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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

Influence of salinity on isoprene production and ispS expression. a Isoprene production rates of selected Synechocystis strains are shown in the presence of 0, 2, or 4 % NaCl. Isoprene production is expressed in relation to optical density (OD750; measure of cell density) over 24 h of phototrophic growth in the Synechocystis strains, which carry various constructs for isoprene synthesis (see Table 1). Statistical significant differences (p ≤ 0.05) to the strain # 642 at 0 % NaCl are marked by asterisk. b Salt (NaCl)-dependent expression of the ispS gene in the different Synechocystis strains. The relative expression (rnpB amount was used as internal loading control) of ispS was estimated by qPCR. Expression of ispS at 0 % NaCl was set to 1
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Fig3: Influence of salinity on isoprene production and ispS expression. a Isoprene production rates of selected Synechocystis strains are shown in the presence of 0, 2, or 4 % NaCl. Isoprene production is expressed in relation to optical density (OD750; measure of cell density) over 24 h of phototrophic growth in the Synechocystis strains, which carry various constructs for isoprene synthesis (see Table 1). Statistical significant differences (p ≤ 0.05) to the strain # 642 at 0 % NaCl are marked by asterisk. b Salt (NaCl)-dependent expression of the ispS gene in the different Synechocystis strains. The relative expression (rnpB amount was used as internal loading control) of ispS was estimated by qPCR. Expression of ispS at 0 % NaCl was set to 1

Mentions: Isoprene productivity decreased in all strains at high NaCl concentrations (Fig. 3a). The relative drop in productivity in cells, supplemented with NaCl, varied between strains harboring different ispS expression cartridges. Strain # 704 showed a significant decline of 68 %, while productivity of strain # 642 decreased by only 29 % in the presence of 4 % NaCl compared to 0 % NaCl. Surprisingly, expression of the ispS gene was stimulated by NaCl. For example, the ispS mRNA level is twofold higher in strain # 642 in the presence of 4 % NaCl compared to standard medium (Fig. 3b). Moreover, an increased ispS expression was also found for strain # 704 at 4 % NaCl; however, it showed a slightly lower expression at 2 % NaCl. These findings are consistent with the reported slight stimulation by NaCl of rbcL and psbA expression in Synechocystis WT cells at the mRNA level (see: http://www.cyanoexpress.sysbiolab.eu/). However, the increased mRNA levels of ispS are not always translated into higher protein amounts. Therefore, future measurements of enzyme activities would be necessary to support the mRNA data.Fig. 3


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)

Influence of salinity on isoprene production and ispS expression. a Isoprene production rates of selected Synechocystis strains are shown in the presence of 0, 2, or 4 % NaCl. Isoprene production is expressed in relation to optical density (OD750; measure of cell density) over 24 h of phototrophic growth in the Synechocystis strains, which carry various constructs for isoprene synthesis (see Table 1). Statistical significant differences (p ≤ 0.05) to the strain # 642 at 0 % NaCl are marked by asterisk. b Salt (NaCl)-dependent expression of the ispS gene in the different Synechocystis strains. The relative expression (rnpB amount was used as internal loading control) of ispS was estimated by qPCR. Expression of ispS at 0 % NaCl was set to 1
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig3: Influence of salinity on isoprene production and ispS expression. a Isoprene production rates of selected Synechocystis strains are shown in the presence of 0, 2, or 4 % NaCl. Isoprene production is expressed in relation to optical density (OD750; measure of cell density) over 24 h of phototrophic growth in the Synechocystis strains, which carry various constructs for isoprene synthesis (see Table 1). Statistical significant differences (p ≤ 0.05) to the strain # 642 at 0 % NaCl are marked by asterisk. b Salt (NaCl)-dependent expression of the ispS gene in the different Synechocystis strains. The relative expression (rnpB amount was used as internal loading control) of ispS was estimated by qPCR. Expression of ispS at 0 % NaCl was set to 1
Mentions: Isoprene productivity decreased in all strains at high NaCl concentrations (Fig. 3a). The relative drop in productivity in cells, supplemented with NaCl, varied between strains harboring different ispS expression cartridges. Strain # 704 showed a significant decline of 68 %, while productivity of strain # 642 decreased by only 29 % in the presence of 4 % NaCl compared to 0 % NaCl. Surprisingly, expression of the ispS gene was stimulated by NaCl. For example, the ispS mRNA level is twofold higher in strain # 642 in the presence of 4 % NaCl compared to standard medium (Fig. 3b). Moreover, an increased ispS expression was also found for strain # 704 at 4 % NaCl; however, it showed a slightly lower expression at 2 % NaCl. These findings are consistent with the reported slight stimulation by NaCl of rbcL and psbA expression in Synechocystis WT cells at the mRNA level (see: http://www.cyanoexpress.sysbiolab.eu/). However, the increased mRNA levels of ispS are not always translated into higher protein amounts. Therefore, future measurements of enzyme activities would be necessary to support the mRNA data.Fig. 3

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