Study On Breeding Of High-yield Gellan Mutant Strains And Its Biosynthesis Mechanism Of Gellan In Sphingomonas Paucimobilis

Gellan gum is an extracellular polysaccharide produced by the aerobic fermentation of Sphingomonas paucimobilis,has a good stability,acid resistance,high temperature resistant,heat reversible reversible and less dosage.It is gradually replacing AGAR and carrageenan in industrial applications.However,the low yield of gellan gum produced by the wild strain of Sphingomonas paucimobilis leads to a high price of gellan gum in the market,which limits the large number of applications of gellan gum in the food industry.Therefore,the selection of high-yield strains to improve production efficiency and reduce production cost is conducive to the wide application of gellan gum in the food industry.In this study,Sphingomonas paucimobilis(ATCC31461)was induced by Atmospheric and Room Temperature Plasma(ARTP)to screen the mutant strains with high-yield gellan gum.Single factor and response surface optimization tests were carried out to obtain the optimal fermentation conditions;Genome resequencing and transcriptomics sequencing were used to explore the mechanism of yield increase of high-yield gellan gum mutants.The near-infrared spectroscopy(NIR)technique was used to detect the yield of gellan gum rapidly and nondestructive,and to screen the mutant strains with high yield of gellan gum rapidly.The main research results of this paper are as follows:(1)The content of gellan gum in fermentation broth was detected by near infrared spectroscopy(NIR).The yield of gellan gum in the fermentation broth of Sphingomonas paucimobilis was measured by alcohol precipitation method.At the same time,near infrared spectroscopy was used to scan the fermentation broth.And the original spectra were pretreated by standard normal transformation,multiple scattering correction,smoothing filtering,normalization and derivative processing.The joint interval partial least squares method was used to establish the quantitative model of gellan gum.After optimization,the optimal pretreatment method was normalization method,and the model training set r_c was 0.923,RMSECV was 0.479,the prediction set r_p was 0.9328,and RMSEP was 0.485,indicating that the prediction results of NIR modeling had good correlation with minimal error,which could realize the effective prediction of the production of gellan gum in the fermentation broth of Sphingomonas paucimobilis.(2)Breeding of high-yield strain of gellan gum by ARTP.Using Sphingomonas paucimobilis(ATCC31461)as the original strain,a strain with high yield of gellan gum was selected by ARTP mutagenesis.The results showed that under the conditions of 100 W,10 SLM(Standard Litre per Minute)and 25 s mutagenesis time,the lethality rate of ARTP to the strain was 91.5%.Through the screening of ampicillin,shaking-flask fermentation combined with the online detection technology of near infrared spectroscopy,570 mutant strains were obtained in the preliminary screening,and 5 strains with stable inheritance and high yield were obtained in the re-screening,among which the mutant strain 519 had the highest yield of gellan gum,which increased 33%compared with the original strain.Compared with the original strain,the morphology of the mutant strain 519 has changed significantly,which mainly showed that the length of the mutant strain 519 increased significantly,and part of the end of the mutant strain 519 was flat;Fluo-4/AM assay found that the concentration of intracellular calcium ions increased,indicating that the membrane permeability of mutant strain 519 was improved.Through single factor test and response surface test design,the optimal fermentation conditions of gellan gum high-yield mutant strain 519 were obtained as follows:Glucose 3%(W/V),soybean meal 0.46%(W/V),KH₂PO₄ 0.1%(W/V),K₂HPO₄ 0.15%(W/V),Mg SO₄ 0.06%(W/V),inoculation amount 6.3%(V/V),initial p H 7.5.Under the optimal fermentation conditions,the yield of Gellan gum was 9.43 g/L,1.87 times that of the original strain.(3)Genome resequencing technique was used to analyze the molecular mechanism of high yield of Gellan gum by mutant strain 519.Compared with the original strain,mutant 519 had a total of 25,691 Single Nucleotide Polymorphism(SNP)and 8622 insertions and deletions(Indel).Through GO(Gene Ontology)and KEGG(Kyoto Encyclopedia of Genes and Genomes)enrichment analysis,Mutant genes are mainly involved in biological processes such as carbon metabolism,two-component system,hypoxia-inducible factor signal transduction,oxidative phosphorylation,amino acid synthesis and metabolism.It is speculated that ARTP mutagenesis may change the genes related to the formation and regulation pathway of gellan gum,thus increasing the yield of gellan gum.(4)The biosynthesis mechanism of high yield of Gellan Gellan mutant strain 519 was studied by transcriptome sequencing technology.Compared with the original strain,there were128 differentially expressed genes(DEGs)in the mutant strain 519,among which 38 genes were up-regulated and 90 genes were down-regulated.The DEGs were enriched into the corresponding GO function and KEGG metabolic pathway,the results showed that gellan gum High-yield mutant strain 519 in two-component system(DEGs:23),bacterial chemotaxis(DEGs:20),flagellum assembly(DEGs:23),oxidative phosphorylation and sugar metabolism(DEGs:6)and other metabolic pathways changed dramatically,which likely play an important role on gellan gum yield synthesis.(5)Combined genomic resequencing and transcriptome analysis,gellan gum high-yield mutant 519 was enriched in the combined analysis into the two-component system and carbohydrate metabolism compared with the original strain.7 two-component transcription factors and 5 carbohydrate-related genes were mutated in the genome of mutant strain 519,and the expression levels of 22 two-component genes and 4 carbohydrate-biosynthesis and metabolism-related genes were significantly different.These gene mutations and differential expression genes promote the increase of gellan gum production.