Minos and Restless transposon insertion mutagenesis of psychrotrophic fungus for red pigment synthesis adaptive to normal temperature.

The polar psychrotrophic fungus Geomyces sp. WNF-15A can produce high-quality natural red pigment for the potential use as edible pigment. However, it shows low-temperature-dependent synthesis of red pigment, which limits its large-scale industrial applications due to the difficult and high-cost bioprocess control. This study aims to develop transposon-mediated mutagenesis methods to generate mutants that are able to synthesize red pigment at normal temperature. Four transposable systems, including single and dual transposable systems, were established in this fungus based on the Minos from Drosophila hydei and the Restless from Tolypocladium inflatum. A total of 23 production-dominant mutants and 12 growth-dominant mutants were thus obtained by constructed transposable systems. At 14 °C and 20 °C, the MPS1 mutant strain achieved the highest level of red pigment (OD of 43.3 and 29.7, respectively), which was increased by 78.4% and 128.7% compared to the wild-type, respectively. Of note, 4 mutants (MPS1, MPS3, MPS4 and MPD1) successfully synthesized red pigment (OD of 5.0, 5.3, 4.7 and 4.9, respectively) at 25 °C, which broke the limit of the wild-type production under normal temperature. Generally, the dual transposable systems of Minos and Restless were more efficient than their single transposable systems for mutagenesis in this fungus. However, the positive mutation ratios were similar between the dual and single transposable systems for either Minos or Restless. This study provides alternative tools for genetic mutagenesis breeding of fungi from extreme environments.