Production of Fatty Acids and Derivatives Using Cyanobacteria.

Fatty acids and their derivatives are highly valuable chemicals that can be produced through chemical or enzymatic processes using plant lipids. This may compete with human food sources. Therefore, there has been an urge to create a new method for synthesizing these chemicals. One approach is to use microbial cells, specifically cyanobacteria, as a factory platform. Engineering may need to be implemented in order to allow a cost-competitive production and to enable a production of a variety of different fatty acids and derivatives. In this chapter, we explain in details the importance of fatty acids and their derivatives, including fatty aldehydes, fatty alcohols, hydrocarbons, fatty acid methyl esters, and hydroxy fatty acids. The production of these chemicals using cyanobacterial native metabolisms together with strategies to engineer them are also explained. Moreover, recent examples of fatty acid and fatty acid derivative production from engineered cyanobacteria are gathered and reported. Commercial opportunities to manufacture fatty acids and derivatives are also discussed in this chapter. Altogether, it is clear that fatty acids and their derivatives are important chemicals, and with recent advancements in genetic engineering, a cyanobacterial platform for bio-based production is feasible. However, there are regulations and guidelines in place for the use of genetically modified organisms (GMOs) and some further developments are still needed before commercialization can be reached.

A Plasmid-Based Fluorescence Reporter System for Monitoring Oxidative Damage in E. coli.

Quantitating intracellular oxidative damage caused by reactive oxygen species (ROS) is of interest in many fields of biological research. The current systems primarily rely on supplemented oxygen-sensitive substrates that penetrate the target cells, and react with ROS to produce signals that can be monitored with spectroscopic or imaging techniques. The objective here was to design a new non-invasive analytical strategy for measuring ROS-induced damage inside living cells by taking advantage of the native redox sensor system of E. coli. The developed plasmid-based sensor relies on an oxygen-sensitive transcriptional repressor IscR that controls the expression of a fluorescent marker in vivo. The system was shown to quantitatively respond to oxidative stress induced by supplemented H2O2 and lowered cultivation temperatures. Comparative analysis with fluorescence microscopy further demonstrated that the specificity of the reporter system was equivalent to the commercial chemical probe (CellROX). The strategy introduced here is not dependent on chemical probes, but instead uses a fluorescent expression system to detect enzyme-level oxidative damage in microbial cells. This provides a cheap and simple means for analysing enzyme-level oxidative damage in a biological context in E. coli.

Synthetic metabolic pathways for conversion of CO2 into secreted short-to medium-chain hydrocarbons using cyanobacteria.

The objective of this study was to implement direct sunlight-driven conversion of CO2 into a naturally excreted ready-to-use fuel. We engineered four different synthetic metabolic modules for biosynthesis of short-to medium-chain length hydrocarbons in the model cyanobacterium Synechocystis sp. PCC 6803. In module 1, the combination of a truncated clostridial n-butanol pathway with over-expression of the native cyanobacterial aldehyde deformylating oxygenase resulted in small quantities of propane when cultured under closed conditions. Direct conversion of CO2 into propane was only observed in strains with CRISPRi-mediated repression of three native putative aldehyde reductases. In module 2, three different pathways towards pentane were evaluated based on the polyunsaturated fatty acid linoleic acid as an intermediate. Through combinatorial evaluation of reaction ingredients, it was concluded that linoleic acid undergoes a spontaneous non-enzymatic reaction to yield pentane and hexanal. When Synechocystis was added to the reaction, hexanal was converted into 1-hexanol, but there was no further stimulation of pentane biosynthesis even in the Synechocystis strains expressing GmLOX1. For modules 3 and 4, several different acyl-ACP thioesterases were evaluated in combination with two different decarboxylases. Small quantities of 1-heptene and 1-nonene were observed in strains expressing the desaturase-like enzyme UndB from Pseudomonas mendocina in combination with C8-C10 preferring thioesterases ('CaFatB3.5 and 'ChoFatB2.2). When UndB instead was combined with a C12-specific 'UcFatB1 thioesterase, this resulted in a ten-fold increase of alkene biosynthesis. When UndB was replaced with the light-dependent FAP decarboxylase, both undecane and tridecane accumulated, albeit with a 10-fold drop in productivity. Preliminary optimization of the RBS, promoter and gene order in some of the synthetic operons resulted in improved 1-alkene productivity, reaching a titer of 230 mg/L after 10 d with 15% carbon partitioning. In conclusion, the direct bioconversion of CO2 into secreted and ready-to-use hydrocarbon fuel was implemented with several different metabolic systems. Optimal productivity was observed with UndB and a C12 chain-length specific thioesterase, although further optimization of the entire biosynthetic system is still possible.

