The color characteristics imparted by pigments can significantly contribute to the attractiveness and commercial value of food.Due to concerns about various food safety issues caused by synthetic pigments,consumers tend to choose natural pigments with higher safety performance.Natural red pigment is widely used as an additive in food and cosmetics.However,due to the problems of toxic byproduct and allergen of natural red pigments used in the market,researchers are still engaged in finding alternatives of the current natural red pigments.Antarctic fungus Geomyces sp.WNF-15A can synthesize a natural red pigment with high color value and good water solubility.The acute toxicity test confirmed that the pigment was actually non-toxic,indicating that it had the potential to be developed as an alternative to the currently used commercial natural red pigments.This study aims to optimize the fermentation process of Antarctic fungus Geomyces sp.through various strategies to increase the production of the new kind of natural red pigments and promote its industrial development.First,a fermentation medium for the production of red pigments by Geomyces sp.WNF-15A was screened from the high-yield media of polyketide and terpene natural products developed in our group.Then,effect value of each component of the fermentation medium was analyzed through the Plackett-Burman experimental design,and the concentration of each component was further optimized combined with the steepest descent analysis to obtain a high-yield medium for the red pigments,which contains 34 g/L sucrose,27.6 g/L maltose,28 g/L soluble starch,1.85 g/L tryptone,0.91 g/l.soybean cake powder,0.8 g/L yeast extract,0.3 g/L magnesium sulfate heptahydrate,1.49 g/L sodium glutamate,0.5 g/L potassium dihydrogen phosphate.The resulted red pigments production(OD520)reached 74.5,which was 178%higher than that from the original PDB medium.For industrial application,complex medium is easy to produce other by-products,and some nitrogen sources in the fermentation medium are quality-unstable,so it needs to be simplified.Through a single factor experiment,a simplified fermentation medium containing 28 g/L soluble starch and 1.85 g/L tryptone was obtained step by step,which was suitable for red pigments synthesis.The output of red pigments(OD520)reached 40.7,which was 52%higher than that from the original PDB medium.Further,adding 20 glass beads with diameter of 4 mm was helpful to the fermentation of the Antarctic fungus,which increased the production of red pigments by 8.3%.Second,microbial mutation breeding is an efficient strategy for obtaining dominant mutant strains,which has been widely used in industrial microbial breeding.In this study,the Geomyces sp.WNF-15A was used as the parent strain,and it was mutagenized by the atmospheric pressure room temperature plasma(ARTP)system.A mutant strain with high red pigments production and shortened fermentation phase was obtained and named as Geomyces sp.WNF-15A-M210(M210).When cultured in the simplified medium at 14℃,the maximum production of red pigments(OD520)by M210 reached 50.6,24.4%higher than the production of wild type,and its fermentation phase was also shortened by 33.3%.When cultured at 20℃,M210 still maintained its dominant characteristics.Its production of red pigments was 3.92 times as that of the wild type,and the fermentation phase was shortened by 25%.Based on this,the fermentation conditions of the M210 were further optimized.It was found that pH control strategy effectively increased the production of red pigments.The strategy was to control the initial pH of the simplified medium to 8.5 by adding 6.67 mmol/L phosphate buffer,which maintained a mildly alkaline environment during fermentation process,and ultimately increased production of red pigments by 19%.The effects of dissolved oxygen on the red pigments production were studied through different shaking speeds and liquid volumes.The results showed that the gentle rotation speed and large liquid volume were suitable for the synthesis of red pigments,indicating that it required only mild demand for dissolved oxygen.Adding different concentrations and types of antifoams had no significant effect on the production of red pigments by M210.Furthermore,analysis of the biosynthetic pathway of red pigments will facilitate rational metabolic regulation.The synthesis pathway of Antarctic fungus red pigments was analyzed by feeding specific precursors and inhibitors of different secondary metabolic pathways.The results showed that the addition of sodium acetate,the precursor of polyketide pathway well supported the production of red pigments.Feeding sodium acetate with a final concentration of 3 mmol/L increased red pigments production by 36.6%and productivity by 34.6%.Meanwhile,the addition of polyketide pathway inhibitor iodoacetamide significantly reduced the output and productivity of red pigments.Taken together,the Antarctic fungus red pigments were mainly synthesized by the polyketide pathway.Based on this,a precursor regulation strategy was developed,that is,sodium acetate feeding strategy in the simplified medium was optimized to further increase the red pigments production of M210.Adding sodium acetate with a final concentration of 6 mmol/L in three aliquots at three time points during day 3～5 showed the best effect on increasing the production of red pigments,which increased the final output of red pigments by 27%.Finally,the fermentation process control of red pigments production by M210 was tested in a 5-L bioreactor.The simplified medium was used to cultivate the mutant strain M210.The initial fermentation control parameters were as follows:inoculation volume 6%(v/v),liquid volume 3.5 L,fermentation temperature 14℃,stirring speed at 250 r/min,initial ventilation volume 1.8 L/min,tank pressure at 0.02～0.03 Mpa,2.0 ml defoamer,and the dissolved oxygen(DO)level above 40%.By this,the maximum output of red pigments(OD520)reached 36.7.Further,the pH control strategy and the sodium acetate feeding strategy developed in shake flask were involved in bioreactor.The results showed that the pH control strategy worked well in bioreactor fermentation.The maximum output(OD520)by pH control reached 40.8,which was increased by 11.1%compared to that with natural pH condition.Overall,in this study,the production of Antarctic fungus red pigments was improved through combinatorial optimization strategies.It provides a high-yield mutant,optimum media and effective metabolic regulation strategies for the potential use of the Antarctic fungus red pigments in future.