Salt-tolerance Mechanism Of Penicillium Citrinum YL-1 And Its Safety Analysis In Fish Sauce Fermentation

Penicillium citrinum YL-1 is a filamentous fungus with the highly tolerance of salt(Its highest tolerance is 26%salinity),highly productive of protease and can accelerate the fermentation of fish sauce.It is isolated from the traditional fermented fish sauce in Chaoshan,China.In order to explore the fermentation mechanism of the flavor substances produced by Penicillium citrinum YL-1 and its safety as a fermen-tation strain under high salt concentration of fish sauce,RNA-Seq technique was used to study the difference of transcription under free-salt,10%salt concentration and26%salt concentration,and the mechanism of salt tolerance was analyzed,and provide theoretical basis for its fermentation mechanism.To Analysis the whole genome sequence of Penicillium citrinum YL-1 by using survey sequencing technology,the key synthetic pathways and genes of penicillium toxin and aflatoxin,as well as mycotoxins metabolism which the Penicillium citrinum YL-I may be involved were analyzed,containing non-ribosomal peptide synthetase(NRPS),polyketide synthase(PKS),PKS-NRPS mixed metabolic pathways,terpenoid metabolism and other metabolic pathways to analyze its poten-tial ability to produce mycotoxins and verify its safety.Based on RNA-Seq transcriptome technique to study the differential genes and metabolism of Penicillium citrinum YL-1 in free-salt,low salt(10%salt concentration)and high salt(26%salt concentration),and then analyze its salt tolerance mechanism.The sequencing results showed that 62 differentially expressed genes were obtained in high salt group compared with free-salt group,2189 differentially expressed genes were obtained in high salt group compared with low salt group,18 differentially expressed genes in low salt group compared with free-salt group if 2 times as the threshold for screening gene differential expression.According to the KEGG Pathway analysis,there were 32 gene metabolic pathways that significantly affected when salt-free group been compare with the high-salt group,of which 22 gene metabolic pathways were up-regulated expressed,and 16 gene metabolic pathways were down-regulated expressed;There were 245 gene metabolic pathways significantly affected when hign-salt versus the low-salt group,205 gene metabolic pathways with up-regulated expression,and 177 gene metabolic pathways with down-regulated expression;the gene metabolism pathways significantly affected by the low-salt group compared with the no-salted group were only of the 15 articles,all were presented with down-regulation of gene expression.Penicillium citrinatum YL-1 had more differential genes and enrichment pathways in high-salt treatment compared to low-salt treatment,but there were fewer differential genes and enriched pathways compared normal conditions with low-salt and high-salt treatments.It may be related to the specificity of Penicillium citrinum YL-1 itself,which has different ways to cope with salt stress under high salinity and low salt conditions;meanwhile,after the short-time stress response of Penicillium citrinum YL-1 under high salinity stimulation gene expression in turn remodels the initial(salt-free)level.By analyzing the RNA-Seq transcription data,it was found that the salt tolerance mechanisms of Penicillium citrinum YL-1 under high salt and low salt is different.Under low salinity,Penicillium citrinatumYL-1mainly through changing the expressi-on ofNa~+/H~+antiporter,Na~+/K~+exchanger family,Sodium/calcium exchanger pro-tein,Na~+exporting ATPase,Na~+/H~+exchanger family,and others membrane ion-transporters,such as inorganic ion proteins,promotes the elimination of sodium ions.Under high salinity,Penicillium citrinum YL-1 adapts to a high salt environment through a series of stress responses including:reducting the genes expression of MFS transport protease(HXT),serine/threonine protein phosphatase PP1 subunit catalytic enzyme(PPP1C)and serine/threonine protein kinase RIM15(RIM15)to slow cell division.Strengthening glycolysis synthesis and other pathways to aggregate energy for enhancing cell metabolism to cope with high salt environment.Strengthening the synthesis of series of 40s and 60s ribosomal RNA to promote protein and amino acid synthesis.Especially the genes proA operon associated with high-level expression of proline synthesis transport to synthesize large amounts of proline as a similar soluble small molecule substance to increase cell osmotic pressure against high-salt environment.Regulate alcohol dehydrogenase expression of genes such as(GCY1,AKR1A1/adh),aldehyde dehydrogenase(ALDH),aldehyde reductase(AKR1B)and glycerol kinase(glpK/GK)to enhance synthesis of small molecules such as glycerol to increase intracellular permeability Press.Enhances the expression of genes CatE(a gene encoding catalase),and SOD1(a gene encoding superoxide dismutase)to increase its antioxidant capacity to transporte reactive oxygen species,peroxidation and other toxic substances due to severe metabolism activities.Regulate the expression of cell wall synthesis-related genes such as cell wall anchoring protein(c8097_g1),glucan1,3-?-glucosidase,and xyloglucan endotransglycosylase to alter cell wall structure to adapt high salt environment.Controls the expression genes of serine proteases such as serA(c5647_g1),PHGDH(c1468_g1),and c5863_g3(E1.2.1.3)to secrete salt-tolerant proteases to increase their salt tolerance.To analyze the safety of Penicillium citrinum YL-1 Based on Whole Genome Sequencing Technology.Survey genome sequencing results showed that the whole genome size of Penicillium citrinum YL-1 was 31.92 Mb,and GC content was46.27%.Using the Maker2 gene prediction technique,11980 predicted genes were obtained.Among them,there were 5,417 genes annotated by KOG and 4946 genes annotated by COG,323 pathways were annotated by KEGG database,including 3,525genes.there is annotated to possible mycotoxin-related metabolic pathways include the microcystins synthesis pathway,ubiquinone and other terpenoid-quinone metabo-lic pathways,synthetic of terpenoids backbond,triterpene and sesquiterpenoid syn-thesis pathways,and aflatoxins synthesis pathways and so on,metabolic analysis showed that only a homologous gene of aflatoxin metabolic pathway Afld and five other related genes were annotated,but its complete metabolic pathways were not annotated.Current data indicate that there is no mycotoxin-producing metabolic pathway in Penicillium citrinum YL-1,it is safe to use it in fish sauce fermentation.