Dataset Information

Ethylene production with engineered Synechocystis sp PCC 6803 strains.

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

SUBMITTER: Veetil VP

PROVIDER: S-EPMC5324202 | biostudies-literature | 2017 Feb

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Ethylene production with engineered Synechocystis sp PCC 6803 strains.

Veetil Vinod Puthan VP Angermayr S Andreas SA Hellingwerf Klaas J KJ

Microbial cell factories 20170223 1

<h4>Background</h4>Metabolic engineering and synthetic biology of cyanobacteria offer a promising sustainable alternative approach for fossil-based ethylene production, by using sunlight via oxygenic photosynthesis, to convert carbon dioxide directly into ethylene. Towards this, both well-studied cyanobacteria, i.e., Synechocystis sp PCC 6803 and Synechococcus elongatus PCC 7942, have been engineered to produce ethylene by introducing the ethylene-forming enzyme (Efe) from Pseudomonas syringae p ...[more]

PMID: 28231787

Similar Datasets

Project description:In recent years, there has been an increased interest in the research and development of sustainable alternatives to fossil fuels. Using photosynthetic microorganisms to produce such alternatives is advantageous, since they can achieve direct conversion of carbon dioxide from the atmosphere into the desired product, using sunlight as the energy source. Squalene is a naturally occurring 30-carbon isoprenoid, which has commercial use in cosmetics and in vaccines. If it could be produced sustainably on a large scale, it could also be used instead of petroleum as a raw material for fuels and as feedstock for the chemical industry. The unicellular cyanobacterium Synechocystis PCC 6803 possesses a gene, slr2089, predicted to encode squalene hopene cyclase (Shc), an enzyme converting squalene into hopene, the substrate for forming hopanoids. Through inactivation of slr2089 (shc), we explored the possibility to produce squalene using cyanobacteria. The inactivation led to accumulation of squalene, to a level over 70 times higher than in wild type cells, reaching 0.67 mg OD750(-1) L(-1). We did not observe any significant growth deficiency in the Δshc strain compared to the wild type Synechocystis, even at high light conditions, suggesting that the observed squalene accumulation was not detrimental to growth, and that formation of hopene by Shc is not crucial for growth under normal conditions, nor for high-light stress tolerance. Effects of different light intensities and growth stages on squalene accumulation in the Δshc strain were investigated. We also identified a gene, sll0513, as a putative squalene synthase in Synechocystis, and verified its function by inactivation. In this work, we show that it is possible to use the cyanobacterium Synechocystis to generate squalene, a hydrocarbon of commercial interest and a potential biofuel. We also report the first identification of a squalene hopene cyclase, and the second identification of squalene synthase, in cyanobacteria.

Project description:BackgroundCyanobacteria are phototrophic prokaryotes that convert inorganic carbon as CO2 into organic compounds at the expense of light energy. They need only inorganic nutrients and can be cultivated to high densities using non-arable land and seawater. This has made cyanobacteria attractive organisms for the production of biofuels and chemical feedstock. Synechocystis sp. PCC 6803 is one of the most widely used cyanobacterial model strains. Based on its available genome sequence and genetic tools, Synechocystis has been genetically modified to produce different biotechnological products. Efficient isoprene production is an attractive goal because this compound is widely used as chemical feedstock.ResultsHere, we report on our attempts to generate isoprene-producing strains of Synechocystis using a plasmid-based strategy. As previously reported, a codon-optimized plant isoprene synthase (IspS) was expressed under the control of different Synechocystis promoters that ensure strong constitutive or light-regulated ispS expression. The expression of the ispS gene was quantified by qPCR and Western blotting, while the amount of isoprene was quantified using GC-MS. In addition to isoprene measurements in the headspace of closed culture vessels, single photon ionization time-of-flight mass spectrometry (SPI-MS) was applied, which allowed online measurements of isoprene production in open-cultivation systems under various conditions. Under standard conditions, a good correlation existed between ispS expression and isoprene production rate. The cultivation of isoprene production strains under NaCl-supplemented conditions decreased isoprene production despite enhanced ispS mRNA levels. The characterization of the metabolome of isoprene-producing strains indicated that isoprene production might be limited by insufficient precursor levels. Transcriptomic analysis revealed the upregulation of mRNA and regulatory RNAs characteristic of acclimation to metabolic stress.ConclusionsOur best production strains produced twofold higher isoprene amounts in the presence of low NaCl concentrations than previously reported strains. These results will guide future attempts to establish isoprene production in cyanobacterial hosts.

