Transposon insertion mutation of Antarctic psychrotrophic fungus for red pigment production adaptive to normal temperature

Abstract

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.

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

“…Protoplasts preparation and PEG-mediated transformation of Geomyces sp. WNF-15A were performed as described previously (Ding et al 2021 ). The transposable plasmid was introduced into protoplasts of the wild-type strain, and the protoplasts were regenerated by incubation at solid seed medium plate with 20 g/L agar in the dark and, at 80% humidity and 20 °C or 25 °C for 36–48 h. Then the upper layer medium was covered using solid seed medium plate with 7.5 g/L agar and 30 μg/mL hygromycin B under the same conditions for 5–7 days.…”

“…Then transformants losing plasmids were identified by spore PCR with primer pairs of Am-F/Am-R. Genomic DNA was extracted using the Plant Genomic DNA Extraction Kit (Tiangen). The spore PCR were performed as described previously (Ding et al 2021 ). Transformants without carrying plasmids were further used for genotypic verification.…”

“…And there was no harmful byproduct detected from Geomyces sp. WNF-15A culture compared with the widely used Monascus red pigment in Asia (Ding et al 2021 ). Therefore, the Antarctic Geomyces red pigment has a great potential for industrial use.…”

“…WNF-15A is a typical polar psychrotrophic fungus. It was apt to produce the red pigment at temperature lower than 14 °C, but its synthesis was severely impaired at above 20 °C (even absolutely blocked at 25 °C) (Huang et al 2020 ; Ding et al 2021 ). This cold-adaptive production trait brings difficult and high-cost bioprocess control in large-scale fermentation.…”

“…Although random mutagenesis by chemical and physical methods is easy-to-use, it is generally hard to track the mutation sites in microbial genomes. Transposon insertion mutation represents an alternative mutation method for gene inactivation and phenotype change, which is user-friendly, economically competitive, traceable, and almost no preferences for any special locus, thus aiding in screening, analyzing and identifying gene functions in species with unclear genetic background (Ding et al 2021 ; Kim and Pyykko 2011 ; Paun and Kempken 2015 ; Sandoval-Villegas et al 2021 ). Unlike the wide use in bacteria, identification and application of transposons in fungi are limited.…”