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Engineering Limonene and Bisabolene Production in Wild Type and a Glycogen-Deficient Mutant of Synechococcus sp. PCC 7002.

ABSTRACT: The plant terpenoids limonene (C10H16) and ?-bisabolene (C15H24) are hydrocarbon precursors to a range of industrially relevant chemicals. High-titer microbial synthesis of limonene and ?-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) ?-bisabolene through heterologous expression of the Mentha spicatal-limonene synthase or the Abies grandis (E)-?-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 ?-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 ?glgC genetic background, where carbon partitioning is redirected at the expense of glycogen biosynthesis. The Synechococcus sp. PCC 7002 ?glgC mutant excreted a suite of overflow metabolites (?-ketoisocaproate, pyruvate, ?-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 ?glgC 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.

SUBMITTER: Davies FK

PROVIDER: S-EPMC4126464 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Project description:BackgroundOxygenic photosynthetic microorganisms such as cyanobacteria and microalgae have attracted attention as an alternative carbon source for the next generation of biofuels. Glycogen abundantly accumulated in cyanobacteria is a promising feedstock which can be converted to ethanol through saccharification and fermentation processes. In addition, the utilization of marine cyanobacteria as a glycogen producer can eliminate the need for a freshwater supply. Synechococcus sp. strain PCC 7002 is a fast-growing marine coastal euryhaline cyanobacteria, however, the glycogen yield has not yet been determined. In the present study, the effects of light intensity, CO2 concentration, and salinity on the cell growth and glycogen content were investigated in order to maximize glycogen production in Synechococcus sp. strain PCC 7002.ResultsThe optimal culture conditions for glycogen production in Synechococcus sp. strain PCC 7002 were investigated. The maximum glycogen production of 3.5 g L(-1) for 7 days (a glycogen productivity of 0.5 g L(-1) d(-1)) was obtained under a high light intensity, a high CO2 level, and a nitrogen-depleted condition in brackish water. The glycogen production performance in Synechococcus sp. strain PCC 7002 was the best ever reported in the α-polyglucan (glycogen or starch) production of cyanobacteria and microalgae. In addition, the robustness of glycogen production in Synechococcus sp. strain PCC 7002 to salinity was evaluated in seawater and freshwater. The peak of glycogen production of Synechococcus sp. strain PCC 7002 in seawater and freshwater were 3.0 and 1.8 g L(-1) in 7 days, respectively. Glycogen production in Synechococcus sp. strain PCC 7002 maintained the same level in seawater and half of the level in freshwater compared with the optimal result obtained in brackish water.ConclusionsWe conclude that Synechococcus sp. strain PCC 7002 has high glycogen production activity and glycogen can be provided from coastal water accompanied by a fluctuation of salinity. This work supports Synechococcus sp. strain PCC 7002 as a promising carbohydrate source for biofuel production.

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Engineering Limonene and Bisabolene Production in Wild Type and a Glycogen-Deficient Mutant of Synechococcus sp. PCC 7002.

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