, C. necator H16 is known for its natural capacity to fix carbon dioxide (CO₂) using hydrogen as an electron donor. Here, we report engineering of this facultative lithoautotrophic bacterium for heterotrophic and autotrophic production of (R)-1,3-BDO. Implementation of (R)-3-hydroxybutyraldehyde-CoA- and pyruvate-dependent biosynthetic pathways in combination with abolishing PHB biosynthesis and reducing flux through the tricarboxylic acid cycle enabled to engineer strain, which produced 2.97 g/L of (R)-1,3-BDO and achieved production rate of nearly 0.4 Cmol Cmol⁻¹ h⁻¹ autotrophically. This is first report of (R)-1,3-BDO production from CO₂.     

Engineering a platform for photosynthetic isoprene production in cyanobacteria,using Synechocystis as the model organism

The concept of “photosynthetic biofuels” envisions application of a single organism, acting both as photo-catalyst and producer of ready-made fuel. This concept was applied upon genetic engineering of the cyanobacterium Synechocystis, conferring the ability to generate volatile isoprene hydrocarbons from CO₂ and H₂O. Heterologous expression of the Pueraria montana (kudzu) isoprene synthase (IspS) gene in Synechocystis enabled photosynthetic isoprene generation in these cyanobacteria. Codon-use optimization of the kudzu IspS gene improved expression of the isoprene synthase in Synechocystis. Use of the photosynthesis psbA2 promoter, to drive the expression of the IspS gene, resulted in a light-intensity-dependent isoprene synthase expression. Results showed that oxygenic photosynthesis can be re-directed to generate useful small volatile hydrocarbons, while consuming CO₂, without a prior requirement for the harvesting, dewatering and processing of the respective biomass.     

Genetically engineering cyanobacteria to convert CO2, water,and light into the long-chain hydrocarbon farnesene

Genetically engineered cyanobacteria offer a shortcut to convert CO₂ and H₂O directly into biofuels and high value chemicals for societal benefits. Farnesene, a long-chained hydrocarbon (C₁₅H₂₄), has many applications in lubricants, cosmetics, fragrances, and biofuels. However, a method for the sustainable, photosynthetic production of farnesene has been lacking. Here, we report the photosynthetic production of farnesene by the filamentous cyanobacterium Anabaena sp. PCC 7120 using only CO₂, mineralized water, and light. A codon-optimized farnesene synthase gene was chemically synthesized and then expressed in the cyanobacterium, enabling it to synthesize farnesene through its endogenous non-mevalonate (MEP) pathway. Farnesene excreted from the engineered cyanobacterium volatilized into the flask head space and was recovered by adsorption in a resin column. The maximum photosynthetic productivity of farnesene was 69.1?±?1.8 μg·L^?1·O.D.^?1·d^?1. Compared to the wild type, the farnesene-producing cyanobacterium also exhibited a 60 % higher PSII activity under high light, suggesting increased farnesene productivity in such conditions. We envision genetically engineered cyanobacteria as a bio-solar factory for photosynthetic production of a wide range of biofuels and commodity chemicals.     

Metabolic Engineering of Escherichia coli for Enhanced Production of (R)- and (S)-3-Hydroxybutyrate