Computational Enzyme Engineering Pipelines for Optimized Production of Renewable Chemicals.

To enable a sustainable supply of chemicals, novel biotechnological solutions are required that replace the reliance on fossil resources. One potential solution is to utilize tailored biosynthetic modules for the metabolic conversion of CO2 or organic waste to chemicals and fuel by microorganisms. Currently, it is challenging to commercialize biotechnological processes for renewable chemical biomanufacturing because of a lack of highly active and specific biocatalysts. As experimental methods to engineer biocatalysts are time- and cost-intensive, it is important to establish efficient and reliable computational tools that can speed up the identification or optimization of selective, highly active, and stable enzyme variants for utilization in the biotechnological industry. Here, we review and suggest combinations of effective state-of-the-art software and online tools available for computational enzyme engineering pipelines to optimize metabolic pathways for the biosynthesis of renewable chemicals. Using examples relevant for biotechnology, we explain the underlying principles of enzyme engineering and design and illuminate future directions for automated optimization of biocatalysts for the assembly of synthetic metabolic pathways.

Improved Bioproduction of 1-Octanol Using Engineered Synechocystis sp. PCC 6803.

1-Octanol has gained interest as a chemical precursor for both high and low value commodities including fuel, solvents, surfactants, and fragrances. By harnessing the power from sunlight and CO2 as carbon source, cyanobacteria has recently been engineered for renewable production of 1-octanol. The productivity, however, remained low. In the present work, we report efforts to further improve the 1-octanol productivity. Different N-terminal truncations were evaluated on three thioesterases from different plant species, resulting in several candidate thioesterases with improved activity and selectivity toward octanoyl-ACP. The structure/function trials suggest that current knowledge and/or state-of-the art computational tools are insufficient to determine the most appropriate cleavage site for thioesterases in Synechocystis. Additionally, by tuning the inducer concentration and light intensity, we further improved the 1-octanol productivity, reaching up to 35% (w/w) carbon partitioning and a titer of 526 ± 5 mg/L 1-octanol in 12 days. Long-term cultivation experiments demonstrated that the improved strain can be stably maintained for at least 30 days and/or over ten times serial dilution. Surprisingly, the improved strain was genetically stable in contrast to earlier strains having lower productivity (and hence a reduced chance of reaching toxic product concentrations). Altogether, improved enzymes and environmental conditions (e.g., inducer concentration and light intensity) substantially increased the 1-octanol productivity. When cultured under continuous conditions, the bioproduction system reached an accumulative titer of >3.5 g/L 1-octanol over close to 180 days.

Calm on the surface, dynamic on the inside. Molecular homeostasis of Anabaena sp. PCC 7120 nitrogen metabolism.

Nitrogen sources are all converted into ammonium/ia as a first step of assimilation. It is reasonable to expect that molecular components involved in the transport of ammonium/ia across biological membranes connect with the regulation of both nitrogen and central metabolism. We applied both genetic (i.e., Δamt mutation) and environmental treatments to a target biological system, the cyanobacterium Anabaena sp PCC 7120. The aim was to both perturb nitrogen metabolism and induce multiple inner nitrogen states, respectively, followed by targeted quantification of key proteins, metabolites and enzyme activities. The absence of AMT transporters triggered a substantial whole-system response, affecting enzyme activities and quantity of proteins and metabolites, spanning nitrogen and carbon metabolisms. Moreover, the Δamt strain displayed a molecular fingerprint indicating nitrogen deficiency even under nitrogen replete conditions. Contrasting with such dynamic adaptations was the striking near-complete lack of an externally measurable altered phenotype. We conclude that this species evolved a highly robust and adaptable molecular network to maintain homeostasis, resulting in substantial internal but minimal external perturbations. This analysis provides evidence for a potential role of AMT transporters in the regulatory/signalling network of nitrogen metabolism and the existence of a novel fourth regulatory mechanism controlling glutamine synthetase activity.

