Metabolic engineering and directed evolution of the isoprenoid pathway in Escherichia coli

The isoprenoid pathway is a versatile biosynthetic network leading to over 23,000 compounds in nature. Similar to other metabolic pathways, the production of isoprenoids in organisms is limited by the supply of precursors, among other factors. In this study, we use carotenoids, a family of colorful compounds, as the example to investigate the biosynthesis of isoprenoids in Escherichia coli.; First, we used the typical methods in metabolic engineering to identify the rate-determining steps and remove the bottlenecks by overexpression of the key enzymes in this pathway. We engineered an E. coli host that has the capability to supply geranylgeranyl diphosphate (GGPP), a common precursor of isoprenoids, by cloning and overexpressing isopentenyl diphosphate (IPP) isomerase (encoded by idi) from E. coli and GGPP synthase (encoded by gps) from the thermophilic archaebacterium Archaeoglobus fulgidus. These two genes and the gene cluster (crtBIYZW) of the marine bacterium Agrobacterium aurantiacum were introduced into E. coli to produce high levels of astaxanthin, an orange pigment and antioxidant.; Second, we applied directed evolution in the metabolic engineering of the isoprenoid biosynthetic pathways. We performed random mutagenesis on the A. fulgidus GGPP synthase (GPS) to enhance the production of the downstream carotenoids, e.g. lycopene, in our metabolically engineered E. coli. Taking advantage of the color screening system, sixteen GPS mutants were isolated. The sequence information of these mutants showed that the mutations might modulate both gene expression and enzymatic properties.; Finally, directed evolution was used to alter the product specificity of Rhodobacter sphaeroides phytoene desaturase for carotenoid production in E. coli. Two generations of random mutagenesis were performed on the wild type and three mutants producing lycopene in addition to neurosporene were isolated from our color screening system. Site-directed mutagenesis was also performed to determine the effect of each amino acid change.