Aspergillus niger is one of the most common fungus species of the genus Aspergillus. It can produce more than 30 kinds of enzyme products such as amylase, acid protease, cellulase, pectinase and glucose oxidase, and has been applied in the field of food production for a long time.

Due to its high expression and secretory capacity, A. niger has a long tradition in the production of enzymes and organic acids. Many of these products have obtained GRAS (generally regarded as safe) status, and are widely used in biotechnology for the production of food ingredients, pharmaceuticals and industrial enzymes.

A. niger

Comparing to E. coli, eukaryotic post-translational protein processing machinery of A. niger is an added bonus, with a view of using fungi as expression hosts for proteins requiring elaborate posttranslational modification – for example, in the form of protein glycosylation, proteolytic cleavage or formation of multiple disulfide bonds.

Besides, A. niger exhibits a remarkably versatile metabolism, enabling growth on a wide range of substrates under various environmental conditions.
Furthermore, it has become a powerful approach to express heterologous protein taking advantage of its sequenced genome. In a word, A. niger can be used as a perfect heterologous protein expression host as cell factory.

The targeted gene replacement system mainly adopts the principle of homologous gene recombination. The method can be used to delete a gene, remove exons, add a gene and introduce point mutations. In the process of target gene replacement, the construction of vectors is of great importance, which typically includes two arms respectively in homology with the target gene’s homologous sequences on both sides and one selection marker gene which is used to eliminate noise bacteria.

In A.niger, to obtain highly heterologous expression, GlaA5 (promoter of highly expressed glaA enzyme coding gene) and GlaA3 (the terminator of glaA) can be used as the homologous arms on either side of the target gene. Hph (hygromycin) is commonly used as selective marker gene which is promoted by pgPDA and terminated by GlaA3. After several generations, homologous recombination will happen between the two GlaA3 sequences, and leads to the loss of Hph. The deletion of selective marker gene enhances the safety of transgenic A. niger and make continuous transformation of exogenous genes possible.

Target gene replacement

Selective marker gene deletion

Based on the above-mentioned method, exogenous genes can be effectively expressed in A. niger by Agrobacterium-mediated transformation and the selective marker gene can be deleted. However the screening process of positive transformants is time-consuming and laborious including a lot of PCR detection and numerous generations of A. niger culture.

We aim to establish a visual operation system for continuous target genes replacement into the high expression sites in A. niger, which is called “gene tracer”.