In silico co-factor balance estimation using constraint-based modelling informs metabolic engineering in Escherichia coli.

In the growing field of metabolic engineering, where cells are treated as 'factories' that synthesize industrial compounds, it is essential to consider the ability of the cells' native metabolism to accommodate the demands of synthetic pathways, as these pathways will alter the homeostasis of cellular energy and electron metabolism. From the breakdown of substrate, microorganisms activate and reduce key co-factors such as ATP and NAD(P)H, which subsequently need to be hydrolysed and oxidized, respectively, in order to restore cellular balance. A balanced supply and consumption of such co-factors, here termed co-factor balance, will influence biotechnological performance. To aid the strain selection and design process, we used stoichiometric modelling (FBA, pFBA, FVA and MOMA) and the Escherichia coli (E.coli) core stoichiometric model to investigate the network-wide effect of butanol and butanol precursor production pathways differing in energy and electron demand on product yield. An FBA-based co-factor balance assessment (CBA) algorithm was developed to track and categorise how ATP and NAD(P)H pools are affected in the presence of a new pathway. CBA was compared to the balance calculations proposed by Dugar et al. (Nature Biotechnol. 29 (12), 1074-1078). Predicted solutions were compromised by excessively underdetermined systems, displaying greater flexibility in the range of reaction fluxes than experimentally measured by 13C-metabolic flux analysis (MFA) and the appearance of unrealistic futile co-factor cycles. With the assumption that futile cycles are tightly regulated in reality, the FBA models were manually constrained in a step-wise manner. Solutions with minimal futile cycling diverted surplus energy and electrons towards biomass formation. As an alternative, the use of loopless FBA or constraining the models with measured flux ranges were tried but did not prevent futile co-factor cycles. The results highlight the need to account for co-factor imbalance and confirm that better-balanced pathways with minimal diversion of surplus towards biomass formation present the highest theoretical yield. The analysis also suggests that ATP and NAD(P)H balancing cannot be assessed in isolation from each other, or even from the balance of additional co-factors such as AMP and ADP. We conclude that, through revealing the source of co-factor imbalance CBA can facilitate pathway and host selection when designing new biocatalysts for implementation by metabolic engineering.

Bioderivatization as a concept for renewable production of chemicals that are toxic or poorly soluble in the liquid phase.

Bio-based production technologies may complement or replace petroleum-based production of chemicals, but they face a number of technical challenges, including product toxicity and/or water insolubility. Plants and microorganisms naturally biosynthesize chemicals that often are converted into derivatives with reduced toxicity or enhanced solubility. Inspired by this principle, we propose a bioderivatization strategy for biotechnological chemicals production, defined as purposeful biochemical derivatization of intended target molecules. As proof of principle, the effects of hydrophobic (e.g., esterification) and hydrophilic (e.g., glycosylation) bioderivatization strategies on the biosynthesis of a relatively toxic and poorly soluble chemical, 1-octanol, were evaluated in Escherichia coli and Synechocystis sp. PCC 6803. The 1-octanol pathway was first optimized to reach product titers at which the host displayed symptoms of toxicity. Solvent overlay used to capture volatile products partially masked product toxicity. Regardless of whether solvent overlay was used, most strains with bioderivatization had a higher molar product titer and product yield, as well as improved cellular growth and glucose consumption, compared with strains without bioderivatization. The positive effect on bioproduction was observed with both the hydrophobic and hydrophilic strategies. Interestingly, in several combinations of genotype/induction strength, bioderivatization had a positive effect on productivity without any apparent effect on growth. We attribute this to enhanced product solubility in the aqueous or solvent fraction of the bioreactor liquid phase (depending on the derivative and medium used), with consequent enhanced product removal. Overall, under most conditions, a benefit of bioproduction was observed, and the bioderivatization strategy could be considered for other similar chemicals as well.

Methanol-free biosynthesis of fatty acid methyl ester (FAME) in Synechocystis sp. PCC 6803.

