Description

Aspergillus Niger is one species of filamentous fungi which have been widely employed in the fermentation industry, and become a principal source of enzymes and metabolites. With features such as low cost and high productivity and Generally Regarded As Safe (GRAS) status in the food and food processing industry, Aspergillus Niger has achieved increased attention as host for the production of heterologous proteins, such as amylase, acid protease, cellulase, pectinase and glucose oxidase, etc.

In this study, we aim to establish one visual operation system of continuous target gene replacement using Aspergillus Niger. Firstly, a construct contains amilCP gene, which produces blue chromoproteins, and cjBlue, which produces green chromoproteins, fused selective marker gene HPH was made and transformed into the high expression site through homologous recombination in the Aspergillus Niger genome. In this construct, amilCP gene was driven by GlaA5 promoter, and followed by GlaA3 terminator; the HPH and cjBlue fusion gene was driven by pgpdA promoter, and followed by GlaA3 terminator. GlaA5 and GlaA3 can be used as homologous arm sequences that can mediate specific recombination. The selected HPH resistant Aspergillus Niger cell will highly express amilCP and cjBlue protein which can be detected by blue color and cjBlue signal. After several generations, homologous recombination will happen between the two GlaA3 sequences, and lead to the loss of HPH and cjBlue, then get the transgenic Aspergillus Niger cells only express amilCP gene which can be easily detected by blue color. These transgenic Aspergillus Niger cells with blue color can be used as original strain for target gene transformation. Those colonies without blue color will be the real transformants in which amilCP gene is replaced with the target gene. In a word, the visual gene operation system in Aspergillus Niger will be established with which the homologous recombination of target genes will be easily traced by detecting the color of colonies.

So far we have finished the expression vectors construction. The genetic transformation work is in the process.

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