Disruption of Aokap6 near the kojic acid gene cluster affects the growth and kojic acid production in Aspergillus oryzae.

Abstract

The kojic acid gene cluster of Aspergillus oryzae plays a key role in kojic acid synthesis. Although the kojic acid gene cluster has been found in 2010, there is little information on the function of the genes located near thekojic acid gene cluster of A. oryzae and whether these genes affect the kojic acid gene cluster containing kojA, kojR and kojT. Here, Aokap6 near thekojic acid gene cluster of A. oryzae was identified and characterized. The Aokap6 disrupted mutants were constructed by the CRISPR/Cas9 system, which exhibited increased mycelium growth and conidial formation. Disruption of Aokap6 enhanced the tolerance to cell wall, oxidative and heat stress but not osmotic stress. Deletion of Aokap6 repressed kojic acid production, together with the reduced expression of kojA, kojR and kojT. Meanwhile, knockout of kojA, kojR and kojT led to the declined expression of Aokap6, indicating that Aokap6 is required for kojic acid production in coordination with kojA, kojR and kojT. Furthermore, overexpression of kojA, kojR and kojT had no effects on the transcript level of Aokap6, and overexpression of kojA in Aokap6 deletion strain could rescue the reduced yield of kojic acid, suggesting that Aokap6 is involved in kojic acid synthesis acting upstream of kojA. These findings provide new insight for the further understanding of kojic acid gene cluster and kojic acid production in A. oryzae.