Stephen K. Picataggio

Asymmetrically acting lycopene β-cyclases (CrtLm) from non-photosynthetic bacteria

Carotenoids have important functions in photosynthesis, nutrition, and protection against oxidative damage. Some natural carotenoids are asymmetrical molecules that are difficult to produce chemically. Biological production of carotenoids using specific enzymes is a potential alternative to extraction from natural sources. Here we report the isolation of lycopene β-cyclases that selectively cyclize only one end of lycopene or neurosporene. The crtLm genes encoding the asymmetrically acting lycopene β-cyclases were isolated from non-photosynthetic bacteria that produced monocyclic carotenoids. Co-expression of these crtLm genes with the crtEIB genes from Pantoea stewartii (responsible for lycopene synthesis) resulted in the production of monocyclic γ-carotene in Escherichia coli. The asymmetric cyclization activity of CrtLm could be inhibited by the lycopene β-cyclase inhibitor 2-(4-chlorophenylthio)-triethylamine (CPTA). Phylogenetic analysis suggested that bacterial CrtL-type lycopene β-cyclases might represent an evolutionary link between the common bacterial CrtY-type of lycopene β-cyclases and plant lycopene β- and ε-cyclases. These lycopene β-cyclases may be used for efficient production of high-value asymmetrically cyclized carotenoids.

Metabolic engineering of an oleaginous yeast for the production of omega-3 fatty acids.

Publisher Summary Numerous clinical studies have demonstrated that the omega-3 fatty acids in fish oil significantly reduce the risk of cardiovascular disease in adults. This chapter discusses the approach to introduce the genes encoding an omega-3 fatty acid biosynthesis pathway into an oleaginous yeast that synthesizes and stores triglycerides as an energy reserve when starved for nitrogen in the presence of an excess carbon source, such as glucose. It explains the development of a clean and sustainable source of omega-3 fatty acids by fermentation, which uses a metabolically engineered strain of the oleaginous yeast Y. lipolytica. While certain strains of Y. lipolytica can accumulate oil up to 40% of the dry cell weight, the only Polyunsaturated Fatty Acid (PUFA) normally synthesized by the organism is Linoleic Acid (LA). Coordinate expression of desaturase genes and elongase genes comprising a “delta6 pathway” was sufficient to demonstrate the synthesis of Eicosapentaenoic Acid (EPA). However, only an integrated strategy, based on the use of strong promoters, an increase in gene copy numbers, the push and pull of carbon into the engineered pathway, and the use of oleaginous condition, resulted in the generation of a high EPA production strain.