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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

Isoprene production rate and ispS expression in standard medium (0 % NaCl) of the different Synechocystis strains. a Isoprene production is expressed in relation to optical density (OD750; a measure of cell number) over 24 h of phototrophic growth in the Synechocystis strains carrying various constructs for isoprene synthesis (see Table 1). Mean values and standard deviation from three independent growth experiments with each two technical replicates are given. Statistical significant differences (p ≤ 0.05) between strain # 642 and other are marked by asterisk. b 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. The expression in strain # 643 was set to 1. c Accumulation of the IspS protein in the different Synechocystis strains. The protein amounts were visualized by immune-blotting. Equal amounts of soluble protein (10 μg) were loaded on gels. The blot was incubated with a specific IspS antibody and the IspS protein was visualized by chemoluminescence. d Coomassie-stained SDS-PAGE of proteins which correspond to the Western Blot above
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Fig1: Isoprene production rate and ispS expression in standard medium (0 % NaCl) of the different Synechocystis strains. a Isoprene production is expressed in relation to optical density (OD750; a measure of cell number) over 24 h of phototrophic growth in the Synechocystis strains carrying various constructs for isoprene synthesis (see Table 1). Mean values and standard deviation from three independent growth experiments with each two technical replicates are given. Statistical significant differences (p ≤ 0.05) between strain # 642 and other are marked by asterisk. b 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. The expression in strain # 643 was set to 1. c Accumulation of the IspS protein in the different Synechocystis strains. The protein amounts were visualized by immune-blotting. Equal amounts of soluble protein (10 μg) were loaded on gels. The blot was incubated with a specific IspS antibody and the IspS protein was visualized by chemoluminescence. d Coomassie-stained SDS-PAGE of proteins which correspond to the Western Blot above

Mentions: The initial cultivation in NaCl-free, standard medium resulted in highly reproducible, specific isoprene production rates for each strain (Fig. 1a). Strain # 642, in which the ispS is under the control of the strong rbcL promoter, showed the highest productivity of 1.16 ng/ml h OD750. Similar isoprene production rates were observed with strain # 704 (1.02 ng/ml h OD750), in which two copies of the ispS are present, one under the control of PrbcL and the other controlled by Ptac. Strain # 731, in which ispS is co-expressed with dxs, showed intermediate isoprene production. Strains # 643 (ispS driven by the E. coli tac promoter), # 796 and # 816 showed significantly lower isoprene production rates (approximately ten times less than strain # 642). Strain # 816 expresses ispS under the control of PpsaA*, and the dxs gene is controlled by PrbcL (Fig. 1a), whereas in strain # 796 these two genes are controlled by PpsaA*. Growth and pigmentation of the different isoprene-producing strains did not differ from WT under these cultivation methods (Additional file 5 A/B).Fig. 1


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)

Isoprene production rate and ispS expression in standard medium (0 % NaCl) of the different Synechocystis strains. a Isoprene production is expressed in relation to optical density (OD750; a measure of cell number) over 24 h of phototrophic growth in the Synechocystis strains carrying various constructs for isoprene synthesis (see Table 1). Mean values and standard deviation from three independent growth experiments with each two technical replicates are given. Statistical significant differences (p ≤ 0.05) between strain # 642 and other are marked by asterisk. b 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. The expression in strain # 643 was set to 1. c Accumulation of the IspS protein in the different Synechocystis strains. The protein amounts were visualized by immune-blotting. Equal amounts of soluble protein (10 μg) were loaded on gels. The blot was incubated with a specific IspS antibody and the IspS protein was visualized by chemoluminescence. d Coomassie-stained SDS-PAGE of proteins which correspond to the Western Blot above
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Related In: Results  -  Collection

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Fig1: Isoprene production rate and ispS expression in standard medium (0 % NaCl) of the different Synechocystis strains. a Isoprene production is expressed in relation to optical density (OD750; a measure of cell number) over 24 h of phototrophic growth in the Synechocystis strains carrying various constructs for isoprene synthesis (see Table 1). Mean values and standard deviation from three independent growth experiments with each two technical replicates are given. Statistical significant differences (p ≤ 0.05) between strain # 642 and other are marked by asterisk. b 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. The expression in strain # 643 was set to 1. c Accumulation of the IspS protein in the different Synechocystis strains. The protein amounts were visualized by immune-blotting. Equal amounts of soluble protein (10 μg) were loaded on gels. The blot was incubated with a specific IspS antibody and the IspS protein was visualized by chemoluminescence. d Coomassie-stained SDS-PAGE of proteins which correspond to the Western Blot above
Mentions: The initial cultivation in NaCl-free, standard medium resulted in highly reproducible, specific isoprene production rates for each strain (Fig. 1a). Strain # 642, in which the ispS is under the control of the strong rbcL promoter, showed the highest productivity of 1.16 ng/ml h OD750. Similar isoprene production rates were observed with strain # 704 (1.02 ng/ml h OD750), in which two copies of the ispS are present, one under the control of PrbcL and the other controlled by Ptac. Strain # 731, in which ispS is co-expressed with dxs, showed intermediate isoprene production. Strains # 643 (ispS driven by the E. coli tac promoter), # 796 and # 816 showed significantly lower isoprene production rates (approximately ten times less than strain # 642). Strain # 816 expresses ispS under the control of PpsaA*, and the dxs gene is controlled by PrbcL (Fig. 1a), whereas in strain # 796 these two genes are controlled by PpsaA*. Growth and pigmentation of the different isoprene-producing strains did not differ from WT under these cultivation methods (Additional file 5 A/B).Fig. 1

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