Project description:BackgroundCyanobacteria are promising hosts for the production of various industrially important compounds such as succinate. This study focuses on introduction of the glyoxylate shunt, which is naturally present in only a few cyanobacteria, into Synechocystis PCC 6803. In order to test its impact on cell metabolism, engineered strains were evaluated for succinate accumulation under conditions of light, darkness and anoxic darkness. Each condition was complemented by treatments with 2-thenoyltrifluoroacetone, an inhibitor of succinate dehydrogenase enzyme, and acetate, both in nitrogen replete and deplete medium.ResultsWe were able to introduce genes encoding the glyoxylate shunt, aceA and aceB, encoding isocitrate lyase and malate synthase respectively, into a strain of Synechocystis PCC 6803 engineered to overexpress phosphoenolpyruvate carboxylase. Our results show that complete expression of the glyoxylate shunt results in higher extracellular succinate accumulation compared to the wild type control strain after incubation of cells in darkness and anoxic darkness in the presence of nitrate. Addition of the inhibitor 2-thenoyltrifluoroacetone increased succinate titers in all the conditions tested when nitrate was available. Addition of acetate in the presence of the inhibitor further increased the succinate accumulation, resulting in high levels when phosphoenolpyruvate carboxylase was overexpressed, compared to control strain. However, the highest succinate titer was obtained after dark incubation of an engineered strain with a partial glyoxylate shunt overexpressing isocitrate lyase in addition to phosphoenolpyruvate carboxylase, with only 2-thenoyltrifluoroacetone supplementation to the medium.ConclusionsHeterologous expression of the glyoxylate shunt with its central link to the tricarboxylic acid cycle (TCA) for acetate assimilation provides insight on the coordination of the carbon metabolism in the cell. Phosphoenolpyruvate carboxylase plays an important role in directing carbon flux towards the TCA cycle.

Project description:BackgroundCyanobacteria are considered potential photosynthetic microbial cell factories for biofuel and biochemical production. Ethylene, one of the most important organic chemicals, has been successfully synthesized in cyanobacteria by introducing an exogenous ethylene-forming enzyme (Efe). However, it remains challenging to significantly improve the biosynthetic efficiency of cyanobacterial ethylene. Genetic modification of transcription factors is a powerful strategy for reprogramming cellular metabolism toward target products. In cyanobacteria, nitrogen control A (NtcA), an important global transcription regulator of primary carbon/nitrogen metabolism, is expected to play a crucial role in ethylene biosynthesis.ResultsThe partial deletion of ntcA (MH021) enhanced ethylene production by 23%, while ntcA overexpression (MH023) in a single-copy efe recombinant Synechocystis (XX76) reduced ethylene production by 26%. Compared to XX76, the Efe protein content increased 1.5-fold in MH021. This result may be due to the release of the negative regulation of NtcA on promoter P cpcB , which controls efe expression. Glycogen content showed a 23% reduction in MH021, and the ratio of intracellular succinate to 2-oxoglutarate (2-OG) increased 4.8-fold. In a four-copy efe recombinant strain with partially deleted ntcA and a modified tricarboxylic acid (TCA) cycle (MH043), a peak specific ethylene production rate of 2463 ± 219 μL L-1 h-1 OD730-1 was achieved, which is higher than previously reported.ConclusionsThe effects of global transcription factor NtcA on ethylene synthesis in genetically engineered Synechocystis sp. PCC 6803 were evaluated, and the partial deletion of ntcA enhanced ethylene production in both single-copy and multi-copy efe recombinant Synechocystis strains. Increased Efe expression, accelerated TCA cycling, and redirected carbon flux from glycogen probably account for this improvement. The results show great potential for improving ethylene synthetic efficiency in cyanobacteria by modulating global regulation factors.