Synthetic metabolic pathways have been constructed for the production of enantiopure (R)- and (S)-3-hydroxybutyrate (3HB) from glucose in recombinant Escherichia coli strains. To promote maximal activity, we profiled three thiolase homologs (BktB, Thl, and PhaA) and two coenzyme A (CoA) removal mechanisms (Ptb-Buk and TesB). Two enantioselective 3HB-CoA dehydrogenases, PhaB, producing the (R)-enantiomer, and Hbd, producing the (S)-enantiomer, were utilized to control the 3HB chirality across two E. coli backgrounds, BL21Star(DE3) and MG1655(DE3), representing E. coli B- and K-12-derived strains, respectively. MG1655(DE3) was found to be superior for the production of each 3HB stereoisomer, although the recombinant enzymes exhibited lower in vitro specific activities than BL21Star(DE3). Hbd in vitro activity was significantly higher than PhaB activity in both strains. The engineered strains achieved titers of enantiopure (R)-3HB and (S)-3HB as high as 2.92 g liter⁻¹ and 2.08 g liter⁻¹, respectively, in shake flask cultures within 2 days. The NADPH/NADP⁺ ratio was found to be two- to three-fold higher than the NADH/NAD⁺ ratio under the culture conditions examined, presumably affecting in vivo activities of PhaB and Hbd and resulting in greater production of (R)-3HB than (S)-3HB. To the best of our knowledge, this study reports the highest (S)-3HB titer achieved in shake flask E. coli cultures to date.The synthesis of chiral molecules is of significant interest in the pharmaceutical industry because frequently one stereoisomer of a drug has efficacy while the other has either substantially reduced or no activity or may even have adverse effects (20, 23). Additionally, chiral molecules serve as building blocks for many pharmaceuticals and high-value compounds. Thus, the ability to prepare chiral molecules with high optical purity is important. Stereoselective chemical processes generally employ expensive chiral catalysts, require harsh physical conditions and solvents, and suffer from extensive by-product formation. In contrast, enzyme-catalyzed reactions are highly stereoselective and can be performed in aqueous solutions under mild conditions (21). As a result, the use of biological processes for chiral molecule production has been extensively investigated (4, 28, 32, 36). One example of such a process is the biosynthesis of 3-hydroxybutyric acid (3HB), a versatile chiral molecule containing one hydroxyl group and one carboxyl group, used as a building block for the synthesis of optically active fine chemicals, such as vitamins, antibiotics, pheromones, and flavor compounds (5, 6, 18, 27).The biosynthesis of 3HB has typically been achieved by two different mechanisms: depolymerization (in vitro or in vivo) of microbially synthesized poly-(R)-3-hydroxybutyric acid (PHB) (8, 13) or direct synthesis of 3HB without a PHB intermediate (9, 12, 15). However, due to the stereospecific constraints of PHB synthesis, in which polymers are composed exclusively of (R)-3HB monomer units, the synthesis of (S)-3HB from PHB is effectively impossible. In contrast, direct synthesis of both enantiopure (R)-3HB and (S)-3HB is possible. Pathways facilitating (R)-3HB synthesis have been constructed in Escherichia coli by simultaneous expression of phaA (encoding acetoacetyl coenzyme A [CoA] thiolase) and phaB [encoding (R)-3HB-CoA dehydrogenase] from Ralstonia eutropha H16, and ptb (encoding phosphotransbutyrylase) and buk (encoding butyrate kinase) from Clostridium acetobutylicum ATCC 824 (9). In addition to the use of ptb and buk to catalyze the conversion of (R)-3HB-CoA to (R)-3HB, tesB (encoding thioesterase II from E. coli) has also been used for the direct hydrolysis of (R)-3HB-CoA to yield (R)-3HB (15). The production of (S)-3HB in E. coli has recently been reported using a biosynthetic pathway consisting of phaA from R. eutropha H16, hbd [encoding (S)-3HB-CoA dehydrogenase] from C. acetobutylicum ATCC 824, and bch (encoding 3-hydroxyisobutyryl-CoA hydrolase) from Bacillus cereus ATCC 14579 (12).In E. coli, the synthesis of both enantiomers of 3HB begins with the condensation of two molecules of acetyl-CoA, catalyzed by a thiolase, to give acetoacetyl-CoA (Fig. ​(Fig.1).1). The acetoacetyl-CoA is then reduced either to (R)-3HB-CoA via ketone reduction mediated by an NADPH-dependent (R)-3HB-CoA dehydrogenase (PhaB) or to (S)-3HB-CoA via an NADH-dependent (S)-3-HB-CoA dehydrogenase (Hbd). (R)-3HB-CoA and (S)-3HB-CoA can each be further modified via a suitable CoA removal reaction to form (R)-3HB and (S)-3HB, respectively. In an effort to increase chiral 3HB production, it is essential to identify a thiolase capable of efficiently catalyzing the first reaction in the 3HB biosynthetic pathways, to draw acetyl-CoA from competing endogenous pathways. Thus, we examined three different thiolases (BktB and PhaA from R. eutropha H16 and Thl from C. acetobutylicum ATCC 824) to determine which is most proficient for 3HB synthesis. (R)-3HB-CoA and (S)-3HB-CoA synthesized via the reduction reaction catalyzed by PhaB and Hbd, respectively, must be converted to their respective free acid forms before transport or diffusion out of the cell. We have compared two sets of CoA-removing enzyme mechanisms, including the phosphotransbutyrylase (Ptb) and butyrate kinase (Buk) system encoded by the ptb-buk operon from C. acetobutylicum ATCC 824 and acyl-CoA thioesterase II (TesB) from E. coli MG1655. Moreover, it has long been argued whether B strains or K-12 strains of E. coli would serve as better hosts for the biosynthesis of small molecules. Microarrays and Northern blot analyses have suggested that several metabolic pathways, including the tricarboxylic acid (TCA) cycle, glyoxylate shunt, glycolysis, and fatty acid degradation are different between these two strains (22, 25, 34, 35), implying that they may differ significantly in their abilities to supply significant levels of acetyl-CoA as the precursor for 3HB synthesis. Thus, we have also compared 3HB synthesis across two representative E. coli strains: BL21Star(DE3) (B strain) and MG1655(DE3) (K-12 strain). 3HB chirality was examined and verified by high-performance liquid chromatography (HPLC) analysis using a chiral stationary phase to provide separation.Open in a separate windowFIG. 1.Schematic representation of (S)-3HB or (R)-3HB synthesis from glucose in engineered E. coli. BktB, acetoacetyl-CoA thiolase from R. eutropha H16; Thl, acetoacetyl-CoA thiolase from C. acetobutylicum ATCC 824; PhaA, acetoacetyl-CoA thiolase from R. eutropha H16; Hbd, (S)-3HB-CoA dehydrogenase from C. acetobutylicum ATCC 824; PhaB, (R)-3HB-CoA dehydrogenase from R. eutropha H16; Ptb, phosphotransbutyrylase from C. acetobutylicum ATCC 824; Buk, butyrate kinase from C. acetobutylicum ATCC 824; TesB, acyl-CoA thioesterase II from E. coli MG1655.Altogether, we have explored the production of each stereoisomer of 3HB across different strains of E. coli, different thiolases, and different CoA removal systems to engineer E. coli strains for enhanced chiral 3HB production.     

