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 psy chrotrophic 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 twofold 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, eight 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.

Keywords: Geomyces sp; Antarctic fungus; Natural red pigment; Protoplast transformation; Transposon mutagenesis.

Fig. 1

The protoplast preparation of Geomyces sp. WNF-15A. (a) Effects of mycelial culture time on protoplast preparation. Wet mycelia (1.5 g) were digested for 1.5 h at 26°C by 2% cellulase and 1.5% snailase with pH adjusted to 7.0. (b) Effects of enzyme compositions on protoplast preparation. C, cellulase; S, snailase. Mycelia were digested for 1.5 h at 26°C with different enzymes with pH adjusted to 7.0. (c) Effects of digesting temperature on protoplast preparation. Mycelia were digested for 1.5 h by 3% cellulase and 1.5% snailase with pH adjusted to 7.0. (d) Effects of digesting time on protoplast preparation. The mycelia were digested at 28°C by 3% cellulase and 1.5% snailase pH adjusted to 7. (e) Effects of digesting pH on protoplast preparation. Mycelia were digested for 1.5 h at 28°C by 3% cellulase and 1.5% snailase.

Fig. 2

Optimization of protoplast transformation in Geomyces sp. WNF-15A. (a) Effects of incubation time on protoplast preparation. Two micrograms of DNA was transformed with 45% PEG. (b) Effects of DNA amount on protoplast preparation. The protoplasts were transformed with 45% PEG and incubated for 36 h. (c) Effects of PEG concentration on protoplast preparation. Two micrograms of DNA was transformed and incubated for 36 h. The calculation of transformation efficiency is shown as Equation 2.

Fig. 3

Construction diagram of transposable systems. Promoter of P_trpC was used. TIRs, terminal inverted repeats of transposons. The transposase recognizes the TIRs to excise the sequence and insert it into a new genomic locus. LTS, left terminal sequence of transposons; RTS, right terminal sequence of transposons. The transposase recognizes the LTS and RTS to carry the rolling-circle transposition. hyg, hygromycin resistance gene. The short arrows represent the primer locations used for verification. Box lengths in the figure do not represent actual relative motif sizes.

Fig. 4

Liquid culture of mutants for red pigment production in shake flask. (a) The highest red pigment yield and culture time of mutant strains at 14°C. (b) The maximum cell growth and culture time of mutant strains at 14°C. (c) The highest red pigment yield and culture time of mutant strains at 20°C. (d) The maximum cell growth and culture time of mutant strains at 20°C. Red pigment production was presented as absorbance at OD₅₂₀.

Fig. 5

Liquid culture of mutants for mycelial growth in shake flask. WT, wild type. Mutants of MG2, MG3, MG4, MG5, MG8, MG9, MG13, MG14, and MG15 were analyzed.