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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

Changes of the metabolism in cells of the wild type (WT) compared to three isoprene-producing strains (see Table 1) of Synechocystis sp. PCC 6803, which were cultivated for 24 h in a closed flask system under salt-free (without NaCl) conditions. Bars correspond to normalized values of two biological replicates measured by at least two technical replicates. Factors are calculated relative to WT. Error bars represent standard error
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Fig4: Changes of the metabolism in cells of the wild type (WT) compared to three isoprene-producing strains (see Table 1) of Synechocystis sp. PCC 6803, which were cultivated for 24 h in a closed flask system under salt-free (without NaCl) conditions. Bars correspond to normalized values of two biological replicates measured by at least two technical replicates. Factors are calculated relative to WT. Error bars represent standard error

Mentions: To analyze how isoprene production affects the overall metabolism of Synechocystis, we performed a non-targeted metabolome analysis. To this end, the metabolome of selected high to medium isoprene producer strains, namely # 642, # 704, and # 731 (see Fig. 1a), was analyzed by GC—MS-based metabolite profiling [33] in comparison to WT cells (PCC 6803 in Fig. 4). The samples were obtained under isoprene-producing conditions in the closed-cultivation system, i.e., 24 h after isoprene production initiation under NaCl-free conditions in the presence of added bicarbonate as inorganic carbon source. In total, 154 analytes were annotated in this dataset; however, the majority of these compounds could not be assigned to specific intermediates. Unfortunately, none of the intermediates of the MEP pathway were detected. Nevertheless, we were able to quantify 58 metabolites of carbon and nitrogen metabolism (Fig. 4; Additional file 6 shows total metabolite data), which allowed us to investigate the effects of isoprene production on cyanobacterial primary metabolism.Fig. 4


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)

Changes of the metabolism in cells of the wild type (WT) compared to three isoprene-producing strains (see Table 1) of Synechocystis sp. PCC 6803, which were cultivated for 24 h in a closed flask system under salt-free (without NaCl) conditions. Bars correspond to normalized values of two biological replicates measured by at least two technical replicates. Factors are calculated relative to WT. Error bars represent standard error
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig4: Changes of the metabolism in cells of the wild type (WT) compared to three isoprene-producing strains (see Table 1) of Synechocystis sp. PCC 6803, which were cultivated for 24 h in a closed flask system under salt-free (without NaCl) conditions. Bars correspond to normalized values of two biological replicates measured by at least two technical replicates. Factors are calculated relative to WT. Error bars represent standard error
Mentions: To analyze how isoprene production affects the overall metabolism of Synechocystis, we performed a non-targeted metabolome analysis. To this end, the metabolome of selected high to medium isoprene producer strains, namely # 642, # 704, and # 731 (see Fig. 1a), was analyzed by GC—MS-based metabolite profiling [33] in comparison to WT cells (PCC 6803 in Fig. 4). The samples were obtained under isoprene-producing conditions in the closed-cultivation system, i.e., 24 h after isoprene production initiation under NaCl-free conditions in the presence of added bicarbonate as inorganic carbon source. In total, 154 analytes were annotated in this dataset; however, the majority of these compounds could not be assigned to specific intermediates. Unfortunately, none of the intermediates of the MEP pathway were detected. Nevertheless, we were able to quantify 58 metabolites of carbon and nitrogen metabolism (Fig. 4; Additional file 6 shows total metabolite data), which allowed us to investigate the effects of isoprene production on cyanobacterial primary metabolism.Fig. 4

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