Team:NEAU-Harbin/application.html

• Background
• Design
• Application

The principal advantages of the Gene Tracer—i.e. the Visual Gene Replacement System are to select the transgenic A. niger by observing the color changing provided by the reporter gene and to delete the selective marker gene by homologous recombination. The deletion of selective marker gene makes it possible to transfer multiple foreign genes continuously to A. niger without using multiple selective markers and to manufacture safer products. Our aim is to import the whole astaxanthin metabolic pathway which contains 6 enzymes into A. niger.

Astaxanthin is one important kind of Carotenoids. Carotenoids are organic pigments that are found in the chloroplasts and chromoplasts of plants and other photosynthetic organisms, including some bacteria and some fungi. Carotenoids can be produced from fats and other basic organic metabolic building blocks by all these organisms. Carotenoids generally cannot be manufactured by species in the animal kingdom so animals obtain carotenoids in their diets, and may employ them in various ways in metabolism. Astaxanthin is the strongest antioxidants in the nature, which can effectively remove oxygen free radicals within the cell and enhance cell regeneration. For human beings, it is helpful to protect the skin health, promote hair growth, delay aging, alleviate sports fatigue and increase body energy.

The Astaxanthin biosynthetic pathway contains 6 enzymes (Figure1). CrtE (geranylgeranyl diphosphate synthase), CrtB (phytoene synthase) and CrtI (phytoene desaturase) catalyze the first 3 steps reaction from FPP (farnesyl diphosphate) to lycopene. CrtY (lycopene β-cyclse), CrtZ (3,(3)- β-ionone hydroxylase) and CrtW (β-carotene ketolase) catalyze the last 3 steps reaction from lycopene to astaxanthin.

Using the parts provided by the IGEM, which contain CrtE, CrtB, CrtI, CrtY, CrtZ and CrtW genes, we respectively fused the first 3 enzymes coding genes crtE,crtB, and crtI together (the fusion gene is named crtEBI) and the last 3 genes crtY,crtZ and crtW together (the fusion gene is named crtYZW) by proper linkers. The two fusion genes will then be transferred respectively to GlaA and AmyA high expression site of A. niger through our Visual Gene Replacement System (Figure 2). If the fused proteins work well, we will be able to effectively produce astaxanthin in A. niger.