To meet the increasing global demand of biodiesel over the next decades, alternative methods for producing one of the key constituents of biodiesel (e.g. fatty acid methyl esters (FAMEs)) are needed. Algal biodiesel has been a long-term target compromised by excessive costs for harvesting and processing. In this work, we engineered cyanobacteria to convert carbon dioxide into excreted FAME, without requiring methanol as a methyl donor. To produce FAME, acyl-ACP, a product of the fatty acid biosynthesis pathway, was first converted into free fatty acid (FFA) by a thioesterase, namely 'UcFatB1 from Umbellularia californica. Next, by employing a juvenile hormone acid O-methyltransferase (DmJHAMT) from Drosophila melanogaster and S-adenosylmethionine (SAM) as a methyl donor, FFAs were converted into corresponding FAMEs. The esters were naturally secreted extracellularly, allowing simple product separation by solvent overlay as opposed to conventional algae biodiesel production where the algae biomass must first be harvested and processed for transesterification of extracted triacylglycerols (TAGs). By optimizing both the promoter and RBS elements, up to 120 mg/L of FAMEs were produced in 10 days. Quantification of key proteins and metabolites, together with constructs over-expressing SAM synthetase (MetK), indicated that 'UcFatB1, MetK, and DmJHAMT were the main factors limiting pathway flux. In order to solve the latter limitation, two reconstructed ancestral sequences of DmJHAMT were also tried, resulting in strains showing a broader methyl ester chain-length profile in comparison to the native DmJHAMT. Altogether, this work demonstrates a promising pathway for direct sunlight-driven conversion of CO2 into excreted FAME.

The effect of modulating the quantity of enzymes in a model ethanol pathway on metabolic flux in Synechocystis sp. PCC 6803.

Synthetic metabolism allows new metabolic capabilities to be introduced into strains for biotechnology applications. Such engineered metabolic pathways are unlikely to function optimally as initially designed and native metabolism may not efficiently support the introduced pathway without further intervention. To develop our understanding of optimal metabolic engineering strategies, a two-enzyme ethanol pathway consisting of pyruvate decarboxylase and acetaldehyde reductase was introduced into Synechocystis sp. PCC 6803. We characteriseda new set of ribosome binding site sequences in Synechocystis sp. PCC 6803 providing a range of translation strengths for different genes under test. The effect of ribosome-bindingsite sequence, operon design and modifications to native metabolism on pathway flux was analysed by HPLC. The accumulation of all introduced proteins was also quantified using selected reaction monitoring mass spectrometry. Pathway productivity was more strongly dependent on the accumulation of pyruvate decarboxylase than acetaldehyde reductase. In fact, abolishment of reductase over-expression resulted in the greatest ethanol productivity, most likely because strains harbouringsingle-gene constructs accumulated more pyruvate decarboxylase than strains carrying any of the multi-gene constructs. Overall, several lessons were learned. Firstly, the expression level of the first gene in anyoperon influenced the expression level of subsequent genes, demonstrating that translational coupling can also occur in cyanobacteria. Longer operons resulted in lower protein abundance for proximally-encoded cistrons. And, implementation of metabolic engineering strategies that have previously been shown to enhance the growth or yield of pyruvate dependent products, through co-expression with pyruvate kinase and/or fructose-1,6-bisphosphatase/sedoheptulose-1,7-bisphosphatase, indicated that other factors had greater control over growth and metabolic flux under the tested conditions.

Economic feasibility and long-term sustainability criteria on the path to enable a transition from fossil fuels to biofuels.

Currently the production of liquid biofuels relies on plant biomass, which in turn depends on the photosynthetic conversion of light and CO2 into chemical energy. As a consequence, the process is renewable on a far shorter time-scale than its fossil counterpart, thus rendering a potential to reduce the environmental impact of the transportation sector. However, the global economy is not intensively pursuing this route, as current generation biofuel production does not meet two key criteria: (1) economic feasibility and (2) long-term sustainability. Herein, we argue that microalgal systems are valuable alternatives to consider, although it is currently technologically immature and therefore not possible to reach criterion 1, nor evaluate criterion 2. In this review we discuss the major limiting factors for this technology and highlight how further research efforts could be deployed to concretize an industrial reality.

Synthetic metabolic pathways for photobiological conversion of CO2 into hydrocarbon fuel.