Ethoxyzolamide Inhibition of CO(2)-Dependent Photosynthesis in the Cyanobacterium Synechococcus PCC7942

Cells of the cyanobacterium, Synechococcus PCC7942, grown under high inorganic carbon (C_i) conditions (1% CO₂; pH 8) were found to be photosynthetically dependent on exogenous CO₂. This was judged by the fact that they had a similar photosynthetic affinity for CO₂ (K_0.5[CO₂] of 3.4-5.4 micromolar) over the pH range 7 to 9 and that the low photosynthetic affinity for C_i measured in dense cell suspensions was improved by the addition of exogenous carbonic anhydrase (CA). The CA inhibitor, ethoxyzolamide (EZ), was shown to reduce photosynthetic affinity for CO₂ in high C_i cells. The addition of 200 micromolar EZ to high C_i cells increased K_0.5(CO₂) from 4.6 micromolar to more than 155 micromolar at pH 8.0, whereas low C_i cells (grown at 30 microliters CO₂ per liter of air) were less sensitive to EZ. EZ inhibition in high and low C_i cells was largely relieved by increasing exogenous C_i up to 100 millimolar. Lipid soluble CA inhibitors such as EZ and chlorazolamide were shown to be the most effective inhibitors of CO₂ usage, whereas water soluble CA inhibitors such as methazolamide and acetazolamide had little or no effect. EZ was found to cause a small drop in photosystem II activity, but this level of inhibition was not sufficient to explain the large effect that EZ had on CO₂ usage. High C_i cells of Anabaena variabilis M3 and Synechocystis PCC6803 were also found to be sensitive to 200 micromolar EZ. We discuss the possibility that the inhibitory effect of EZ on CO₂ usage in high C_i cells of Synechococcus PCC7942 may be due to inhibition of a `CA-like' function associated with the CO₂ utilizing C_i pump or due to inhibition of an internal CA activity, thus affecting CO₂ supply to ribulose bisphosphate carboxylase-oxygenase.     

