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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

Rates of limonene and α-bisabolene production in transformant lines. (A) Limonene accumulation in three independent LS transformant lines (LS.1, LS.2, and LS.3). Yields were quantified by GC–FID analysis of limonene extracted by a dodecane overlay during the 96-h growth phase (Figure 5A, +dodecane). (B) α-Bisabolene extracted from three independent BIS lines (BIS.1, BIS.2, and BIS.3) over the 96-h growth phase (Figure 5B, +dodecane) using a dodecane overlay. (C) Yields of limonene and α-bisabolene extracted from cells harvested after 96 h of photoautotrophic growth (Figure 5) in the presence (solid bars) or absence (hatched bars) of a dodecane overlay. Terpenoids were extracted from cells using a methanol:chloroform extraction method, and quantified by GC–FID. Error bars represent standard deviation from at least three biological replicates.
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Figure 6: Rates of limonene and α-bisabolene production in transformant lines. (A) Limonene accumulation in three independent LS transformant lines (LS.1, LS.2, and LS.3). Yields were quantified by GC–FID analysis of limonene extracted by a dodecane overlay during the 96-h growth phase (Figure 5A, +dodecane). (B) α-Bisabolene extracted from three independent BIS lines (BIS.1, BIS.2, and BIS.3) over the 96-h growth phase (Figure 5B, +dodecane) using a dodecane overlay. (C) Yields of limonene and α-bisabolene extracted from cells harvested after 96 h of photoautotrophic growth (Figure 5) in the presence (solid bars) or absence (hatched bars) of a dodecane overlay. Terpenoids were extracted from cells using a methanol:chloroform extraction method, and quantified by GC–FID. Error bars represent standard deviation from at least three biological replicates.

Mentions: Three independent lines of the LS transformant consistently produced yields over 4 mg limonene L culture−1, with the highest rate of 50 μg L culture−1 h−1 recorded over the exponential growth phase when cells were actively dividing (Figure 6A). As cultures reached higher cell densities, light limitation due to cell shading began to limit photosynthetic growth (Figure 5A), which likely translated into reduced rates of limonene biosynthesis. Assuming that carbon comprised ~50% of the total biomass, the carbon partitioning to limonene was estimated at 0.3%. Yields of α-bisabolene were considerably lower, where average yields in BIS transformant lines ranged between 0.5 and 0.7 mg α-bisabolene L culture−1 (Figure 6B). The highest α-bisabolene production rate was 6 μg L culture−1 h−1 over the active growth phase, equivalent to ~0.06% of assimilated carbon partitioning as α-bisabolene.


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)

Rates of limonene and α-bisabolene production in transformant lines. (A) Limonene accumulation in three independent LS transformant lines (LS.1, LS.2, and LS.3). Yields were quantified by GC–FID analysis of limonene extracted by a dodecane overlay during the 96-h growth phase (Figure 5A, +dodecane). (B) α-Bisabolene extracted from three independent BIS lines (BIS.1, BIS.2, and BIS.3) over the 96-h growth phase (Figure 5B, +dodecane) using a dodecane overlay. (C) Yields of limonene and α-bisabolene extracted from cells harvested after 96 h of photoautotrophic growth (Figure 5) in the presence (solid bars) or absence (hatched bars) of a dodecane overlay. Terpenoids were extracted from cells using a methanol:chloroform extraction method, and quantified by GC–FID. Error bars represent standard deviation from at least three biological replicates.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: Rates of limonene and α-bisabolene production in transformant lines. (A) Limonene accumulation in three independent LS transformant lines (LS.1, LS.2, and LS.3). Yields were quantified by GC–FID analysis of limonene extracted by a dodecane overlay during the 96-h growth phase (Figure 5A, +dodecane). (B) α-Bisabolene extracted from three independent BIS lines (BIS.1, BIS.2, and BIS.3) over the 96-h growth phase (Figure 5B, +dodecane) using a dodecane overlay. (C) Yields of limonene and α-bisabolene extracted from cells harvested after 96 h of photoautotrophic growth (Figure 5) in the presence (solid bars) or absence (hatched bars) of a dodecane overlay. Terpenoids were extracted from cells using a methanol:chloroform extraction method, and quantified by GC–FID. Error bars represent standard deviation from at least three biological replicates.
Mentions: Three independent lines of the LS transformant consistently produced yields over 4 mg limonene L culture−1, with the highest rate of 50 μg L culture−1 h−1 recorded over the exponential growth phase when cells were actively dividing (Figure 6A). As cultures reached higher cell densities, light limitation due to cell shading began to limit photosynthetic growth (Figure 5A), which likely translated into reduced rates of limonene biosynthesis. Assuming that carbon comprised ~50% of the total biomass, the carbon partitioning to limonene was estimated at 0.3%. Yields of α-bisabolene were considerably lower, where average yields in BIS transformant lines ranged between 0.5 and 0.7 mg α-bisabolene L culture−1 (Figure 6B). The highest α-bisabolene production rate was 6 μg L culture−1 h−1 over the active growth phase, equivalent to ~0.06% of assimilated carbon partitioning as α-bisabolene.

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