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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

Metabolic redistribution in Synechococcus sp. PCC 7002 upon simultaneous blocks to protein and carbohydrate sinks. Protein (amino acids), carbohydrates (mainly glycogen), and lipids represent the major metabolic sinks in Synechococcus sp. PCC 7002. When metabolic flux to glycogen and amino acids were simultaneously blocked, through inactivation of ΔglgC and elimination of nitrogen from the culture medium (red crosses), a suite of organic acids were secreted from the cell as overflow metabolites from central metabolism (red boxes). There is potential to redirect these spill-over intermediates toward biotechnologically relevant pathways to increase yields of engineered products, such as limonene and α-bisabolene via the terpenoid biosynthetic pathway.
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Figure 10: Metabolic redistribution in Synechococcus sp. PCC 7002 upon simultaneous blocks to protein and carbohydrate sinks. Protein (amino acids), carbohydrates (mainly glycogen), and lipids represent the major metabolic sinks in Synechococcus sp. PCC 7002. When metabolic flux to glycogen and amino acids were simultaneously blocked, through inactivation of ΔglgC and elimination of nitrogen from the culture medium (red crosses), a suite of organic acids were secreted from the cell as overflow metabolites from central metabolism (red boxes). There is potential to redirect these spill-over intermediates toward biotechnologically relevant pathways to increase yields of engineered products, such as limonene and α-bisabolene via the terpenoid biosynthetic pathway.

Mentions: Targeted metabolic flux redistribution among the major carbon sinks is the key to producing novel end-products from engineered microbes at commercially available quantities. The goal remains to increase product yield at the expense of biomass accumulation (cell growth and division), without negatively impacting metabolisms associated with the assimilation and direction of carbon to the product. Although disruption of glycogen biosynthesis in Synechococcus sp. PCC 7002 produced an oversupply of central metabolites (α-ketoisocaproate, α-ketoglutarate, pyruvate, succinate, and acetate) (Figure 10), there was no apparent increase in flux though the MEP terpenoid pathway as conveyed by yields of limonene and α-bisabolene reporter products. Furthermore, such a severe perturbation of central carbon metabolism had a detrimental effect on the organism fitness as manifested by a complete growth inhibition. To that end, introduction of alternative routes for carbon partitioning have proven successful in cyanobacteria as carbon fixation rates are stimulated by providing alternative sinks for photosynthetically derived NADPH and ATP (Ducat et al., 2012; Li et al., 2014). While the MEP terpenoid pathway utilizes both NADPH and ATP as cofactors, growth and pathway optimization are required so that photosynthesis and terpenoid biosynthesis remain active despite major metabolic redistributions.


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)

Metabolic redistribution in Synechococcus sp. PCC 7002 upon simultaneous blocks to protein and carbohydrate sinks. Protein (amino acids), carbohydrates (mainly glycogen), and lipids represent the major metabolic sinks in Synechococcus sp. PCC 7002. When metabolic flux to glycogen and amino acids were simultaneously blocked, through inactivation of ΔglgC and elimination of nitrogen from the culture medium (red crosses), a suite of organic acids were secreted from the cell as overflow metabolites from central metabolism (red boxes). There is potential to redirect these spill-over intermediates toward biotechnologically relevant pathways to increase yields of engineered products, such as limonene and α-bisabolene via the terpenoid biosynthetic pathway.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 10: Metabolic redistribution in Synechococcus sp. PCC 7002 upon simultaneous blocks to protein and carbohydrate sinks. Protein (amino acids), carbohydrates (mainly glycogen), and lipids represent the major metabolic sinks in Synechococcus sp. PCC 7002. When metabolic flux to glycogen and amino acids were simultaneously blocked, through inactivation of ΔglgC and elimination of nitrogen from the culture medium (red crosses), a suite of organic acids were secreted from the cell as overflow metabolites from central metabolism (red boxes). There is potential to redirect these spill-over intermediates toward biotechnologically relevant pathways to increase yields of engineered products, such as limonene and α-bisabolene via the terpenoid biosynthetic pathway.
Mentions: Targeted metabolic flux redistribution among the major carbon sinks is the key to producing novel end-products from engineered microbes at commercially available quantities. The goal remains to increase product yield at the expense of biomass accumulation (cell growth and division), without negatively impacting metabolisms associated with the assimilation and direction of carbon to the product. Although disruption of glycogen biosynthesis in Synechococcus sp. PCC 7002 produced an oversupply of central metabolites (α-ketoisocaproate, α-ketoglutarate, pyruvate, succinate, and acetate) (Figure 10), there was no apparent increase in flux though the MEP terpenoid pathway as conveyed by yields of limonene and α-bisabolene reporter products. Furthermore, such a severe perturbation of central carbon metabolism had a detrimental effect on the organism fitness as manifested by a complete growth inhibition. To that end, introduction of alternative routes for carbon partitioning have proven successful in cyanobacteria as carbon fixation rates are stimulated by providing alternative sinks for photosynthetically derived NADPH and ATP (Ducat et al., 2012; Li et al., 2014). While the MEP terpenoid pathway utilizes both NADPH and ATP as cofactors, growth and pathway optimization are required so that photosynthesis and terpenoid biosynthesis remain active despite major metabolic redistributions.

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