Liquid fuels sourced from fossil sources are the dominant energy form for mobile transport today. The consumption of fossil fuels is still increasing, resulting in a continued search for more sustainable methods to renew our supply of liquid fuel. Photosynthetic microorganisms naturally accumulate hydrocarbons that could serve as a replacement for fossil fuel, however productivities remain low. We report successful introduction of five synthetic metabolic pathways in two green cell factories, prokaryotic cyanobacteria and eukaryotic algae. Heterologous thioesterase expression enabled high-yield conversion of native fatty acyl-acyl carrier protein (ACP) into free fatty acids (FFA) in Synechocystis sp. PCC 6803 but not in Chlamydomonas reinhardtii where the polar lipid fraction instead was enhanced. Despite no increase in measurable FFA in Chlamydomonas, genetic recoding and over-production of the native fatty acid photodecarboxylase (FAP) resulted in increased accumulation of 7-heptadecene. Implementation of a carboxylic acid reductase (CAR) and aldehyde deformylating oxygenase (ADO) dependent synthetic pathway in Synechocystis resulted in the accumulation of fatty alcohols and a decrease in the native saturated alkanes. In contrast, the replacement of CAR and ADO with Pseudomonas mendocina UndB (so named as it is responsible for 1-undecene biosynthesis in Pseudomonas) or Chlorella variabilis FAP resulted in high-yield conversion of thioesterase-liberated FFAs into corresponding alkenes and alkanes, respectively. At best, the engineering resulted in an increase in hydrocarbon accumulation of 8- (from 1 to 8.5 mg/g cell dry weight) and 19-fold (from 4 to 77 mg/g cell dry weight) for Chlamydomonas and Synechocystis, respectively. In conclusion, reconstitution of the eukaryotic algae pathway in the prokaryotic cyanobacteria host generated the most effective system, highlighting opportunities for mix-and-match synthetic metabolism. These studies describe functioning synthetic metabolic pathways for hydrocarbon fuel synthesis in photosynthetic microorganisms for the first time, moving us closer to the commercial implementation of photobiocatalytic systems that directly convert CO2 into infrastructure-compatible fuels.

Photosynthesis-dependent biosynthesis of medium chain-length fatty acids and alcohols.

Cyanobacteria can directly channel atmospheric CO2 into a wide range of versatile carbon products such as fatty acids and fatty alcohols with applications including fuel, cosmetics, and health products. Works on alcohol production in cyanobacteria have so far focused on either long (C12-C18) or short (C2-C4) chain-length products. In the present work, we report the first synthetic pathway for 1-octanol (C8) biosynthesis in Synechocystis sp. PCC 6803, employing a carboxylic acid reductase and C8-preferring fatty acyl-ACP thioesterase. The first engineered strain produced 1-octanol but exhibited poor productivity and cellular health issues. We therefore proceeded to systematically optimize the strain and cultivation conditions in order to understand what the limiting factors were. The identification of optimal promoters and ribosomal binding sites, in combination with isopropyl myristate solvent overlay, resulted in a combined (C8-OH and C10-OH) titer of more than 100 mg/L (a 25-fold improvement relative to the first engineered strain) and a restoration of cellular health. Additionally, more than 905 mg/L 1-octanol was produced when the strain expressing sfp (phosphopantetheinyl transferase) and car (carboxylic acid reductase) was fed with octanoic acid. A combination of feeding experiments and protein quantification indicated that the supply of octanoic acid from the introduced thioesterase, and possibly also native fatty acid synthesis pathway, were the main bottlenecks of the pathway.

Finding novel relationships with integrated gene-gene association network analysis of Synechocystis sp. PCC 6803 using species-independent text-mining.

The increasing move towards open access full-text scientific literature enhances our ability to utilize advanced text-mining methods to construct information-rich networks that no human will be able to grasp simply from 'reading the literature'. The utility of text-mining for well-studied species is obvious though the utility for less studied species, or those with no prior track-record at all, is not clear. Here we present a concept for how advanced text-mining can be used to create information-rich networks even for less well studied species and apply it to generate an open-access gene-gene association network resource for Synechocystis sp. PCC 6803, a representative model organism for cyanobacteria and first case-study for the methodology. By merging the text-mining network with networks generated from species-specific experimental data, network integration was used to enhance the accuracy of predicting novel interactions that are biologically relevant. A rule-based algorithm (filter) was constructed in order to automate the search for novel candidate genes with a high degree of likely association to known target genes by (1) ignoring established relationships from the existing literature, as they are already 'known', and (2) demanding multiple independent evidences for every novel and potentially relevant relationship. Using selected case studies, we demonstrate the utility of the network resource and filter to (i) discover novel candidate associations between different genes or proteins in the network, and (ii) rapidly evaluate the potential role of any one particular gene or protein. The full network is provided as an open-source resource.

