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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

Photoautotrophic growth of transformant lines in the presence and absence of a dodecane overlay. (A) Chlorophyll and biomass accumulation [as measured by dry cell weight (DCW)] over 96 h in the YFP control strain, and three independent LS lines (LS.1, LS.2, and LS.3) in comparison to wild type. Left panels show growth in the absence of a solvent overlay, while right panels display growth in the presence of a dodecane overlay. (B) Comparison of chlorophyll and biomass accumulation (DCW) between wild type and three independent BIS transformant lines (BIS.1, BIS.2, and BIS.3), in the absence (left panels) and presence (right panels) of a dodecane overlay. Error bars represent standard deviation from at least three biological replicates.
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Figure 5: Photoautotrophic growth of transformant lines in the presence and absence of a dodecane overlay. (A) Chlorophyll and biomass accumulation [as measured by dry cell weight (DCW)] over 96 h in the YFP control strain, and three independent LS lines (LS.1, LS.2, and LS.3) in comparison to wild type. Left panels show growth in the absence of a solvent overlay, while right panels display growth in the presence of a dodecane overlay. (B) Comparison of chlorophyll and biomass accumulation (DCW) between wild type and three independent BIS transformant lines (BIS.1, BIS.2, and BIS.3), in the absence (left panels) and presence (right panels) of a dodecane overlay. Error bars represent standard deviation from at least three biological replicates.

Mentions: Photoautotrophic growth rates, as measured by chlorophyll and biomass accumulation, were comparable between wild type and the YFP control strain (Figure 5A, left panels). This verified that NSI is a suitable “neutral” site for transgene integration in Synechococcus sp. PCC 7002 under the defined experimental conditions, because cell growth rate was not adversely affected by the genetic manipulation at this locus. Rates of chlorophyll and biomass accumulation in the LS and BIS transformants were also comparable to that of the wild type (Figure 5, left panels), confirming that expression of the MsLS and AgBIS transgenes, and subsequent intracellular accumulation of limonene and α-bisabolene, did not adversely affect cell fitness under our experimental conditions.


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)

Photoautotrophic growth of transformant lines in the presence and absence of a dodecane overlay. (A) Chlorophyll and biomass accumulation [as measured by dry cell weight (DCW)] over 96 h in the YFP control strain, and three independent LS lines (LS.1, LS.2, and LS.3) in comparison to wild type. Left panels show growth in the absence of a solvent overlay, while right panels display growth in the presence of a dodecane overlay. (B) Comparison of chlorophyll and biomass accumulation (DCW) between wild type and three independent BIS transformant lines (BIS.1, BIS.2, and BIS.3), in the absence (left panels) and presence (right panels) of a dodecane overlay. Error bars represent standard deviation from at least three biological replicates.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Photoautotrophic growth of transformant lines in the presence and absence of a dodecane overlay. (A) Chlorophyll and biomass accumulation [as measured by dry cell weight (DCW)] over 96 h in the YFP control strain, and three independent LS lines (LS.1, LS.2, and LS.3) in comparison to wild type. Left panels show growth in the absence of a solvent overlay, while right panels display growth in the presence of a dodecane overlay. (B) Comparison of chlorophyll and biomass accumulation (DCW) between wild type and three independent BIS transformant lines (BIS.1, BIS.2, and BIS.3), in the absence (left panels) and presence (right panels) of a dodecane overlay. Error bars represent standard deviation from at least three biological replicates.
Mentions: Photoautotrophic growth rates, as measured by chlorophyll and biomass accumulation, were comparable between wild type and the YFP control strain (Figure 5A, left panels). This verified that NSI is a suitable “neutral” site for transgene integration in Synechococcus sp. PCC 7002 under the defined experimental conditions, because cell growth rate was not adversely affected by the genetic manipulation at this locus. Rates of chlorophyll and biomass accumulation in the LS and BIS transformants were also comparable to that of the wild type (Figure 5, left panels), confirming that expression of the MsLS and AgBIS transgenes, and subsequent intracellular accumulation of limonene and α-bisabolene, did not adversely affect cell fitness under our experimental conditions.

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