The Cyanobacterium Synechocystis sp. Strain PCC 6803 Expresses a DNA Methyltransferase Specific for the Recognition Sequence of the Restriction Endonuclease PvuI

By use of restriction endonucleases, the DNA of the cyanobacterium Synechocystis sp. strain PCC 6803 was analyzed for DNA-specific methylation. Three different recognition sites of methyltransferases, a dam-like site including N⁶-methyladenosine and two other sites with methylcytosine, were identified, whereas no activities of restriction endonucleases could be detected in this strain. slr0214, a Synechocystis gene encoding a putative methyltransferase that shows significant similarities to C⁵-methylcytosine-synthesizing enzymes, was amplified by PCR and cloned for further characterization. Mutations in slr0214 were generated by the insertion of an aphII gene cassette. Analyses of chromosomal DNAs of such mutants demonstrated that the methylation pattern was changed. The recognition sequence of the methyltransferase was identified as 5′-CGATCG-3′, corresponding to the recognition sequence of PvuI. The specific methyltransferase activity was significantly reduced in protein extracts obtained from mutant cells. Mutation of slr0214 also led to changed growth characteristics of the cells compared to wild-type cells. These alterations led to the conclusion that the methyltransferase Slr0214 might play a regulatory role in Synechocystis. The Slr0214 protein was also overexpressed in Escherichia coli, and the purified protein demonstrated methyltransferase activity and specificity for PvuI recognition sequences in vitro. We propose the designation SynMI (Synechocystis methyltransferase I) for the slr0214-encoded enzyme.     

Reconstruction and analysis of genome-scale metabolic model of a photosynthetic bacterium

Background  

Synechocystis sp. PCC6803 is a cyanobacterium considered as a candidate photo-biological production platform - an attractive cell factory capable of using CO₂ and light as carbon and energy source, respectively. In order to enable efficient use of metabolic potential of Synechocystis sp. PCC6803, it is of importance to develop tools for uncovering stoichiometric and regulatory principles in the Synechocystis metabolic network.     

Identification of a plausible biosynthetic enzyme for the IM-2-type autoregulator in Streptomyces antibioticus

Virginiae butanolides (VBs) and IM-2 are members of Streptomyces hormones called ‘butyrolactone autoregulators’ which regulate the antibiotic production in Streptomyces species at nanomolar concentrations. Cell-free extract of a VB-A overproducer, Streptomyces antibioticus NF-18, is capable of catalyzing the final step of the autoregulator biosynthesis, namely, the NADPH-dependent reduction of 6-dehydroVB-A. However, physico-chemical analyses of the purified enzymatic products revealed that, in addition to the VB-type isomer [(2R,3R,6S)-enantiomer], IM-2-type isomers [(2R,3R,6R)- and (2S,3S,6S)-enantiomers] were also produced from (±)-6-dehydroVB-A, suggesting the existence of several 6-dehydroVB-A reductases with respective stereoselectivities. The reductase activity of the crude extracts was separated into two activity peaks, peak I (major) and peak II (minor), by DEAE-5PW HPLC. Chiral HPLC analyses demonstrated that peak I enzyme and peak II enzyme catalyzed the production of (2R,3R,6S), (2R,3R,6R) and (2S,3S,6S) isomers at ratios of 46:1:3.2 and 4.9:1:1.5, respectively, indicating clearly that S. antibioticus NF-18 possesses at least two 6-dehydroVB-A reductases: one much favored toward VB-A biosynthesis, the other with relaxed stereoselectivity capable of synthesizing both VB-type and IM-2-type autoregulators.     

Efficient (R)-3-hydroxybutyrate production using acetyl CoA-regenerating pathway catalyzed by coenzyme A transferase

(R)-3-hydroxybutyrate [(R)-3HB] is a useful precursor in the synthesis of value-added chiral compounds such as antibiotics and vitamins. Typically, (R)-3HB has been microbially produced from sugars via modified (R)-3HB-polymer-synthesizing pathways in which acetyl CoA is converted into (R)-3-hydroxybutyryl-coenzyme A [(R)-3HB-CoA] by β-ketothiolase (PhaA) and acetoacetyl CoA reductase (PhaB). (R)-3HB-CoA is hydrolyzed into (R)-3HB by modifying enzymes or undergoes degradation of the polymerized product. In the present study, we constructed a new (R)-3HB-generating pathway from glucose by using propionyl CoA transferase (PCT). This pathway was designed to excrete (R)-3HB by means of a PCT-catalyzed reaction coupled with regeneration of acetyl CoA, the starting substance for synthesizing (R)-3HB-CoA. Considering the equilibrium reaction of PCT, the PCT-catalyzed (R)-3HB production would be expected to be facilitated by the addition of acetate since it acts as an acceptor of CoA. As expected, the engineered Escherichia coli harboring the phaAB and pct genes produced 1.0 g?L^?1 (R)-3HB from glucose, and with the addition of acetate into the medium, the concentration was increased up to 5.2 g?L^?1, with a productivity of 0.22 g?L^?1 h^?1. The effectiveness of the extracellularly added acetate was evaluated by monitoring the conversion of ¹³C carbonyl carbon-labeled acetate into (R)-3HB using gas chromatography/mass spectrometry. The enantiopurity of (R)-3HB was determined to be 99.2% using chiral liquid chromatography. These results demonstrate that the PCT pathway achieved a rapid co-conversion of glucose and acetate into (R)-3HB.     