Overexpression of bifunctional fructose-1,6-bisphosphatase/sedoheptulose-1,7-bisphosphatase leads to enhanced photosynthesis and global reprogramming of carbon metabolism in Synechococcus sp. PCC 7002.

Cyanobacteria fix atmospheric CO2 to biomass and through metabolic engineering can also act as photosynthetic factories for sustainable productions of fuels and chemicals. The Calvin Benson cycle is the primary pathway for CO2 fixation in cyanobacteria, algae and C3 plants. Previous studies have overexpressed the Calvin Benson cycle enzymes, ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) and bifunctional sedoheptulose-1,7-bisphosphatase/fructose-1,6-bisphosphatase (hereafter BiBPase), in both plants and algae, although their impacts on cyanobacteria have not yet been rigorously studied. Here, we show that overexpression of BiBPase and RuBisCO have distinct impacts on carbon metabolism in the cyanobacterium Synechococcus sp. PCC 7002 through physiological, biochemical, and proteomic analyses. The former enhanced growth, cell size, and photosynthetic O2 evolution, and coordinately upregulated enzymes in the Calvin Benson cycle including RuBisCO and fructose-1,6-bisphosphate aldolase. At the same time it downregulated enzymes in respiratory carbon metabolism (glycolysis and the oxidative pentose phosphate pathway) including glucose-6-phosphate dehydrogenase (G6PDH). The content of glycogen was also significantly reduced while the soluble carbohydrate content increased. These results indicate that overexpression of BiBPase leads to global reprogramming of carbon metabolism in Synechococcus sp. PCC 7002, promoting photosynthetic carbon fixation and carbon partitioning towards non-storage carbohydrates. In contrast, whilst overexpression of RuBisCO had no measurable impact on growth and photosynthetic O2 evolution, it led to coordinated increase in the abundance of proteins involved in pyruvate metabolism and fatty acid biosynthesis. Our results underpin that singular genetic modifications in the Calvin Benson cycle can have far broader cellular impact than previously expected. These features could be exploited to more efficiently direct carbons towards desired bioproducts.

Synthetic metabolism: metabolic engineering meets enzyme design.

Metabolic engineering aims at modifying the endogenous metabolic network of an organism to harness it for a useful biotechnological task, for example, production of a value-added compound. Several levels of metabolic engineering can be defined and are the topic of this review. Basic 'copy, paste and fine-tuning' approaches are limited to the structure of naturally existing pathways. 'Mix and match' approaches freely recombine the repertoire of existing enzymes to create synthetic metabolic networks that are able to outcompete naturally evolved pathways or redirect flux toward non-natural products. The space of possible metabolic solution can be further increased through approaches including 'new enzyme reactions', which are engineered on the basis of known enzyme mechanisms. Finally, by considering completely 'novel enzyme chemistries' with de novo enzyme design, the limits of nature can be breached to derive the most advanced form of synthetic pathways. We discuss the challenges and promises associated with these different metabolic engineering approaches and illuminate how enzyme engineering is expected to take a prime role in synthetic metabolic engineering for biotechnology, chemical industry and agriculture of the future.

Pyridine nucleotide transhydrogenase PntAB is essential for optimal growth and photosynthetic integrity under low-light mixotrophic conditions in Synechocystis sp. PCC 6803.

Pyridine nucleotide transhydrogenase (PntAB) is an integral membrane protein complex participating in the regulation of NAD(P)+ :NAD(P)H redox homeostasis in various prokaryotic and eukaryotic organisms. In the present study we addressed the function and biological role of PntAB in oxygenic photosynthetic cyanobacteria capable of both autotrophic and heterotrophic growth, with support from structural three-dimensional (3D)-modeling. The pntA gene encoding the α subunit of heteromultimeric PntAB in Synechocystis sp. PCC 6803 was inactivated, followed by phenotypic and biophysical characterization of the ΔpntA mutant under autotrophic and mixotrophic conditions. Disruption of pntA resulted in phenotypic growth defects observed under low light intensities in the presence of glucose, whereas under autotrophic conditions the mutant did not differ from the wild-type strain. Biophysical characterization and protein-level analysis of the ΔpntA mutant revealed that the phenotypic defects were accompanied by significant malfunction and damage of the photosynthetic machinery. Our observations link the activity of PntAB in Synechocystis directly to mixotrophic growth, implicating that under these conditions PntAB functions to balance the NADH: NADPH equilibrium specifically in the direction of NADPH. The results also emphasize the importance of NAD(P)+ :NAD(P)H redox homeostasis and associated ATP:ADP equilibrium for maintaining the integrity of the photosynthetic apparatus under low-light glycolytic metabolism.

