56 results found
Sattayawat P, Yunus IS, Jones PR, 2023, Production of Fatty Acids and Derivatives Using Cyanobacteria., Adv Biochem Eng Biotechnol, Vol: 183, Pages: 145-169, ISSN: 0724-6145
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.
Yunus IS, Anfelt J, Sporre E, et al., 2022, Synthetic metabolic pathways for conversion of CO2 into secreted short-to medium-chain hydrocarbons using cyanobacteria, METABOLIC ENGINEERING, Vol: 72, Pages: 14-23, ISSN: 1096-7176
Yunus IS, Anfelt J, Hudson EP, et al., 2021, Synthetic metabolic pathways for conversion of CO<sub>2</sub> into secreted short-to medium-chain hydrocarbons using cyanobacteria
<jats:title>Abstract</jats:title><jats:p>The objective of this study was to implement direct sunlight-driven conversion of CO<jats:sub>2</jats:sub> 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 <jats:italic>Synechocystis</jats:italic> 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 CO<jats:sub>2</jats:sub> 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 bioreaction ingredients it was concluded that linoleic acid undergoes a spontaneous non-enzymatic reaction to yield pentane and hexanal. When <jats:italic>Synechocystis</jats:italic> was added to the bioreaction, hexanal was converted into 1-hexanol, but there was no further stimulation of pentane biosynthesis. 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 <jats:italic>Pseudomonas mendocina</jats:italic> in combination with C8-C10 preferring thioestersaes. When UndB instead was combined with a C12-specific ‘<jats:italic>Uc</jats:italic>FatB1 thioesterase, this resulted in ten-fold increase of alkene biosynthesis. When UndB was replaced with the light-dependent FAP decarboxylase, both undecane and tridecane accumulated, a
Yunus IS, Wang Z, Sattayawat P, et al., 2021, Improved Bioproduction of 1-Octanol Using Engineered <i>Synechocystis</i> sp. PCC 6803, ACS SYNTHETIC BIOLOGY, Vol: 10, Pages: 1417-1428, ISSN: 2161-5063
Perin G, Fletcher T, Sagi-Kiss V, et al., 2021, Calm on the surface, dynamic on the inside. Molecular homeostasis of <i>Anabaena</i> sp. PCC 7120 nitrogen metabolism, PLANT CELL AND ENVIRONMENT, Vol: 44, Pages: 1885-1907, ISSN: 0140-7791
Wichmann J, Lauersen KJ, Biondi N, et al., 2021, Engineering Biocatalytic Solar Fuel Production: The PHOTOFUEL Consortium, TRENDS IN BIOTECHNOLOGY, Vol: 39, Pages: 323-327, ISSN: 0167-7799
Fuente D, Lazar D, Oliver-Villanueva JV, et al., 2021, Reconstruction of the absorption spectrum of <i>Synechocystis</i> sp. PCC 6803 optical mutants from the in vivo signature of individual pigments, PHOTOSYNTHESIS RESEARCH, Vol: 147, Pages: 75-90, ISSN: 0166-8595
Garcia LDA, Jones PR, 2020, <i>In silico</i>co-factor balance estimation using constraint-based modelling informs metabolic engineering in<i>Escherichia coli</i>, PLOS COMPUTATIONAL BIOLOGY, Vol: 16, ISSN: 1553-734X
Perin G, Fletcher T, Sagi-Kiss V, et al., 2020, Calm on the surface, dynamic on the inside. Molecular homeostasis in response to regulatory and metabolic perturbation of<i>Anabaena</i>sp. PCC 7120 nitrogen metabolism
<jats:title>Abstract</jats:title><jats:p>Nitrogen is a key macro-nutrient required for the metabolism and growth of biological systems. Although multiple nitrogen sources can serve this purpose, they are all converted into ammonium/ammonia as a first step of assimilation. It is thus reasonable to expect that molecular parts involved in the transport of ammonium/ammonia across biological membranes (i.e. catalysed by AMT transporters) connect with the regulation of both nitrogen and central carbon metabolism. In order to test this hypothesis, we applied both (1) genetic (i.e. Δ<jats:italic>amt</jats:italic>mutation) and (2) environmental treatments to a target biological system, the cyanobacterium Anabaena sp. PCC 7120. Cyanobacteria have a key role in the global nitrogen cycle and thus represent a useful model system. The aim was to both (1) perturb sensing and low-affinity uptake of ammonium/ammonia and (2) induce multiple inner N states, followed by targeted quantification of key proteins, metabolites and enzyme activities, with experiments intentionally designed over a longer time-scale than the available studies in literature. We observed that the absence of AMT transporters triggered a substantial response at a whole-system level, affecting enzyme activities and the quantity of both proteins and metabolites, spanning both N and C metabolism. Moreover, the absence of AMT transporters left a molecular fingerprint indicating N-deficiency even under N replete conditions (i.e. greater GS activity, lower 2-OG content and faster nitrogenase activation upon N deprivation). Contrasting with all of the above dynamic adaptations was the striking near-complete lack of any externally measurable phenotype (i.e. growth, photosynthesis, pigments, metabolites). We thus conclude that this species evolved a highly robust and adaptable molecular network to maintain homeostasis, resulting in substantial internal but minimal external perturbations.
