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Engineering Limonene and Bisabolene Production in Wild Type and a Glycogen-Deficient Mutant of Synechococcus sp. PCC 7002.

Davies FK, Work VH, Beliaev AS, Posewitz MC - Front Bioeng Biotechnol (2014)

Bottom Line: None of the excreted metabolites, however, appeared to be effectively utilized for terpenoid metabolism.Overall, Synechococcus sp.PCC 7002 provides a highly promising platform for terpenoid biosynthetic and metabolic engineering efforts.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Geochemistry, Colorado School of Mines , Golden, CO , USA.

ABSTRACT
The plant terpenoids limonene (C10H16) and α-bisabolene (C15H24) are hydrocarbon precursors to a range of industrially relevant chemicals. High-titer microbial synthesis of limonene and α-bisabolene could pave the way for advances in in vivo engineering of tailor-made hydrocarbons, and production at commercial scale. We have engineered the fast-growing unicellular euryhaline cyanobacterium Synechococcus sp. PCC 7002 to produce yields of 4 mg L(-1) limonene and 0.6 mg L(-1) α-bisabolene through heterologous expression of the Mentha spicatal-limonene synthase or the Abies grandis (E)-α-bisabolene synthase genes, respectively. Titers were significantly higher when a dodecane overlay was applied during culturing, suggesting either that dodecane traps large quantities of volatile limonene or α-bisabolene that would otherwise be lost to evaporation, and/or that continuous product removal in dodecane alleviates product feedback inhibition to promote higher rates of synthesis. We also investigate limonene and bisabolene production in the ΔglgC genetic background, where carbon partitioning is redirected at the expense of glycogen biosynthesis. The Synechococcus sp. PCC 7002 ΔglgC mutant excreted a suite of overflow metabolites (α-ketoisocaproate, pyruvate, α-ketoglutarate, succinate, and acetate) during nitrogen-deprivation, and also at the onset of stationary growth in nutrient-replete media. None of the excreted metabolites, however, appeared to be effectively utilized for terpenoid metabolism. Interestingly, we observed a 1.6- to 2.5-fold increase in the extracellular concentration of most excreted organic acids when the ΔglgC mutant was conferred with the ability to produce limonene. Overall, Synechococcus sp. PCC 7002 provides a highly promising platform for terpenoid biosynthetic and metabolic engineering efforts.

No MeSH data available.


Related in: MedlinePlus

High-performance liquid chromatography and NMR identification of secreted organic acids from ΔglgC. (A) HPLC-generated chromatograms showing the presence of secreted metabolites in ΔglgC, which are absent from the wild type culture media, when grown for 48 h under nitrogen-deplete (left panels) and nitrogen-replete (right panels) conditions. (B) The retention times of the metabolites is shown relative to a mixture of 4 mM α-ketoglutarate, pyruvate, succinate, acetate, and α-ketoisocaproate standards. (C) Proton NMR spectrum of the secreted metabolites in ΔglgC, showing chemical shifts and peak splitting patterns that correspond to α-ketoglutarate, pyruvate, succinate, acetate, and α-ketoisocaproate.
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Figure 8: High-performance liquid chromatography and NMR identification of secreted organic acids from ΔglgC. (A) HPLC-generated chromatograms showing the presence of secreted metabolites in ΔglgC, which are absent from the wild type culture media, when grown for 48 h under nitrogen-deplete (left panels) and nitrogen-replete (right panels) conditions. (B) The retention times of the metabolites is shown relative to a mixture of 4 mM α-ketoglutarate, pyruvate, succinate, acetate, and α-ketoisocaproate standards. (C) Proton NMR spectrum of the secreted metabolites in ΔglgC, showing chemical shifts and peak splitting patterns that correspond to α-ketoglutarate, pyruvate, succinate, acetate, and α-ketoisocaproate.

Mentions: Consistent with studies in Synechocystis sp. PCC 6803 (Carrieri et al., 2012; Grundel et al., 2012) and Synechococcus elongatus PCC 7942 (Hickman et al., 2013), we identified pyruvate, α-ketoglutarate, and succinate in the spent media of nitrogen-deprived Synechococcus sp. PCC 7002 ΔglgC cultures using HPLC analysis (Figure 8A, left panels), through a comparison of elution times with known standards (Figure 8B). However, we also detected α-ketoisocaproate and acetate within the same concentration range, metabolites which were not previously reported in the ΔglgC strains of the other cyanobacterial species. Acetate is a common fermentation product putatively derived from acetyl-CoA in cyanobacterial metabolism (Xu et al., 2013), while α-ketoisocaproate is the immediate precursor to leucine. This highlights subtle metabolic distinctions in the metabolism of the ΔglgC mutants between the model cyanobacterial species. An additional unique observation was the excretion of the same organic acids by ΔglgC in nutrient-replete media at the onset of stationary phase, when wild type cells would normally begin to increase glycogen stores for energy reserves (Figure 8A, right panels). However, on a per-cell basis, these concentrations were much lower relative to the concentrations of those excreted during nitrogen starvation. NMR was used as a second, independent method to verify the identity of these metabolites (Figure 8C). Pyruvate, succinate, and α-ketoisocaproate accumulated at concentrations of ~250 μM, while α-ketoglutarate accumulated at ~200 μM, and acetate ~500 μM under nitrogen starvation.


