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

Abstract

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 for 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 this strategy. At 14℃, the MPS1 mutant achieved the highest level of red pigment (OD520 of 43.3), which was 78.4% higher than the wild-type. Red pigment production of MPS1 also reached the highest level (OD520 of 29.7) among all the mutants at 20℃, which increased by 128.7% compared to the wild-type. Of note, 4 mutants (MPS1, MPS3, MPS4 and MPD1) successfully synthesized red pigment at 25℃, which broke the production repression limit in the wild-type under normal temperature. Generally, the transposition of 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.