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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

MsLS and AgBIS transgene integration to the Synechococcus chromosome. (A) The YFP, MsLS, and AgBIS transgene constructs were introduced to a neutral site (NSI) of genomic DNA (between open-reading frames A0935 and A0936) via double homologous recombination. Constructs contained the Synechocystis sp. PCC 6803 cpcBA (cpc) promoter to drive transgene expression, and a spectinomycin-resistance selectable marker (SmR). Arrows indicate the position of genomic DNA primers used to check for integration and complete chromosomal segregation in transformant lines. (B) Verification of compete chromosomal segregation for the introduced transgenes in YFP, LS, and BIS transformants by colony-PCR using the above-mentioned primers. Three independent transformant lines of LS (LS.1, LS.2, and LS.3) and BIS (BIS.1, BIS.2, and BIS.3) were verified.
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Figure 2: MsLS and AgBIS transgene integration to the Synechococcus chromosome. (A) The YFP, MsLS, and AgBIS transgene constructs were introduced to a neutral site (NSI) of genomic DNA (between open-reading frames A0935 and A0936) via double homologous recombination. Constructs contained the Synechocystis sp. PCC 6803 cpcBA (cpc) promoter to drive transgene expression, and a spectinomycin-resistance selectable marker (SmR). Arrows indicate the position of genomic DNA primers used to check for integration and complete chromosomal segregation in transformant lines. (B) Verification of compete chromosomal segregation for the introduced transgenes in YFP, LS, and BIS transformants by colony-PCR using the above-mentioned primers. Three independent transformant lines of LS (LS.1, LS.2, and LS.3) and BIS (BIS.1, BIS.2, and BIS.3) were verified.

Mentions: Wild type and transformant Synechococcus sp. PCC 7002 strains were maintained on solid A+ media (Stevens et al., 1973) supplemented with 8.25 mM Tris–HCl (pH 8.2) and 0.3% sodium thiosulfate. Where appropriate, spectinomycin was used at a concentration of 50 μg mL−1 and kanamycin at 100 μg mL−1. Liquid cultures were grown in A+ media supplemented with 8.25 mM Tris–HCl (pH 8.2). For nitrogen-deprivation, NaNO3 was omitted from the growth media and replaced mol:mol with NaCl. Nitrogen-deplete media is referred to as A+(−N). Cultures were grown in 250 mL Erlenmeyer flasks on an orbital shaker at 37°C, in an incubator with an atmosphere of 1% CO2 in air, and constant illumination at 250 μmol photons m−2 s−1 of photosynthetically active radiation (PAR). Liquid starter cultures were grown in the presence of antibiotics; however, during experimental procedures antibiotics were eliminated. Transformation of Synechococcus sp. PCC 7002 with the plasmid constructs described above was performed according to established procedures for cyanobacteria (Eaton-Rye, 2011) to generate the strains LS (containing MsLS) and BIS (containing AgBIS). Complete chromosomal segregation for the introduced transgenes was achieved through propagation of multiple generations on selective agar and verified by colony-PCR using the primers: NSI_us_F, 5′-CTAGCACAAATGAAGCCCGAC-3′, and NSI_ds_R, 5′-GCAGATATAAGCAACGGTACAG-3′ (Figure 2). The ΔglgC strain was obtained from D. A. Bryant, which was generated via the insertional disruption of the glgC open reading frame (SYNPCC7002_A0095) with a kanamycin-resistance cassette (KmR) (Guerra et al., 2013). Oligonucleotide primers used to verify glgC disruption in transformant strains are: glgC_F, 5′-TCACGTAGTCGGGTTTGATGTC-3′, and glgC_R, 5′-CACTAAAGTCCACGACACGACC-3′. The ΔglgC strain was also transformed with the MsLS and AgBIS plasmid constructs to generate strains ΔglgC:LS and ΔglgC:BIS, respectively.


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)

MsLS and AgBIS transgene integration to the Synechococcus chromosome. (A) The YFP, MsLS, and AgBIS transgene constructs were introduced to a neutral site (NSI) of genomic DNA (between open-reading frames A0935 and A0936) via double homologous recombination. Constructs contained the Synechocystis sp. PCC 6803 cpcBA (cpc) promoter to drive transgene expression, and a spectinomycin-resistance selectable marker (SmR). Arrows indicate the position of genomic DNA primers used to check for integration and complete chromosomal segregation in transformant lines. (B) Verification of compete chromosomal segregation for the introduced transgenes in YFP, LS, and BIS transformants by colony-PCR using the above-mentioned primers. Three independent transformant lines of LS (LS.1, LS.2, and LS.3) and BIS (BIS.1, BIS.2, and BIS.3) were verified.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: MsLS and AgBIS transgene integration to the Synechococcus chromosome. (A) The YFP, MsLS, and AgBIS transgene constructs were introduced to a neutral site (NSI) of genomic DNA (between open-reading frames A0935 and A0936) via double homologous recombination. Constructs contained the Synechocystis sp. PCC 6803 cpcBA (cpc) promoter to drive transgene expression, and a spectinomycin-resistance selectable marker (SmR). Arrows indicate the position of genomic DNA primers used to check for integration and complete chromosomal segregation in transformant lines. (B) Verification of compete chromosomal segregation for the introduced transgenes in YFP, LS, and BIS transformants by colony-PCR using the above-mentioned primers. Three independent transformant lines of LS (LS.1, LS.2, and LS.3) and BIS (BIS.1, BIS.2, and BIS.3) were verified.
Mentions: Wild type and transformant Synechococcus sp. PCC 7002 strains were maintained on solid A+ media (Stevens et al., 1973) supplemented with 8.25 mM Tris–HCl (pH 8.2) and 0.3% sodium thiosulfate. Where appropriate, spectinomycin was used at a concentration of 50 μg mL−1 and kanamycin at 100 μg mL−1. Liquid cultures were grown in A+ media supplemented with 8.25 mM Tris–HCl (pH 8.2). For nitrogen-deprivation, NaNO3 was omitted from the growth media and replaced mol:mol with NaCl. Nitrogen-deplete media is referred to as A+(−N). Cultures were grown in 250 mL Erlenmeyer flasks on an orbital shaker at 37°C, in an incubator with an atmosphere of 1% CO2 in air, and constant illumination at 250 μmol photons m−2 s−1 of photosynthetically active radiation (PAR). Liquid starter cultures were grown in the presence of antibiotics; however, during experimental procedures antibiotics were eliminated. Transformation of Synechococcus sp. PCC 7002 with the plasmid constructs described above was performed according to established procedures for cyanobacteria (Eaton-Rye, 2011) to generate the strains LS (containing MsLS) and BIS (containing AgBIS). Complete chromosomal segregation for the introduced transgenes was achieved through propagation of multiple generations on selective agar and verified by colony-PCR using the primers: NSI_us_F, 5′-CTAGCACAAATGAAGCCCGAC-3′, and NSI_ds_R, 5′-GCAGATATAAGCAACGGTACAG-3′ (Figure 2). The ΔglgC strain was obtained from D. A. Bryant, which was generated via the insertional disruption of the glgC open reading frame (SYNPCC7002_A0095) with a kanamycin-resistance cassette (KmR) (Guerra et al., 2013). Oligonucleotide primers used to verify glgC disruption in transformant strains are: glgC_F, 5′-TCACGTAGTCGGGTTTGATGTC-3′, and glgC_R, 5′-CACTAAAGTCCACGACACGACC-3′. The ΔglgC strain was also transformed with the MsLS and AgBIS plasmid constructs to generate strains ΔglgC:LS and ΔglgC:BIS, respectively.

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