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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

Volcano plot of the microarray results. Only data of protein encoding genes and of intergenic encoded sRNAs are shown. Significantly differential expressed genes (log2 fold change ≥ 1 and adjusted p value ≤0.05) are in the upper left and upper right sector. Functional related features are color-coded and selected features are named. Intergenic encoded sRNAs are indicated by a gray circle with a black border
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Fig5: Volcano plot of the microarray results. Only data of protein encoding genes and of intergenic encoded sRNAs are shown. Significantly differential expressed genes (log2 fold change ≥ 1 and adjusted p value ≤0.05) are in the upper left and upper right sector. Functional related features are color-coded and selected features are named. Intergenic encoded sRNAs are indicated by a gray circle with a black border

Mentions: We concentrated on differentially regulated sRNAs and mRNAs (Fig. 5). Interestingly, three of the strongest downregulated genes (slr1668, slr1667, ssr2848) in strain # 642 are possibly controlled by the cAMP-controlled transcriptional regulator SYCRP1 because the ORFs slr1668 and slr1667 showed deregulated expression in the corresponding mutant [34], whereas the upstream region of ssr2848 was identified in an in silico target prediction for SYCRP1 [35]. Genes slr1668 and slr1667 encode proteins that are involved in the construction of cell surface components [36], but the precise function of these proteins is unknown. The gene ssr2048 encodes a small protein of only 72 amino acids that has no matches to other proteins in the entire database except a single protein in Synechocystis sp. PCC 6714. The transcriptome data indicated that isoprene production is stressful for Synechocystis cells because many genes that are known to be induced under environmental stress showed increased RNA levels. For example, we found induction of the mRNA of the serine protease HtrA, the small heat shock protein HspA and several transposases. Four genes encoding a bacterial ubiquitin system (sll6052–sll6055), which might be involved in degradation of misfolded proteins or proteome remodeling, also showed increased transcription in the isoprene producer cells (Fig. 5). The iron stress-activated sRNA IsaR1 and the high light stress-induced sRNA Syr1/PsrR1 [36] were also upregulated. The observed repression of petE and the induction of petJ indicate disturbed internal copper availability because these genes are known to respond to changing copper concentrations in Synechocystis [37, 38]. Furthermore, the demand for some macronutrients appeared to be changed. Nearly the complete pho regulon involved in phosphate uptake (pst1 operon: sll0680–sll0684; pst2 operon: sll1248 + sll1249, phoA) was downregulated, whereas the sulfate transport operon was induced (slr1452–slr1455). Nitrogen-related genes were also differentially transcribed. The sRNA NsiR4 [39], the mRNAs nblA1 and nblA2, and the glutamine synthetase inactivating factors gifA and gifB were upregulated. We observed no differential expression of genes encoding proteins involved in inorganic carbon uptake, but the expression of the carboxysome shell protein operon (sll1028–sll1032) decreased. The genes for the core photosynthetic proteins were largely unaffected. Only psbZ was significantly downregulated, and psbM and psaM were upregulated, whereas the full atp1 operon (sll1321–sll1327) encoding ATP synthase subunits was repressed.Fig. 5


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)

Volcano plot of the microarray results. Only data of protein encoding genes and of intergenic encoded sRNAs are shown. Significantly differential expressed genes (log2 fold change ≥ 1 and adjusted p value ≤0.05) are in the upper left and upper right sector. Functional related features are color-coded and selected features are named. Intergenic encoded sRNAs are indicated by a gray circle with a black border
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig5: Volcano plot of the microarray results. Only data of protein encoding genes and of intergenic encoded sRNAs are shown. Significantly differential expressed genes (log2 fold change ≥ 1 and adjusted p value ≤0.05) are in the upper left and upper right sector. Functional related features are color-coded and selected features are named. Intergenic encoded sRNAs are indicated by a gray circle with a black border
Mentions: We concentrated on differentially regulated sRNAs and mRNAs (Fig. 5). Interestingly, three of the strongest downregulated genes (slr1668, slr1667, ssr2848) in strain # 642 are possibly controlled by the cAMP-controlled transcriptional regulator SYCRP1 because the ORFs slr1668 and slr1667 showed deregulated expression in the corresponding mutant [34], whereas the upstream region of ssr2848 was identified in an in silico target prediction for SYCRP1 [35]. Genes slr1668 and slr1667 encode proteins that are involved in the construction of cell surface components [36], but the precise function of these proteins is unknown. The gene ssr2048 encodes a small protein of only 72 amino acids that has no matches to other proteins in the entire database except a single protein in Synechocystis sp. PCC 6714. The transcriptome data indicated that isoprene production is stressful for Synechocystis cells because many genes that are known to be induced under environmental stress showed increased RNA levels. For example, we found induction of the mRNA of the serine protease HtrA, the small heat shock protein HspA and several transposases. Four genes encoding a bacterial ubiquitin system (sll6052–sll6055), which might be involved in degradation of misfolded proteins or proteome remodeling, also showed increased transcription in the isoprene producer cells (Fig. 5). The iron stress-activated sRNA IsaR1 and the high light stress-induced sRNA Syr1/PsrR1 [36] were also upregulated. The observed repression of petE and the induction of petJ indicate disturbed internal copper availability because these genes are known to respond to changing copper concentrations in Synechocystis [37, 38]. Furthermore, the demand for some macronutrients appeared to be changed. Nearly the complete pho regulon involved in phosphate uptake (pst1 operon: sll0680–sll0684; pst2 operon: sll1248 + sll1249, phoA) was downregulated, whereas the sulfate transport operon was induced (slr1452–slr1455). Nitrogen-related genes were also differentially transcribed. The sRNA NsiR4 [39], the mRNAs nblA1 and nblA2, and the glutamine synthetase inactivating factors gifA and gifB were upregulated. We observed no differential expression of genes encoding proteins involved in inorganic carbon uptake, but the expression of the carboxysome shell protein operon (sll1028–sll1032) decreased. The genes for the core photosynthetic proteins were largely unaffected. Only psbZ was significantly downregulated, and psbM and psaM were upregulated, whereas the full atp1 operon (sll1321–sll1327) encoding ATP synthase subunits was repressed.Fig. 5

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