Identification of Genes Essential for Growth at High Salt Concentrations Using Salt-Sensitive Mutants of the Cyanobacterium Synechocystis sp. Strain PCC 6803

A collection of 17 salt-sensitive mutants of the cyanobacterium Synechocystis sp. strain PCC 6803 was obtained by random cartridge mutagenesis. The genes coding for proteins essential for growth at high salt concentrations were mapped on the completely known genome sequence of this strain. The two genes coding for enzymes involved in biosynthesis of the osmolyte glucosylglycerol were affected in nine mutants. Two mutants defective in a glycoprotease encoding gene gcp showed a reduced salt resistance. Four genes were identified not previously known to be essential for salt tolerance in cyanobacteria. These genes (slr1799, slr1087, sll1061, and sll1062) code for proteins not yet functionally characterized. Received: 21 May 2001 / Accepted: 27 June 2001     

Respiratory terminal oxidases alleviate photo-oxidative damage in photosystem I during repetitive short-pulse illumination in <Emphasis Type="Italic">Synechocystis</Emphasis> sp. PCC 6803

Oxygenic phototrophs are vulnerable to damage by reactive oxygen species (ROS) that are produced in photosystem I (PSI) by excess photon energy over the demand of photosynthetic CO₂ assimilation. In plant leaves, repetitive short-pulse (rSP) illumination produces ROS to inactivate PSI. The production of ROS is alleviated by oxidation of the reaction center chlorophyll in PSI, P700, during the illumination with the short-pulse light, which is supported by flavodiiron protein (FLV). In this study, we found that in the cyanobacterium Synechocystis sp. PCC 6803 P700 was oxidized and PSI was not inactivated during rSP illumination even in the absence of FLV. Conversely, the mutant deficient in respiratory terminal oxidases was impaired in P700 oxidation during the illumination with the short-pulse light to suffer from photo-oxidative damage in PSI. Interestingly, the other cyanobacterium Synechococcus sp. PCC 7002 could not oxidize P700 without FLV during rSP illumination. These data indicate that respiratory terminal oxidases are critical to protect PSI from ROS damage during rSP illumination in Synechocystis sp. PCC 6803 but not Synechococcus sp. PCC 7002.     

Biosynthesis of flavan-3-ols and other secondary plant products from (2S)phenylalanine

(2S)-Phenyl[2-¹⁴C,3R-³H₁]alanine and (2S)-phenyl[2-¹⁴C,3S-³H₁]alanine have been employed as substrates to study procyanidin and flavan-3-ol biosynthesis. Parallel studies with the cyanogenic glucosides prunasin and sambunigrin, Winterstein's acid [(3R)-3-dimethylaminophenylpropionic acid] and tropic acid show these to be derived by stereospecific processes from (2S)-phenylalanine. New proposals for procyanidin biosynthesis are briefly commented upon.     

Characterisation of CO2 and HCO3 − uptake in the cyanobacterium Synechocystis sp. PCC6803

The availability of a complete genome database for the cyanobacterium Synechocystissp. PCC6803 (glucose-tolerant strain) has raised expectations that this organism would become a reference strain for work aimed at understanding the CO₂-concentrating mechanism (CCM) in cyanobacteria. However, the amount of physiological data available has been relatively limited. In this report we provide data on the relative contributions of net HCO₃ ⁻ uptake and CO₂ uptake under steady state photosynthetic conditions. Cells were compared after growth at high CO₂ (2% v/v in air) or limiting CO₂ conditions (20 ppm CO₂). Synechocystishas a very high dependence on net HCO₃ ⁻ uptake at low to medium concentrations of inorganic carbon (Ci). At high Ci concentrations net CO₂ uptake became more important but did not contribute more than 40% to the rate of photosynthetic O₂ evolution. The data also confirm that high Ci cells of Synechocystissp. PCC6803 possess a strong capacity for net HCO₃ ⁻ uptake under steady state photosynthetic conditions. Time course experiments show that induction of maximal Ci uptake capacity on a shift from high CO₂ to low CO₂ conditions was near completion by four hours. By contrast, relaxation of the induced state on return of cells to high CO₂, takes in excess of 230 h. Experiments were conducted to determine if Synechocystissp. PCC6803 is able to exhibit a `fast induction' response under severe Ci limitation and whether glucose was capable of causing a rapid inactivation in Ci uptake capacity. Clear evidence for either response was not found. This revised version was published online in August 2006 with corrections to the Cover Date.     