A Comprehensively Curated Genome-Scale Two-Cell Model for the Heterocystous Cyanobacterium Anabaena sp. PCC 7120.

Anabaena sp. PCC 7120 is a nitrogen-fixing filamentous cyanobacterium. Under nitrogen-limiting conditions, a fraction of the vegetative cells in each filament terminally differentiate to nongrowing heterocysts. Heterocysts are metabolically and structurally specialized to enable O2-sensitive nitrogen fixation. The functionality of the filament, as an association of vegetative cells and heterocysts, is postulated to depend on metabolic exchange of electrons, carbon, and fixed nitrogen. In this study, we compile and evaluate a comprehensive curated stoichiometric model of this two-cell system, with the objective function based on the growth of the filament under diazotrophic conditions. The predicted growth rate under nitrogen-replete and -deplete conditions, as well as the effect of external carbon and nitrogen sources, was thereafter verified. Furthermore, the model was utilized to comprehensively evaluate the optimality of putative metabolic exchange reactions between heterocysts and vegetative cells. The model suggested that optimal growth requires at least four exchange metabolites. Several combinations of exchange metabolites resulted in predicted growth rates that are higher than growth rates achieved by only considering exchange of metabolites previously suggested in the literature. The curated model of the metabolic network of Anabaena sp. PCC 7120 enhances our ability to understand the metabolic organization of multicellular cyanobacteria and provides a platform for further study and engineering of their metabolism.

Development of a Quantitative SRM-Based Proteomics Method to Study Iron Metabolism of Synechocystis sp. PCC 6803.

The cyanobacterium Synechocystis sp. PCC 6803 (S. 6803) is a well-established model species in oxygenic photosynthesis research and a potential host for biotechnological applications. Despite recent advances in genome sequencing and microarray techniques applied in systems biology, quantitative proteomics approaches with corresponding accuracy and depth are scarce for S. 6803. In this study, we developed a protocol to screen changes in the expression of 106 proteins representing central metabolic pathways in S. 6803 with a targeted mass spectrometry method, selected reaction monitoring (SRM). We evaluated the response to the exposure of both short- and long-term iron deprivation. The experimental setup enabled the relative quantification of 96 proteins, with 87 and 92 proteins showing adjusted p-values <0.01 under short- and long-term iron deficiency, respectively. The high sensitivity of the SRM method for S. 6803 was demonstrated by providing quantitative data for altogether 64 proteins that previously could not be detected with the classical data-dependent MS approach under similar conditions. This highlights the effectiveness of SRM for quantification and extends the analytical capability to low-abundance proteins in unfractionated samples of S. 6803. The SRM assays and other generated information are now publicly available via PASSEL and Panorama.

A quantitative evaluation of ethylene production in the recombinant cyanobacterium Synechocystis sp. PCC 6803 harboring the ethylene-forming enzyme by membrane inlet mass spectrometry.

The prediction of the world's future energy consumption and global climate change makes it desirable to identify new technologies to replace or augment fossil fuels by environmentally sustainable alternatives. One appealing sustainable energy concept is harvesting solar energy via photosynthesis coupled to conversion of CO2 into chemical feedstock and fuel. In this work, the production of ethylene, the most widely used petrochemical produced exclusively from fossil fuels, in the model cyanobacterium Synechocystis sp. PCC 6803 is studied. A novel instrumentation setup for quantitative monitoring of ethylene production using a combination of flat-panel photobioreactor coupled to a membrane-inlet mass spectrometer is introduced. Carbon partitioning is estimated using a quantitative model of cyanobacterial metabolism. The results show that ethylene is produced under a wide range of light intensities with an optimum at modest irradiances. The results allow production conditions to be optimized in a highly controlled setup.