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Insights into isoprene production using the cyanobacterium Synechocystis sp. PCC 6803.

Pade N, Erdmann S, Enke H, Dethloff F, Dühring U, Georg J, Wambutt J, Kopka J, Hess WR, Zimmermann R, Kramer D, Hagemann M - Biotechnol Biofuels (2016)

Bottom Line: Transcriptomic analysis revealed the upregulation of mRNA and regulatory RNAs characteristic of acclimation to metabolic stress.Our 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.

View Article: PubMed Central - PubMed

Affiliation: Plant Physiology Department, Institute of Biological Science, University of Rostock, Albert-Einstein-Str. 3, 18059 Rostock, Germany.

ABSTRACT

Background: Cyanobacteria 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.

Results: Here, 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.

Conclusions: Our 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.

No MeSH data available.


Related in: MedlinePlus

Salt (NaCl)-dependent accumulation of the compatible solute glucosylglycerol (GG) in selected Synechocystis strains, which carry different constructs for isoprene synthesis in comparison to the wild type (PCC 6803). For GG analysis, the cells were harvested from the closed-cultivation system after 24 h of isoprene production in the presence of 0, 2, or 4 % NaCl. Data are the mean ± SD of triplicate cultures
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Fig2: Salt (NaCl)-dependent accumulation of the compatible solute glucosylglycerol (GG) in selected Synechocystis strains, which carry different constructs for isoprene synthesis in comparison to the wild type (PCC 6803). For GG analysis, the cells were harvested from the closed-cultivation system after 24 h of isoprene production in the presence of 0, 2, or 4 % NaCl. Data are the mean ± SD of triplicate cultures

Mentions: To analyze the effects of increasing salinity on isoprene productivity of Synechocystis strains, the standard growth medium was supplemented with 2 or 4 % NaCl. The addition of 2 % NaCl had only minor effects on growth, whereas growth of the isoprene producer strains and the WT decreased at 4 % NaCl. Despite the clear effects on growth, pigmentation was not significantly changed at different salinities. The chlorophyll a relative to phycocyanin or to carotenoids ratios was similar in isoprene producer and WT cells at different NaCl concentrations (Additional file 5 A). The major compatible solute, glucosylglycerol (GG), which allows Synechocystis cells to grow at enhanced salinities, accumulated equally with increasing NaCl concentration in isoprene producer and WT cells (Fig. 2). The GG level rose to approximately 150 nmol/ml OD750 in isoprene-producing as well as WT cells at 4 % NaCl.Fig. 2


Insights into isoprene production using the cyanobacterium Synechocystis sp. PCC 6803.

Pade N, Erdmann S, Enke H, Dethloff F, Dühring U, Georg J, Wambutt J, Kopka J, Hess WR, Zimmermann R, Kramer D, Hagemann M - Biotechnol Biofuels (2016)

Salt (NaCl)-dependent accumulation of the compatible solute glucosylglycerol (GG) in selected Synechocystis strains, which carry different constructs for isoprene synthesis in comparison to the wild type (PCC 6803). For GG analysis, the cells were harvested from the closed-cultivation system after 24 h of isoprene production in the presence of 0, 2, or 4 % NaCl. Data are the mean ± SD of triplicate cultures
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4836186&req=5

Fig2: Salt (NaCl)-dependent accumulation of the compatible solute glucosylglycerol (GG) in selected Synechocystis strains, which carry different constructs for isoprene synthesis in comparison to the wild type (PCC 6803). For GG analysis, the cells were harvested from the closed-cultivation system after 24 h of isoprene production in the presence of 0, 2, or 4 % NaCl. Data are the mean ± SD of triplicate cultures
Mentions: To analyze the effects of increasing salinity on isoprene productivity of Synechocystis strains, the standard growth medium was supplemented with 2 or 4 % NaCl. The addition of 2 % NaCl had only minor effects on growth, whereas growth of the isoprene producer strains and the WT decreased at 4 % NaCl. Despite the clear effects on growth, pigmentation was not significantly changed at different salinities. The chlorophyll a relative to phycocyanin or to carotenoids ratios was similar in isoprene producer and WT cells at different NaCl concentrations (Additional file 5 A). The major compatible solute, glucosylglycerol (GG), which allows Synechocystis cells to grow at enhanced salinities, accumulated equally with increasing NaCl concentration in isoprene producer and WT cells (Fig. 2). The GG level rose to approximately 150 nmol/ml OD750 in isoprene-producing as well as WT cells at 4 % NaCl.Fig. 2

Bottom Line: Transcriptomic analysis revealed the upregulation of mRNA and regulatory RNAs characteristic of acclimation to metabolic stress.Our 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.

View Article: PubMed Central - PubMed

Affiliation: Plant Physiology Department, Institute of Biological Science, University of Rostock, Albert-Einstein-Str. 3, 18059 Rostock, Germany.

ABSTRACT

Background: Cyanobacteria 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.

Results: Here, 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.

Conclusions: Our 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.

No MeSH data available.


Related in: MedlinePlus