Amer M, Wojcik EZ, Sun C, et al., 2020, Low carbon strategies for sustainable bio-alkane gas production and renewable energy, Energy and Environmental Science, Vol: 13, Pages: 1818-1831, ISSN: 1754-5692
Propane and butane are the main constituents of liquefied petroleum gas and are used extensively for transport and domestic use. They are clean burning fuels, suitable for the development of low carbon footprint fuel and energy policies. Here, we present blueprints for the production of bio-alkane gas (propane and butane) through the conversion of waste volatile fatty acids by bacterial culture. We show that bio-propane and bio-butane can be produced photo-catalytically by bioengineered strains of E. coli and Halomonas (in non-sterile seawater) using fatty acids derived from biomass or industrial waste, and by Synechocystis (using carbon dioxide as feedstock). Scaled production using available infrastructure is calculated to be economically feasible using Halomonas. These fuel generation routes could be deployed rapidly, in both advanced and developing countries, and contribute to energy security to meet global carbon management targets and clean air directives.
Sattayawat P, Yunus IS, Jones PR, 2020, Bioderivatization as a concept for renewable production of chemicals that are toxic or poorly soluble in the liquid phase, Proceedings of the National Academy of Sciences of USA, Vol: 117, Pages: 1404-1413, ISSN: 0027-8424
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.
Yunus IS, Palma A, Trudeau DL, et al., 2020, Methanol-free biosynthesis of fatty acid methyl ester (FAME) in <i>Synechocystis</i> sp. PCC 6803, METABOLIC ENGINEERING, Vol: 57, Pages: 217-227, ISSN: 1096-7176
Bartasun P, Prandi N, Storch M, et al., 2019, The effect of modulating the quantity of enzymes in a model ethanol pathway on metabolic flux in Synechocystis sp. PCC 6803, PEERJ, Vol: 7, ISSN: 2167-8359
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 con
Perin G, Yunus IS, Valton M, et al., 2019, Sunlight-driven recycling to increase nutrient use-efficiency in agriculture, Algal Research, Vol: 41, ISSN: 2211-9264
Humans unsustainably scavenge massive amounts of nutrients from the environment to feed our agricultural systems, thereby perturbing pre-existing natural re-cycling processes. Only a minor fraction of the nutrients are eventually taken up by crops and converted into food, while the majority runs-off into the environment, causing the release of greenhouse gases (e.g. emission of nitrous and nitrogen oxides from the soil) and threatening water security/biodiversity in several ecosystems. The estimated continued growth in global population in the 21st century is expected to place even greater pressure on nutrient use, with likely consequences for the sustainability of human society. Technologies that are able to balance the requirement for intensification of food production with a mitigation of its impact on the environment will be essential to deploy in the near future. The aim is to substantially increase nutrient use-efficiency in order to lower the pressure on finite resources and lighten the environmental impact of intensive agriculture. In this review, we will discuss one such technology, sunlight-driven prokaryotic and eukaryotic microalgae, as a vehicle for both capture and provision of nutrients leached from and provided to agricultural systems, respectively. This technology has the potential to make a difference, but it remains immature and we need to rapidly enhance our knowledge of its opportunities and challenges in order to exploit it for a sustainable circular nutrient economy.
