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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

GC–FID analyses of solvent overlay-extracted limonene and bisabolene from transformant strains. (A) Hexane-overlay extraction from the LS transformant (LS.1 line as a representative) showed accumulation of a product that was absent in the wild type, and with a similar retention time (4.40 min) to a commercial l-limonene standard. (B) Dodecane overlay extraction from the BIS transformant (BIS.1 line as a representative) revealed a peak with a similar retention time (9.89 min) to a bisabolene standard, which is absent in the wild type. For these experiments, dodecane was used for bisabolene extraction because of the extended period of time required to accumulate detectable levels of bisabolene, whereas hexane was applied for better limonene peak resolution due to the reverse solvent effect observed with dodecane.
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Figure 3: GC–FID analyses of solvent overlay-extracted limonene and bisabolene from transformant strains. (A) Hexane-overlay extraction from the LS transformant (LS.1 line as a representative) showed accumulation of a product that was absent in the wild type, and with a similar retention time (4.40 min) to a commercial l-limonene standard. (B) Dodecane overlay extraction from the BIS transformant (BIS.1 line as a representative) revealed a peak with a similar retention time (9.89 min) to a bisabolene standard, which is absent in the wild type. For these experiments, dodecane was used for bisabolene extraction because of the extended period of time required to accumulate detectable levels of bisabolene, whereas hexane was applied for better limonene peak resolution due to the reverse solvent effect observed with dodecane.

Mentions: The use of an organic solvent overlay has proven to be a successful method for harvesting terpenoids from microbial cultures (Newman et al., 2006; Anthony et al., 2009; Alonso-Gutierrez et al., 2013; Bentley et al., 2013). GC–FID analyses of solvent overlays from LS (Figure 3A) and BIS (Figure 3B) transformants showed prominent peaks with similar retention times to commercial standards of l-limonene (4.40 min) and bisabolene (9.89 min), respectively, which were absent from the wild type. Figure 4 shows the mass spectral analyses of putative l-limonene and α-bisabolene peaks from transformant solvent extracts in comparison to the corresponding reference spectra from the NIST Mass Spectral Library (upper panels). The solvent also extracted additional products common to all strains, including wild type (Figure 3, ~10.5 min retention time), which may be membrane lipids (accumulated levels were below detection limits for mass spectral analysis). A comparison of l-limonene and bisabolene commercially available standards with NIST reference spectra is also shown in Figure 4 (lower panels). The major peak (9.89 min) in the bisabolene standard (Figure 3B) was identified as α-bisabolene, the peak at 9.94 min was bisabolol, while the small peaks with retention times <9.98 min were all sesquiterpenes based on a mass to charge (m/z) of 204.


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)

GC–FID analyses of solvent overlay-extracted limonene and bisabolene from transformant strains. (A) Hexane-overlay extraction from the LS transformant (LS.1 line as a representative) showed accumulation of a product that was absent in the wild type, and with a similar retention time (4.40 min) to a commercial l-limonene standard. (B) Dodecane overlay extraction from the BIS transformant (BIS.1 line as a representative) revealed a peak with a similar retention time (9.89 min) to a bisabolene standard, which is absent in the wild type. For these experiments, dodecane was used for bisabolene extraction because of the extended period of time required to accumulate detectable levels of bisabolene, whereas hexane was applied for better limonene peak resolution due to the reverse solvent effect observed with dodecane.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: GC–FID analyses of solvent overlay-extracted limonene and bisabolene from transformant strains. (A) Hexane-overlay extraction from the LS transformant (LS.1 line as a representative) showed accumulation of a product that was absent in the wild type, and with a similar retention time (4.40 min) to a commercial l-limonene standard. (B) Dodecane overlay extraction from the BIS transformant (BIS.1 line as a representative) revealed a peak with a similar retention time (9.89 min) to a bisabolene standard, which is absent in the wild type. For these experiments, dodecane was used for bisabolene extraction because of the extended period of time required to accumulate detectable levels of bisabolene, whereas hexane was applied for better limonene peak resolution due to the reverse solvent effect observed with dodecane.
Mentions: The use of an organic solvent overlay has proven to be a successful method for harvesting terpenoids from microbial cultures (Newman et al., 2006; Anthony et al., 2009; Alonso-Gutierrez et al., 2013; Bentley et al., 2013). GC–FID analyses of solvent overlays from LS (Figure 3A) and BIS (Figure 3B) transformants showed prominent peaks with similar retention times to commercial standards of l-limonene (4.40 min) and bisabolene (9.89 min), respectively, which were absent from the wild type. Figure 4 shows the mass spectral analyses of putative l-limonene and α-bisabolene peaks from transformant solvent extracts in comparison to the corresponding reference spectra from the NIST Mass Spectral Library (upper panels). The solvent also extracted additional products common to all strains, including wild type (Figure 3, ~10.5 min retention time), which may be membrane lipids (accumulated levels were below detection limits for mass spectral analysis). A comparison of l-limonene and bisabolene commercially available standards with NIST reference spectra is also shown in Figure 4 (lower panels). The major peak (9.89 min) in the bisabolene standard (Figure 3B) was identified as α-bisabolene, the peak at 9.94 min was bisabolol, while the small peaks with retention times <9.98 min were all sesquiterpenes based on a mass to charge (m/z) of 204.

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