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

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

SUBMITTER: Angermayr SA

PROVIDER: S-EPMC4959195 | biostudies-literature | 2016 Jul

REPOSITORIES: biostudies-literature

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

Angermayr S Andreas SA van Alphen Pascal P Hasdemir Dicle D Kramer Gertjan G Iqbal Muzamal M van Grondelle Wilmar W Hoefsloot Huub C HC Choi Young Hae YH Hellingwerf Klaas J KJ

Applied and environmental microbiology 20160630 14

<h4>Unlabelled</h4>Investigating the physiology of cyanobacteria cultured under a diel light regime is relevant for a better understanding of the resulting growth characteristics and for specific biotechnological applications that are foreseen for these photosynthetic organisms. Here, we present the results of a multiomics study of the model cyanobacterium Synechocystis sp. strain PCC 6803, cultured in a lab-scale photobioreactor in physiological conditions relevant for large-scale culturing. Th ...[more]

PMID: 27208121

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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: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:BackgroundMetabolic 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.ResultsThis study focuses on Synechocystis sp PCC 6803 and shows stable ethylene production through the integration of a codon-optimized version of the efe gene under control of the Ptrc promoter and the core Shine-Dalgarno sequence (5'-AGGAGG-3') as the ribosome-binding site (RBS), at the slr0168 neutral site. We have increased ethylene production twofold by RBS screening and further investigated improving ethylene production from a single gene copy of efe, using multiple tandem promoters and by putting our best construct on an RSF1010-based broad-host-self-replicating plasmid, which has a higher copy number than the genome. Moreover, to raise the intracellular amounts of the key Efe substrate, 2-oxoglutarate, from which ethylene is formed, we constructed a glycogen-synthesis knockout mutant (ΔglgC) and introduced the ethylene biosynthetic pathway in it. Under nitrogen limiting conditions, the glycogen knockout strain has increased intracellular 2-oxoglutarate levels; however, surprisingly, ethylene production was lower in this strain than in the wild-type background.ConclusionMaking 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.

Dataset Information

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

Publications

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

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