Project description:Cyanobacteria are phototrophic prokaryotes that can convert inorganic carbon as CO2 into organic carbon compounds at the expense of light energy. In addition, they need only a few inorganic nutrients and can be cultivated in high densities using non-arable land and seawater. This features qualified cyanobacteria as attractive organisms for the production of third generation biofuels as part of the development of future CO2-neutral energy production. Synechocystis sp. PCC 6803 represents one of the most widely used cyanobacterial model strains. On the basis of its available genome sequence and genetic tools, many strains of Synechocystis have been generated that produce different biotechnological products. Efficient isoprene production is an attractive goal, since this compound represents not only an energy-rich biofuel but is also used as chemical feedstock. Here, we report on our attempts to generate isoprene-producing strains of Synechocystis. The cDNA of a codon-optimized plant isoprene synthase (IspS) was cloned under the control of different Synechocystis promoters, which ensure strong constitutive or light-regulated ispS expression. The expression of the ispS gene was quantified by qPCR, whereas the amount of isoprene was quantified using GC-MS. Incubation of our strains at different salt conditions had marked impact on the isoprene production rates. Under low salt conditions, a good correlation was found between ispS expression and isoprene production rate. However, the cultivation of isoprene production strains under salt-supplemented conditions decreased isoprene production despite the fact that ispS expression was salt-stimulated. The characterization of the metabolome of isoprene producing strains indicated that isoprene production might be limited by insufficient precursor levels. Our isoprene production rates under low salt conditions were 2 - 6.5times higher compared to the previous report of Lindberg et al. (2010). These results can be used to guide future attempts establishing the isoprene production with cyanobacterial host systems.

Project description:Cyanobacterial biofuels have the potential to reduce the cost and climate impacts of biofuel production because primary carbon fixation and conversion to fuel are completed together in the cultivation of the cyanobacteria. Cyanobacterial biofuels, therefore, do not rely on costly organic carbon feedstocks that heterotrophs require, which reduces competition for agricultural resources such as arable land and freshwater. However, the published product titer achieved for most molecules of interest using cyanobacteria lag behind what has been achieved using yeast and Escherichia coli (E. coli) cultures. In Synechocystis sp. PCC 6803 (S. 6803), we attempted to increase the product titer of the sesquiterpene, bisabolene, which may be converted to bisabolane, a possible diesel replacement. We tested 19 strains of genetically modified S. 6803 with five different codon usage sequences of the bisabolene synthase from the grand fir tree (Abies grandis). At least three ribosome binding sites (most designed using the RBS Calculator) were tested for each codon usage sequence. We also tested strains with and without the farnesyl pyrophosphate synthase gene from E. coli. Bisabolene titers after five days of growth in continuous light ranged from un-detected to 7.8 ?mg/L. Bisabolene synthase abundance was measured and found to be well correlated with titer. Select strains were also tested in 12:12 light:dark cycles, where similar titers were reached after the same amount of light exposure time. One engineered strain was also tested in photobioreactors exposed to a simulated outdoor light pattern with maximum light intensity of 1600 ??mol photons m-2 s-1. Here, the bisabolene titer reached 22.2 ?mg/L after 36 days of growth. Dramatic improvements in our ability to control gene expression in cyanobacteria such as S. 6803, and the co-utilization of additional metabolic engineering methods, are needed in order for these titers to improve to the levels reported for engineered E. coli.

Dataset Information

Ethylene production with engineered Synechocystis sp PCC 6803 strains.

Publications

Ethylene production with engineered Synechocystis sp PCC 6803 strains.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets