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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

Quantitative online SPI-MS isoprene measurements of strain # 642 under different culture conditions (one data point illustrates an averaged spectrum over 60 s). Line displays values from the fitted data approach. a Shift from medium light conditions (70 µmol photons/m2 s) to high light conditions (175 µmol photons/m2 s); b shift from high light conditions to dark (0 μmol photons/m2 s)
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Fig6: Quantitative online SPI-MS isoprene measurements of strain # 642 under different culture conditions (one data point illustrates an averaged spectrum over 60 s). Line displays values from the fitted data approach. a Shift from medium light conditions (70 µmol photons/m2 s) to high light conditions (175 µmol photons/m2 s); b shift from high light conditions to dark (0 μmol photons/m2 s)

Mentions: All previous isoprene-producing attempts, which are described here or in the literature (e.g., [4]), used closed culture systems to collect the volatile product in the headspace. However, Synechocystis grows slower in such systems than in open-cultivation systems. Therefore, we established an online measuring system via SPI-MS to register continuous isoprene production in the gas phase of an open-cultivation system. The SPI-MS measurements also enabled snapshots during the production, which allows for analysis of production profiles and isoprene amounts with changing parameters. For example, the measurements displayed in Fig. 6 show that isoprene immediately accumulated after switching the light on until a steady-state value was reached. At this point, the maximal production rates at the given light intensity could be quantified. In contrast, the isoprene amount decreased within 2–3 h to the limit of detection after switching the light off. Regarding maximal production rates, similar steady-state isoprene production levels were obtained compared to classical GC–MS measurements for the different isoprene-producing strains. Although there were approximately 4.5-times higher isoprene production rates in all strains in the open-cultivation systems, the relations of the different strains to each other were similar in the closed- and open-cultivation systems. As observed previously, strains # 704 and # 642 showed four times higher isoprene production rates compared to # 643 (Fig. 7).Fig. 6


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)

Quantitative online SPI-MS isoprene measurements of strain # 642 under different culture conditions (one data point illustrates an averaged spectrum over 60 s). Line displays values from the fitted data approach. a Shift from medium light conditions (70 µmol photons/m2 s) to high light conditions (175 µmol photons/m2 s); b shift from high light conditions to dark (0 μmol photons/m2 s)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig6: Quantitative online SPI-MS isoprene measurements of strain # 642 under different culture conditions (one data point illustrates an averaged spectrum over 60 s). Line displays values from the fitted data approach. a Shift from medium light conditions (70 µmol photons/m2 s) to high light conditions (175 µmol photons/m2 s); b shift from high light conditions to dark (0 μmol photons/m2 s)
Mentions: All previous isoprene-producing attempts, which are described here or in the literature (e.g., [4]), used closed culture systems to collect the volatile product in the headspace. However, Synechocystis grows slower in such systems than in open-cultivation systems. Therefore, we established an online measuring system via SPI-MS to register continuous isoprene production in the gas phase of an open-cultivation system. The SPI-MS measurements also enabled snapshots during the production, which allows for analysis of production profiles and isoprene amounts with changing parameters. For example, the measurements displayed in Fig. 6 show that isoprene immediately accumulated after switching the light on until a steady-state value was reached. At this point, the maximal production rates at the given light intensity could be quantified. In contrast, the isoprene amount decreased within 2–3 h to the limit of detection after switching the light off. Regarding maximal production rates, similar steady-state isoprene production levels were obtained compared to classical GC–MS measurements for the different isoprene-producing strains. Although there were approximately 4.5-times higher isoprene production rates in all strains in the open-cultivation systems, the relations of the different strains to each other were similar in the closed- and open-cultivation systems. As observed previously, strains # 704 and # 642 showed four times higher isoprene production rates compared to # 643 (Fig. 7).Fig. 6

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