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 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 spicata l-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.

Dynamics of Photosynthesis in a Glycogen-Deficient glgC Mutant of Synechococcus sp. Strain PCC 7002

“…Synechococcus sp. PCC 7002 wild-type and glgC mutant strains (16,19) were maintained on solid A ϩ medium (20) supplemented with 8.25 mM Tris-HCl (pH 8.2) and 0.3% sodium thiosulfate. This growth medium was also supplemented with 100 g ml Ϫ1 kanamycin for the glgC mutant strain.…”

“…As a gluconeogenic end product, glycogen uses as a precursor ADP-glucose, which is synthesized from glucose 1-phosphate in a reaction catalyzed by ADPglucose pyrophosphorylase (encoded by the glgC gene) (12). The inhibition of glycogen biosynthesis via genetic deletion of the glgC gene eliminates the primary native reductant sink during nitrogen-limited growth, and cyanobacterial glgC mutants have been shown to secrete a suite of organic acids under these conditions (13)(14)(15)(16). Another intriguing phenotype of cyanobacterial glgC mutants is the absence of phycobilisome degradation under nitrogen-limiting conditions (13)(14)(15); however, the impaired signaling mechanism(s) that prevents phycobilisome degradation remains to be determined.…”

Isoprenoid-Based Biofuels: Homologous Expression and Heterologous Expression in Prokaryotes

“…Sesquiterpenes have been proposed as diesel fuels, and their estimated cetane value, cloud point, and energy density are also in the range of those of diesel fuels (2,7,15,75). Recently, metabolic engineering approaches have been utilized in cyanobacteria to exploit their ability to produce isoprenoids from H 2 O and CO 2 (simplest carbon source) (14,22,58,59). The advantages associated with cyanobacteria, such as fast growth compared to that of plants and yeast, high photosynthetic rate, genetic tractability, and sequenced genomes bring them to the forefront of biofuel research.…”

“…Strain engineering efforts have provided an array of new opportunities to engineer alternate microbial strains as hosts for the nonnatural production of isoprenoid-based biofuels (13,15,58,59). Engineering alternate microbial hosts also enables researchers to explore their ability to utilize a vast array of carbon sources for biofuel production.…”

“…Both enzymes have been introduced in microbes either with a heterologous MVA pathway (73,74) or with an optimized endogenous DXP pathway (13). Modulation of the DXP pathway in alternate microbial hosts, such as cyanobacteria, Corynebacterium glutamicum, and Streptomyces sp., has improved biofuel yields in such hosts (13,15,58,59). Combinatorial gene expression of Table 1).…”