Perin G, Jones PR, 2019, Economic feasibility and long-term sustainability criteria on the path to enable a transition from fossil fuels to biofuels., Current Opinion in Biotechnology, Vol: 57, Pages: 175-182, ISSN: 0958-1669
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.
Yunus IS, Wichmann J, Wördenweber R, et al., 2018, Synthetic metabolic pathways for photobiological conversion of CO2 into hydrocarbon fuel, Metabolic Engineering, Vol: 49, Pages: 201-211, ISSN: 1096-7176
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 synt
Yunus IS, Jones PR, 2018, Photosynthesis-dependent biosynthesis of medium chain-length fatty acids and alcohols, Metabolic Engineering, Vol: 49, Pages: 59-68, ISSN: 1096-7176
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.
Kreula SM, Kaewphan S, Ginter F, et al., 2018, Finding novel relationships with integrated gene-gene association network analysis of Synechocystis sp. PCC 6803 using species-independent text-mining, PeerJ, Vol: 6, ISSN: 2167-8359
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.
De Porcellinis AJ, Norgaard H, Brey LMF, et al., 2018, 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, Metabolic Engineering, Vol: 47, Pages: 170-183, ISSN: 1096-7176
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
Kreula S, Kaewphan S, Ginter F, et al., 2017, Finding novel relationships with integrated gene-gene association network analysis of Synechocystis sp. PCC 6803 using species-independent text-mining, Publisher: PeerJ Inc.
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 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 rule-based algorithm 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.
Kreula SM, Kaewphan S, Ginter F, et al., 2017, Finding novel relationships with integrated gene-gene association network analysis of <i>Synechocystis sp. </i>PCC 6803 using species-independent text-mining
<jats:p>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 <jats:italic>Synechocystis sp.</jats:italic> 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 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 rule-based algorithm to (<jats:italic>i</jats:italic>) discover novel candidate associations between different genes or proteins in the network, and (<jats:italic>ii</jats:italic>) rapidly evaluate the potential role of any one particular gene or protein. The full network is provided as an open source resource.</jats:p>
Kamarainen J, Huokko T, Kreula S, et al., 2017, Pyridine nucleotide transhydrogenase PntAB is essential for optimal growth and photosynthetic integrity under low-light mixotrophic conditions in Synechocystis sp PCC 6803, New Phytologist, Vol: 214, Pages: 194-204, ISSN: 1469-8137
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.
Erb TJ, Jones PR, Bar-Even A, 2017, Synthetic metabolism: metabolic engineering meets enzyme design., Current Opinion in Chemical Biology, Vol: 37, Pages: 56-62, ISSN: 1879-0402
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.
