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

Terpenoid biosynthesis via the 2-C-methyl-l-erythritol 4-phosphate (MEP) pathway. The MEP terpenoid biosynthetic pathway converts pyruvate and glyceraldehyde-3-phosphate (GAP) feedstock to the C5 isomers, isopentenyl pyrophosphate (IPP), and dimethylallyl pyrophosphate (DMAPP). The stepwise addition of IPP to DMAPP generates C10 geranyl pyrophosphate (GPP) and C15 farnesyl pyrophosphate (FPP), the respective precursors to monoterpenes and sesquiterpenes. In this study, the heterologous expression of the M. spicatal-limonene synthase (MsLS) and A. grandis (E)-α-bisabolene synthase (AgBIS) in Synechococcus sp. PCC 7002 was designed to draw on GPP and FPP pools, respectively, generated by the native cyanobacterial MEP pathway for the production of the non-native terpenoids l-limonene and α-bisabolene.
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Figure 1: Terpenoid biosynthesis via the 2-C-methyl-l-erythritol 4-phosphate (MEP) pathway. The MEP terpenoid biosynthetic pathway converts pyruvate and glyceraldehyde-3-phosphate (GAP) feedstock to the C5 isomers, isopentenyl pyrophosphate (IPP), and dimethylallyl pyrophosphate (DMAPP). The stepwise addition of IPP to DMAPP generates C10 geranyl pyrophosphate (GPP) and C15 farnesyl pyrophosphate (FPP), the respective precursors to monoterpenes and sesquiterpenes. In this study, the heterologous expression of the M. spicatal-limonene synthase (MsLS) and A. grandis (E)-α-bisabolene synthase (AgBIS) in Synechococcus sp. PCC 7002 was designed to draw on GPP and FPP pools, respectively, generated by the native cyanobacterial MEP pathway for the production of the non-native terpenoids l-limonene and α-bisabolene.

Mentions: In the context of photoautotrophic biotechnology, the terpenoid biosynthetic pathway is of particular interest, as it produces the largest and most diverse array of naturally occurring organic compounds (typically of plant origin) (Davies et al., 2014). The plant terpenoids limonene (C10H16) and bisabolene (C15H24) are recognized as precursors to a range of commercially valuable products, with applications in biofuels, bioplastics, pharmaceutical, nutraceutical, and cosmetic industries (Duetz et al., 2003; Peralta-Yahya et al., 2011). In this study, we engineer the fast-growing euryhaline cyanobacterium Synechococcus sp. PCC 7002 for the production of limonene and α-bisabolene through heterologous expression of the Mentha spicatal-limonene synthase and Abies grandis (E)-α-bisabolene synthase genes. In cyanobacteria, terpenoids are synthesized via the 2-C-methyl-l-erythritol 4-phosphate (MEP) pathway, utilizing photosynthetically generated pyruvate and glyceraldehyde-3-phosphate (GAP) as the primary substrates (Figure 1). Limonene and bisabolene represent new carbon sinks that draw from the native terpenoid pathway at the levels of geranyl pyrophosphate (GPP) and farnesyl pyrophosphate (FPP).


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)

Terpenoid biosynthesis via the 2-C-methyl-l-erythritol 4-phosphate (MEP) pathway. The MEP terpenoid biosynthetic pathway converts pyruvate and glyceraldehyde-3-phosphate (GAP) feedstock to the C5 isomers, isopentenyl pyrophosphate (IPP), and dimethylallyl pyrophosphate (DMAPP). The stepwise addition of IPP to DMAPP generates C10 geranyl pyrophosphate (GPP) and C15 farnesyl pyrophosphate (FPP), the respective precursors to monoterpenes and sesquiterpenes. In this study, the heterologous expression of the M. spicatal-limonene synthase (MsLS) and A. grandis (E)-α-bisabolene synthase (AgBIS) in Synechococcus sp. PCC 7002 was designed to draw on GPP and FPP pools, respectively, generated by the native cyanobacterial MEP pathway for the production of the non-native terpenoids l-limonene and α-bisabolene.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Terpenoid biosynthesis via the 2-C-methyl-l-erythritol 4-phosphate (MEP) pathway. The MEP terpenoid biosynthetic pathway converts pyruvate and glyceraldehyde-3-phosphate (GAP) feedstock to the C5 isomers, isopentenyl pyrophosphate (IPP), and dimethylallyl pyrophosphate (DMAPP). The stepwise addition of IPP to DMAPP generates C10 geranyl pyrophosphate (GPP) and C15 farnesyl pyrophosphate (FPP), the respective precursors to monoterpenes and sesquiterpenes. In this study, the heterologous expression of the M. spicatal-limonene synthase (MsLS) and A. grandis (E)-α-bisabolene synthase (AgBIS) in Synechococcus sp. PCC 7002 was designed to draw on GPP and FPP pools, respectively, generated by the native cyanobacterial MEP pathway for the production of the non-native terpenoids l-limonene and α-bisabolene.
Mentions: In the context of photoautotrophic biotechnology, the terpenoid biosynthetic pathway is of particular interest, as it produces the largest and most diverse array of naturally occurring organic compounds (typically of plant origin) (Davies et al., 2014). The plant terpenoids limonene (C10H16) and bisabolene (C15H24) are recognized as precursors to a range of commercially valuable products, with applications in biofuels, bioplastics, pharmaceutical, nutraceutical, and cosmetic industries (Duetz et al., 2003; Peralta-Yahya et al., 2011). In this study, we engineer the fast-growing euryhaline cyanobacterium Synechococcus sp. PCC 7002 for the production of limonene and α-bisabolene through heterologous expression of the Mentha spicatal-limonene synthase and Abies grandis (E)-α-bisabolene synthase genes. In cyanobacteria, terpenoids are synthesized via the 2-C-methyl-l-erythritol 4-phosphate (MEP) pathway, utilizing photosynthetically generated pyruvate and glyceraldehyde-3-phosphate (GAP) as the primary substrates (Figure 1). Limonene and bisabolene represent new carbon sinks that draw from the native terpenoid pathway at the levels of geranyl pyrophosphate (GPP) and farnesyl pyrophosphate (FPP).

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