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Natural red pigments have been widely used as food and cosmetics additives. However, due to toxic byproducts or allergen issues, it is still necessary to look for some other red pigment products. This study proposed combinatorial strategies to improve production of a new kind of red pigments from the fungus Geomyces WNF-15A, isolated from Antarctica. A high-production medium was developed by statistical experimental design, which was further simplified for industrial use by single-factor experiments. Strain breeding by atmospheric room temperature plasma mutagenesis generated a mutant, Geomyces sp. WNF-15A-M210, which increased production of red pigments by 24.4% and shortened culture phase by 33.3% comparing with the wild-type. The production of red pigments by this mutant favored a weak alkaline condition but required only mild dissolved oxygen tension. Control of initial pH 8.5 (process pH around 7.5) increased red pigments production by 19% comparing with natural condition. Precursor and inhibitor addition experiments indicated that the red pigments were synthesized by polyketide pathway, and feeding 6 mmol/L precursor of sodium acetate by three aliquots at days 3 to 5 improved biosynthesis of red pigments by 27%. Finally, the developed culture process was verified in a 5-L stirred tank bioreactor. The red pigments production of the pH regulation group reached 1.11-fold of the control and 1.95-fold of the precursor regulation group, respectively. This study provides high-production strain, optimized medium, and bioprocess for the possible industrial production of Antarctic Geomyces red pigments in future. PRACTICAL APPLICATION: Antarctic Geomyces red pigments showed high color value, nontoxic characteristic, and good water solubility. It holds potential for industrial use and is under development for food additive in China currently. This study provides an optional manufacturing process for this new kind of red pigments.

Additional file 1: Table S1. Primers used in this study. Figure S1. Mutants screened by single transposable systems of Minos and Restless. (a) Cultured at 20℃. Right in each plate: wild-type (WT); Left in each plate: mutants (MPS1 ~ MPS10). MPS1 ~ MPS7, screened by Minos transposon; MPS8 ~ MPS10, screened by Restless transposon. (b) Cultured at 25 °C. WT, wild-type; MGS1 ~ MGS4, screened by Minos transposon; MGS5 ~ MGS6, screened by Restless transposon. * indicates a significant difference at p < 0.05, ** indicates a significant difference at p < 0.01. The pictures in figure were gained from different incubation time. The mutants were inoculated with the same number of spores as the wild-type. Figure S2. The red pigment production of mutants obtained by single transposable systems in fermentation. (a) Cultured at 14℃. (b) Cultured at 20℃. WT, wild-type; mutants MPS1 ~ MPS10, screened by single transposable systems; DW, biomass dry weight. Determination of absorbance value of red pigment at wavelength of 520 nm (OD520). Figure S3. The growth of mutants obtained by single transposable systems in fermentation at 25 °C. WT, wild-type; (a) MGS1. (b) MGS3. (c) MGS6, screened by single transposable systems; DW, biomass dry weight. Figure S4. The varying amplitude analysis of production and growth of positive mutants in liquid culture. The variation percentage in red pigment production of mutants cultured at 14 °C (a) and 20 °C (c) were shown. The variation percentage in cell growth of mutants cultured at 14 °C (b), 20 °C (d), and 25 °C (e) were shown. WT, wild-type; mutants MPS1 ~ MPS10, MGS1, MGS3 and MGS 6 screened by single transposable systems. Figure S5. Mutants screened by dual transposable systems of Minos and Restless. (a) Cultured at 20℃. Right in each plate, wild-type (WT); Left in each plate: mutants (MPD1 ~ MPD13). MPD1 ~ MPD6, screened by Minos transposon; MPD7 ~ MPD13, screened by Restless transposon. (b) Cultured at 25 °C. Mutants screened by dual transposable systems. WT, wild-type; MGD1 ~ MGD4, screened by Minos transposon; MGD5 ~ MGD8, screened by Restless transposon. * indicates a significant difference at p < 0.05, ** indicates a significant difference at p < 0.01. The pictures in figure were gained from different incubation time. The mutants were inoculated with the same number of spores as the wild-type. Figure S6. The red pigment production of mutants obtained by dual transposable systems in fermentation. a (a) Cultured at 14 °C. (b) Cultured at 20 °C. WT, wild-type; mutants MPD1 ~ MPD13, screened by dual transposable systems; DW, biomass dry weight. Determination of absorbance value of red pigment at wavelength of 520 nm (OD520). Figure S7. The growth of mutants obtained by dual transposable systems in fermentation at 25˚C. WT, wild-type; (a) MGD2. (b) MGD4. (c) MGD6, screened by dual transposable systems; DW, biomass dry weight. Figure S8. The varying amplitude analysis of production and growth of positive mutants in liquid culture. The variation percentage in red pigment production of mutants cultured at 14 °C (a) and 20 °C (c) were shown. The variation percentage in cell growth of mutants cultured at 14 °C (b), 20 °C (d), and 25 °C (e) were shown. WT, wild-type; mutants MPD1 ~ MPD13, MGD2, MGD4 and MGD6 screened by dual transposable systems.

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.

Additional file 1.

Additional file 1: Figure S1. Flowchart of selecting process for the participants.