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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

Physiology of Synechococcus sp. PCC 7002 ΔglgC. (A). Comparison of photoautotrophic growth rates in wild type and ΔglgC cultures in nitrogen-replete and nitrogen-deplete media, as measured by changes of optical density (730 nm) and chlorophyll content over 48 h. (B) Whole cell absorption spectra of wild type and ΔglgC cells after 48 h growth in nitrogen-replete and nitrogen-deplete media. (C) Differences in pigmentation of wild type and ΔglgC cultures after 48 h growth in the presence or absence of nitrogen in the media. (D) Carbohydrate content (reported as glucose equivalents) of wild type and ΔglgC cells at the different stages of growth (0, 24, and 48 h) shown in (A). Error bars represent standard deviation from three biological replicates, and are hidden beneath the marker if not apparent.
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Figure 7: Physiology of Synechococcus sp. PCC 7002 ΔglgC. (A). Comparison of photoautotrophic growth rates in wild type and ΔglgC cultures in nitrogen-replete and nitrogen-deplete media, as measured by changes of optical density (730 nm) and chlorophyll content over 48 h. (B) Whole cell absorption spectra of wild type and ΔglgC cells after 48 h growth in nitrogen-replete and nitrogen-deplete media. (C) Differences in pigmentation of wild type and ΔglgC cultures after 48 h growth in the presence or absence of nitrogen in the media. (D) Carbohydrate content (reported as glucose equivalents) of wild type and ΔglgC cells at the different stages of growth (0, 24, and 48 h) shown in (A). Error bars represent standard deviation from three biological replicates, and are hidden beneath the marker if not apparent.

Mentions: At a light intensity of 250 μmol photons m−2 s−1 PAR, the ΔglgC strain displayed slightly impaired photoautotrophic growth in nutrient-replete media, as measured by OD730 nm and chlorophyll content (Figure 7A, left panels). In contrast to the wild type, ΔglgC did not grow photoautotrophically in nitrogen-deplete medium (Figure 7A, right panels) due to its inability to degrade the light-harvesting phycobilisomes as a source of nitrogen (Carrieri et al., 2012; Grundel et al., 2012; Guerra et al., 2013; Hickman et al., 2013). Accordingly, the absorbance spectrum of ΔglgC cells in nitrogen-deplete media shows the presence of phycobilin, which is absent in the wild type after nitrogen-deprivation (Figure 7B). As a result, the ΔglgC culture retained the blue–green hue under nitrogen-deprivation, while the wild type appeared more yellow–green due to the unmasking of chlorophyll a as the blue-pigmented phycobiliproteins were degraded (Figure 7C). We confirmed that the reducing carbohydrate content of ΔglgC was diminished compared to the wild type, particularly during stationary phase at 48 h in nutrient-replete media, and also during the first 24 h after the onset of nitrogen-deprivation (Figure 7D). These data confirmed that ΔglgC is defective in glycogen biosynthesis, and that our experimental conditions replicated the phenotypes previously observed in ΔglgC mutants of other cyanobacterial species (Carrieri et al., 2012; Grundel et al., 2012; Guerra et al., 2013; Hickman et al., 2013), and in Synechococcus sp. PCC 7002 (Guerra et al., 2013).


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)

Physiology of Synechococcus sp. PCC 7002 ΔglgC. (A). Comparison of photoautotrophic growth rates in wild type and ΔglgC cultures in nitrogen-replete and nitrogen-deplete media, as measured by changes of optical density (730 nm) and chlorophyll content over 48 h. (B) Whole cell absorption spectra of wild type and ΔglgC cells after 48 h growth in nitrogen-replete and nitrogen-deplete media. (C) Differences in pigmentation of wild type and ΔglgC cultures after 48 h growth in the presence or absence of nitrogen in the media. (D) Carbohydrate content (reported as glucose equivalents) of wild type and ΔglgC cells at the different stages of growth (0, 24, and 48 h) shown in (A). Error bars represent standard deviation from three biological replicates, and are hidden beneath the marker if not apparent.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4126464&req=5

Figure 7: Physiology of Synechococcus sp. PCC 7002 ΔglgC. (A). Comparison of photoautotrophic growth rates in wild type and ΔglgC cultures in nitrogen-replete and nitrogen-deplete media, as measured by changes of optical density (730 nm) and chlorophyll content over 48 h. (B) Whole cell absorption spectra of wild type and ΔglgC cells after 48 h growth in nitrogen-replete and nitrogen-deplete media. (C) Differences in pigmentation of wild type and ΔglgC cultures after 48 h growth in the presence or absence of nitrogen in the media. (D) Carbohydrate content (reported as glucose equivalents) of wild type and ΔglgC cells at the different stages of growth (0, 24, and 48 h) shown in (A). Error bars represent standard deviation from three biological replicates, and are hidden beneath the marker if not apparent.
Mentions: At a light intensity of 250 μmol photons m−2 s−1 PAR, the ΔglgC strain displayed slightly impaired photoautotrophic growth in nutrient-replete media, as measured by OD730 nm and chlorophyll content (Figure 7A, left panels). In contrast to the wild type, ΔglgC did not grow photoautotrophically in nitrogen-deplete medium (Figure 7A, right panels) due to its inability to degrade the light-harvesting phycobilisomes as a source of nitrogen (Carrieri et al., 2012; Grundel et al., 2012; Guerra et al., 2013; Hickman et al., 2013). Accordingly, the absorbance spectrum of ΔglgC cells in nitrogen-deplete media shows the presence of phycobilin, which is absent in the wild type after nitrogen-deprivation (Figure 7B). As a result, the ΔglgC culture retained the blue–green hue under nitrogen-deprivation, while the wild type appeared more yellow–green due to the unmasking of chlorophyll a as the blue-pigmented phycobiliproteins were degraded (Figure 7C). We confirmed that the reducing carbohydrate content of ΔglgC was diminished compared to the wild type, particularly during stationary phase at 48 h in nutrient-replete media, and also during the first 24 h after the onset of nitrogen-deprivation (Figure 7D). These data confirmed that ΔglgC is defective in glycogen biosynthesis, and that our experimental conditions replicated the phenotypes previously observed in ΔglgC mutants of other cyanobacterial species (Carrieri et al., 2012; Grundel et al., 2012; Guerra et al., 2013; Hickman et al., 2013), and in Synechococcus sp. PCC 7002 (Guerra et al., 2013).

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