Team:NEAU-Harbin/application.html

• Background
• Design
• Application

The principal advantage of the “Gene Tracer” System is to select the transgenic A. niger without selective marker gene by observing the color changes of transformants. The deletion of selective marker gene makes it possible to transfer multiple foreign genes continuously to A. niger 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 have to obtain carotenoids from their diets, and may employ them in various ways in metabolism process. Astaxanthin is the strongest antioxidant 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 reaction steps 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 reaction steps from lycopene to astaxanthin.

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

Figure1. The biosynthetic pathway of astaxanthin

Figure2. Genetic engineering of astaxanthin biosynthetic pathway in A. niger