Combinatorial assembly platform enabling engineering of genetically stable metabolic pathways in cyanobacteria

Cyanobacteria are simple, efficient, genetically-tractable photosynthetic microorganisms which in principle represent ideal biocatalysts for CO₂ capture and conversion. However, in practice, genetic instability and low productivity are key, linked problems in engineered cyanobacteria. We took a massively parallel approach, generating and characterising libraries of synthetic promoters and RBSs for the cyanobacterium Synechocystis sp. PCC 6803, and assembling a sparse combinatorial library of millions of metabolic pathway-encoding construct variants. Genetic instability was observed for some variants, which is expected when variants cause metabolic burden. Surprisingly however, in a single combinatorial round without iterative optimisation, 80% of variants chosen at random and cultured photoautotrophically over many generations accumulated the target terpenoid lycopene from atmospheric CO₂, apparently overcoming genetic instability. This large-scale parallel metabolic engineering of cyanobacteria provides a new platform for development of genetically stable cyanobacterial biocatalysts for sustainable light-driven production of valuable products directly from CO₂, avoiding fossil carbon or competition with food production.     

Paradigm of Monoterpene (β-phellandrene) Hydrocarbons Production via Photosynthesis in Cyanobacteria

A direct “photosynthesis-to-fuels” approach envisions application of a single organism, absorbing sunlight, photosynthesizing, and converting the primary products of photosynthesis into ready-made fuel. The work reported here applied this concept for the photosynthetic generation of monoterpene (β-phellandrene) hydrocarbons in the unicellular cyanobacteria Synechocystis sp. PCC 6803. Heterologous expression of a codon-optimized Lavandula angustifolia β-phellandrene synthase (β-PHLS) gene in Synechocystis enabled photosynthetic generation of β-phellandrene in these microorganisms. β-phellandrene accumulation occurred constitutively and in tandem with biomass accumulation, generated from sunlight, CO₂, and H₂O. Results showed that β-phellandrene diffused through the plasma membrane and cell wall of the cyanobacteria and accumulated on the surface of the liquid culture. Spontaneous β-phellandrene separation from the biomass and its removal from the liquid phase alleviated product inhibition of cellular metabolism and enabled a continuous production process. The work showed that oxygenic photosynthesis can be directed to generate monoterpene hydrocarbons, while consuming CO₂, without a prior requirement for the harvesting, dewatering, and processing of the respective biomass.     

Identification of two genes, sll0804 and slr1306, as putative components of the CO2-concentrating mechanism in the cyanobacterium Synechocystis sp. strain PCC 6803

Insertional transposon mutations in the sll0804 and slr1306 genes were found to lead to a loss of optimal photoautotrophy in the cyanobacterium Synechocystis sp. strain PCC 6803 grown under ambient CO₂ concentrations (350 ppm). Mutants containing these insertions (4BA2 and 3ZA12, respectively) could grow photoheterotrophically on glucose or photoautotrophically at elevated CO₂ concentrations (50,000 ppm). Both of these mutants exhibited an impaired affinity for inorganic carbon. Consequently, the Sll0804 and Slr1306 proteins appear to be putative components of the carbon-concentrating mechanism in Synechocystis sp. strain PCC 6803.     

Zur biogenese des aza-oxa-spiran-systems der steroidalkaloide vom spirosolan-typ in Solanaceen

5α,6-³H₂-Solacongestidine and 5α,6-³ H₂-(22S)-dihydrosolacongestidine administered to Solanum dulcamara as well as 16-³H₂-(22S: 25R)-22,26-epimino- cholest-5-en-3β-ol (25-isodihydroverazine) and 7α-³H-(22S: 25R)-22,26-epimino-cholest-5-en-3β,16β-diol administered to Solanum laciniatum were converted to coladulcidine and solasodine, respectively. These results are discussed in relation to spirosolane alkaloid biosynthesis.