Developing A Green and Reversiby Regulated Inducible Expression System in Fungus

Aspergillus represents a potential host for expression of recombinant proteins after bacteria, yeast, plant and animal cells. Due to the late start of Aspergillus expression system research, the technology is still immature. And the number of alternative expression vectors and available promoters are limited, which limits the application of Aspergillus expression system. On our previous work, we found a peroxiredoxin from Aspergillus nidulans (AnPrxA) could be expressed at a high level and its expression level responded to H₂O₂ in the medium. Based on the low price and self-decomposition properties of H₂O₂, we explored a novel and efficient Aspergillus nidulans expression system using Aspergillus nidulans as the expression host, AnPrxA promoter as the expression promoter, and H₂O₂ as the expression inducer. Four promoters of different lengths of the 5’untranslated regions (5’-UTR) of Aspergillus nidulans (P_PRX500, P_PRX1000, P_PRX1500 and P_PRX2033) were amplified and cloned in vectors, and the ability of the four promoters to drive GFP expression was evaluated. The results depicted that all four expression systems were sensitive to H₂O₂, and P_PRX500 was sufficient for GFP expression, but the induction ratio of P_PRX2033 is higher. The optimal induction time of H₂O₂ was determined to be 8 h. The optimal induction concentration range was 0.5—2.0 mmol/L. Comparison of the conditions with and without the inducer revealed thatP_prx2033 mediated a 30-fold induction ratio of GFP at the transcriptional level and a 3-fold induction ratio at the protein expression level. In the induction phase of this expression system, the residual concentration could no longer be detected after 2 h of adding 2.0 mmol/L H₂O₂ in the medium, and could no longer be detected after 1 h of adding 0.5 mmol/L H₂O₂.