Lycopene, or provitamin A, has a range of diverse biological functions and actions, especially in relation to human health for its high nutritional value and contribution in disease defense. For example, it is known to be crucial for maintaining normal vision and associated with reduced risk of several degenerative diseases including cancer (Aggarwal and Shishodia, 2006, http://www.lycopene.org). Biosynthesis of Lycopene occurs in all photosynthetic organisms - bacteria, algae and plants, as well as in some non-photosynthetic bacteria and fungi. It belongs to a family of nutrients known as lycopene-natural pigments especially abundant in red, orange and yellow fruits and vegetables, and in dark green, leafy vegetables. Tomatoes hold the highest natural concentration of lycopene, but the pigment can also be found in watermelon, pink grapefruit, red guava, papaya and apricots, as well as in the skins of red grapes (http://www.findarticles.com/ p/articles/mi_m0FKA/is_4_65/ai_98921160, Evangelia et al., 2005).
PLAN OF WORK
E. coli is considered as a convenient bacterial host for heterologous lycopene production (kang et al., 2005). Most of the carotenogenic genes from bacteria, fungi and higher plants can be functionally expressed in this bacterium. Furthermore, plasmids belonging to different incompatibility groups with different antibiotic resistance markers are available. They can all be introduced simultaneously in E. coli for lycopene synthesis, enabling combinations of individual genes to be used. The potential of E. coli as a lycopene production system has been recently reviewed .
Lycopene has been produced successfully by chemical or biological approaches. However, the latter is favored as natural product for safety considerations. Knowledge on the genes and enzymes in the lycopene pathway and the mechanisms by which accumulation or sequestration of lycopene take place in the cell have been figured out . Transformation with the plant phytoene synthase and the bacterial phytoene desaturase genes was carried out and resulted in mediated synthesis of lycopene from the substrate geranyl pyrophosphate. This information allows the possible recovery of bacterial strains with improved lycopene biosynthesis to be used in the development of new lycopene-rich food products for industrial purposes.
- Isolation of crtB, crtE and crtI from Erwinia herbicola using conventional PCR.
- Cloning of the desire sequences in the pCRScript cloning vector.
- Molecular assay for the cloning fragments.
- Growth and induction assay.
- Quantification of the mRNA levels and lycopene production.