Engineering Limonene and Bisabolene Production in Wild Type and a Glycogen-Deficient Mutant of Synechococcus sp. PCC 7002.

Davies FK, Work VH, Beliaev AS, Posewitz MC - Front Bioeng Biotechnol (2014)

High-performance liquid chromatography and NMR identification of secreted organic acids from ΔglgC. (A) HPLC-generated chromatograms showing the presence of secreted metabolites in ΔglgC, which are absent from the wild type culture media, when grown for 48 h under nitrogen-deplete (left panels) and nitrogen-replete (right panels) conditions. (B) The retention times of the metabolites is shown relative to a mixture of 4 mM α-ketoglutarate, pyruvate, succinate, acetate, and α-ketoisocaproate standards. (C) Proton NMR spectrum of the secreted metabolites in ΔglgC, showing chemical shifts and peak splitting patterns that correspond to α-ketoglutarate, pyruvate, succinate, acetate, and α-ketoisocaproate.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4126464&req=5

Figure 8: High-performance liquid chromatography and NMR identification of secreted organic acids from ΔglgC. (A) HPLC-generated chromatograms showing the presence of secreted metabolites in ΔglgC, which are absent from the wild type culture media, when grown for 48 h under nitrogen-deplete (left panels) and nitrogen-replete (right panels) conditions. (B) The retention times of the metabolites is shown relative to a mixture of 4 mM α-ketoglutarate, pyruvate, succinate, acetate, and α-ketoisocaproate standards. (C) Proton NMR spectrum of the secreted metabolites in ΔglgC, showing chemical shifts and peak splitting patterns that correspond to α-ketoglutarate, pyruvate, succinate, acetate, and α-ketoisocaproate.
Mentions: Consistent with studies in Synechocystis sp. PCC 6803 (Carrieri et al., 2012; Grundel et al., 2012) and Synechococcus elongatus PCC 7942 (Hickman et al., 2013), we identified pyruvate, α-ketoglutarate, and succinate in the spent media of nitrogen-deprived Synechococcus sp. PCC 7002 ΔglgC cultures using HPLC analysis (Figure 8A, left panels), through a comparison of elution times with known standards (Figure 8B). However, we also detected α-ketoisocaproate and acetate within the same concentration range, metabolites which were not previously reported in the ΔglgC strains of the other cyanobacterial species. Acetate is a common fermentation product putatively derived from acetyl-CoA in cyanobacterial metabolism (Xu et al., 2013), while α-ketoisocaproate is the immediate precursor to leucine. This highlights subtle metabolic distinctions in the metabolism of the ΔglgC mutants between the model cyanobacterial species. An additional unique observation was the excretion of the same organic acids by ΔglgC in nutrient-replete media at the onset of stationary phase, when wild type cells would normally begin to increase glycogen stores for energy reserves (Figure 8A, right panels). However, on a per-cell basis, these concentrations were much lower relative to the concentrations of those excreted during nitrogen starvation. NMR was used as a second, independent method to verify the identity of these metabolites (Figure 8C). Pyruvate, succinate, and α-ketoisocaproate accumulated at concentrations of ~250 μM, while α-ketoglutarate accumulated at ~200 μM, and acetate ~500 μM under nitrogen starvation.

Bottom Line: None of the excreted metabolites, however, appeared to be effectively utilized for terpenoid metabolism.Overall, Synechococcus sp.PCC 7002 provides a highly promising platform for terpenoid biosynthetic and metabolic engineering efforts.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Geochemistry, Colorado School of Mines , Golden, CO , USA.

ABSTRACT
The plant terpenoids limonene (C10H16) and α-bisabolene (C15H24) are hydrocarbon precursors to a range of industrially relevant chemicals. High-titer microbial synthesis of limonene and α-bisabolene could pave the way for advances in in vivo engineering of tailor-made hydrocarbons, and production at commercial scale. We have engineered the fast-growing unicellular euryhaline cyanobacterium Synechococcus sp. PCC 7002 to produce yields of 4 mg L(-1) limonene and 0.6 mg L(-1) α-bisabolene through heterologous expression of the Mentha spicatal-limonene synthase or the Abies grandis (E)-α-bisabolene synthase genes, respectively. Titers were significantly higher when a dodecane overlay was applied during culturing, suggesting either that dodecane traps large quantities of volatile limonene or α-bisabolene that would otherwise be lost to evaporation, and/or that continuous product removal in dodecane alleviates product feedback inhibition to promote higher rates of synthesis. We also investigate limonene and bisabolene production in the ΔglgC genetic background, where carbon partitioning is redirected at the expense of glycogen biosynthesis. The Synechococcus sp. PCC 7002 ΔglgC mutant excreted a suite of overflow metabolites (α-ketoisocaproate, pyruvate, α-ketoglutarate, succinate, and acetate) during nitrogen-deprivation, and also at the onset of stationary growth in nutrient-replete media. None of the excreted metabolites, however, appeared to be effectively utilized for terpenoid metabolism. Interestingly, we observed a 1.6- to 2.5-fold increase in the extracellular concentration of most excreted organic acids when the ΔglgC mutant was conferred with the ability to produce limonene. Overall, Synechococcus sp. PCC 7002 provides a highly promising platform for terpenoid biosynthetic and metabolic engineering efforts.

No MeSH data available.


Related in: MedlinePlus