Polar regions are rich in microbial and product resources. Geomyces sp. WNF-15A is an Antarctic psychrotrophic filamentous fungus producing high quality red pigment with potential for industrial use. However, efficient biosynthesis of red pigment can only realize at low temperature, which brings difficult control and high cost for the large-scale fermentation. This study aims to develop transposon insertion mutation method to improve cell growth and red pigment production adaptive to normal temperature. Genetic manipulation system of this fungus was firstly developed by antibiotic marker screening, protoplast preparation and transformation optimization, by which transformation efficiency of ∼50% was finally achieved. Then transposable insertion systems were established using Helitron, Fot1 and Impala transposons. The transposition efficiency reached 11.9%, 9.4% and 4.6%, respectively. Mutant MP1 achieved the highest red pigment production (OD520 of 39) at 14°C, which was 40% higher than the wild-type strain. Mutant MP14 reached a maximum red pigment production (OD520 of 14.8) at 20°C, which was about 2-fold of the wild-type strain. Mutants MP2 and MP10 broke the repression mechanism of red pigment biosynthesis in the wild-type and allowed production at 25°C. For cell growth, 8 mutants grew remarkably better (12%∼30% biomass higher) than the wild-type at 25°C. This study established an efficient genetic manipulation and transposon insertion mutation platform for polar filamentous fungus. It provides reference for genetic breeding of psychrotrophic fungi from polar and other regions.

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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 (OD520 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 (OD520 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. Graphical 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.