Malatinszky D, Steuer R, Jones PR, 2017, A comprehensively curated genome-scale two-cell model for the cyanobacterium Anabaena sp. PCC 7120, Plant Physiology, Vol: 173, Pages: 509-523, ISSN: 0032-0889
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 non-growing 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 the present work, 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 multi-cellular cyanobacteria and provides a platform for further study and engineering of their metabolism
Zavrel T, Knoop H, Steuer R, et al., 2016, A quantitative evaluation of ethylene production in the recombinant cyanobacterium <i>Synechocystis</i> sp PCC 6803 harboring the ethylene-forming enzyme by membrane inlet mass spectrometry, BIORESOURCE TECHNOLOGY, Vol: 202, Pages: 142-151, ISSN: 0960-8524
Vuorijoki L, Isojarvi J, Kallio P, et al., 2015, Development of a Quantitative SRM-Based Proteomics Method to Study Iron Metabolism of Synechocystis sp. PCC 6803, Journal of Proteome Research, Vol: 15, Pages: 266-279, ISSN: 1535-3907
Kremer F, Jones PR, Blank LM, et al., 2015, A comparison of the microbial production and combustion characteristics of three alcohol biofuels: ethanol, 1-butanol, and 1-octanol, Frontiers in Bioengineering and Biotechnology, Vol: 3, ISSN: 2296-4185
Over the last decade, microbes have been engineered for the manufacture of a variety of biofuels. Saturated linear-chain alcohols have great potential as transport biofuels. Their hydrocarbon backbones, as well as oxygenated content, confer combustive properties that make it suitable for use in internal combustion engines. Herein, we compared the microbial production and combustion characteristics of ethanol, 1-butanol, and 1-octanol. In terms of productivity and efficiency, current microbial platforms favor the production of ethanol. From a combustion standpoint, the most suitable fuel for spark-ignition engines would be ethanol, while for compression-ignition engines it would be 1-octanol. However, any general conclusions drawn at this stage regarding the most superior biofuel would be premature, as there are still many areas that need to be addressed, such as large-scale purification and pipeline compatibility. So far, the difficulties in developing and optimizing microbial platforms for fuel production, particularly for newer fuel candidates, stem from our poor understanding of the myriad biological factors underpinning them. A great deal of attention therefore needs to be given to the fundamental mechanisms that govern biological processes. Additionally, research needs to be undertaken across a wide range of disciplines to overcome issues of sustainability and commercial viability.
Menon N, Pasztor A, Menon BRK, et al., 2015, A microbial platform for renewable propane synthesis based on a fermentative butanol pathway, Biotechnology for Biofuels, Vol: 8, ISSN: 1754-6834
Akhtar MK, Dandapani H, Thiel K, et al., 2014, Microbial production of 1-octanol: a naturally excreted biofuel with diesel-like properties, Metab Eng Commun, Vol: 2, Pages: 1-5, ISSN: 2214-0301
The development of sustainable, bio-based technologies to convert solar energy and carbon dioxide into fuels is a grand challenge. A core part of this challenge is to produce a fuel that is compatible with the existing transportation infrastructure. This task is further compounded by the commercial desire to separate the fuel from the biotechnological host. Based on its fuel characteristics, 1-octanol was identified as an attractive metabolic target with diesel-like properties. We therefore engineered a synthetic pathway specifically for the biosynthesis of 1-octanol in Escherichia coli BL21(DE3) by over-expression of three enzymes (thioesterase, carboxylic acid reductase and aldehyde reductase) and one maturation factor (phosphopantetheinyl transferase). Induction of this pathway in a shake flask resulted in 4.4 mg 1-octanol L−1 h−1 which exceeded the productivity of previously engineered strains. Furthermore, the majority (73%) of the fatty alcohol was localised within the media without the addition of detergent or solvent overlay. The deletion of acrA reduced the production and excretion of 1-octanol by 3-fold relative to the wild-type, suggesting that the AcrAB–TolC complex may be responsible for the majority of product efflux. This study presents 1-octanol as a potential fuel target that can be synthesised and naturally accumulated within the media using engineered microbes.
Kallio P, Pasztor A, Thiel K, et al., 2014, An engineered pathway for the biosynthesis of renewable propane, Nature Communications, Vol: 5, Pages: 1-8, ISSN: 2041-1723
The deployment of next-generation renewable biofuels can be enhanced by improving their compatibility with the current infrastructure for transportation, storage and utilization. Propane, the bulk component of liquid petroleum gas, is an appealing target as it already has a global market. In addition, it is a gas under standard conditions, but can easily be liquefied. This allows the fuel to immediately separate from the biocatalytic process after synthesis, yet does not preclude energy-dense storage as a liquid. Here we report, for the first time, a synthetic metabolic pathway for producing renewable propane. The pathway is based on a thioesterase specific for butyryl-acyl carrier protein (ACP), which allows native fatty acid biosynthesis of the Escherichia coli host to be redirected towards a synthetic alkane pathway. Propane biosynthesis is markedly stimulated by the introduction of an electron-donating module, optimizing the balance of O2 supply and removal of native aldehyde reductases.
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