Limits...
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

Accumulation of excreted organic acids in the spend media of cells grown under nitrogen starvation for 48 h. Concentrations of organic acids were measured by HPLC analysis and presented relative to cell optical density at 730 nm. Error bars represent standard deviation from three biological replicates.
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Figure 9: Accumulation of excreted organic acids in the spend media of cells grown under nitrogen starvation for 48 h. Concentrations of organic acids were measured by HPLC analysis and presented relative to cell optical density at 730 nm. Error bars represent standard deviation from three biological replicates.

Mentions: Interestingly, however, we observed a higher extracellular concentration of excreted organic acids in the limonene-producing ΔglgC:LS strain, relative to the ΔglgC and ΔglgC:BIS strains (Figure 9). Under nitrogen starvation, ΔglgC:LS accumulated ~2.5-fold more α-ketoglutarate, and almost twofold more α-ketoisocaproate, pyruvate, and succinate in the spent media compared with the ΔglgC strain, despite no detectable photoautotrophic growth in either strains. It is unclear whether the higher concentrations in the ΔglgC:LS strain are due to increased metabolism toward these end-products, or if a greater proportion of the organic acids are excreted from the cell, but provides an interesting line of future investigation.


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)

Accumulation of excreted organic acids in the spend media of cells grown under nitrogen starvation for 48 h. Concentrations of organic acids were measured by HPLC analysis and presented relative to cell optical density at 730 nm. Error bars represent standard deviation from three biological replicates.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 9: Accumulation of excreted organic acids in the spend media of cells grown under nitrogen starvation for 48 h. Concentrations of organic acids were measured by HPLC analysis and presented relative to cell optical density at 730 nm. Error bars represent standard deviation from three biological replicates.
Mentions: Interestingly, however, we observed a higher extracellular concentration of excreted organic acids in the limonene-producing ΔglgC:LS strain, relative to the ΔglgC and ΔglgC:BIS strains (Figure 9). Under nitrogen starvation, ΔglgC:LS accumulated ~2.5-fold more α-ketoglutarate, and almost twofold more α-ketoisocaproate, pyruvate, and succinate in the spent media compared with the ΔglgC strain, despite no detectable photoautotrophic growth in either strains. It is unclear whether the higher concentrations in the ΔglgC:LS strain are due to increased metabolism toward these end-products, or if a greater proportion of the organic acids are excreted from the cell, but provides an interesting line of future investigation.

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