Construction And Optimization Of High Yield Gellan Strains Based On Modification Of Carbon Metabolic Pathways

Gellan gum,produced by Sphingomonas elodea,is a high molecular linear anionic extracellular capsular polysaccharide.Due to its excellent physical and chemical properties,it is widely used in various fields as suspension agent,thickening agent,stabilizer,gel agent,emulsifier,etc.The fermentation of gellan gum is to synthesis of polysaccharide in a high C/N ratio medium with glucose and other carbon substrates,while the primary metabolism also requires a large amount of carbon consumption,competing with polysaccharide synthesis and reducing the yield of gellan.Currently,the main problems of gellan are low yield and high production cost.The selection and utilization of fermentation carbon sources is often neglected,which leads to a large amount of carbon sources being consumed for biomass enhancement rather than product synthesis.In order to solve the problem of efficient carbon source utilization,metabolic engineering methods are used to modify the strains to improve the yield and reduce the production cost of gellan gum,which is of great significance to improve the market of gellan gum.In addition,as a resilient component,high gellan gum production helps to enhance the resilient performance of cells.When cells are in extreme environments,they can protect themselves by regulating the intracellular metabolism to produce certain special substances,including gellan gum,to resist or mitigate the damage caused by the environments.Therefore,using unfavorable environment to stress cells can help to enhance gellan gum yield.To explore the effect of carbon metabolic regulation on gellan gum production,the metabolic network of Sphingomonas elodea ATCC 31461 was modified to optimize the different requirements of carbon source for primary metabolism and polysaccharide synthesis.The knockout strainsΔpgi,Δzwf,Δppc,Δpyk andΔzwf/pgi were obtained by knocking out the genes encoding glucose-6-phosphate dehydrogenase（zwf）and phosphoglucose isomerase（pgi）in the glucose main metabolic pathway,and the genes encoding phosphoenolpyruvate carboxylase（ppc）and pyruvate kinase（pyk）in the PEP depletion pathway,respectively.The growth characteristics,polysaccharide yield and gene expression were analyzed with mixed carbon sources to investigate the effect of gene knockout on gellan gum production.The results showed that the pgi and ppc gene knockout made more carbon source used in gellan synthesis pathway,which led to a improvement of carbon source utilization efficiency.Therefore,the yield of gellan gum increased,with the most obvious increase inΔppc yield,reaching 9.98±0.13 g/L,an increase of 62.5%compared to the original strain.Although the double knockout strainΔzwf/pgi showed a slight increase in gellan gum production,the genetic stability of the strain was poor.After several generations,the cell activity and gellan gum yield decreased significantly.To further enhance the yield of gellan,the culture conditions for carbon co-utilization of constructed highest yield mutantΔppc was optimized.A suitable culture medium is:K₂HPO₄1.5 g/L,KH₂PO₄1 g/L,Mg SO₄·7H₂O 0.6 g/L,yeast extract 0.2 g/L,peptone 2 g/L,sucrose 30 g/L,glycerol 10 g/L.The suitable fermentation conditions is:initial p H 7.2,10%inoculation（v/v）,incubation temperature 30°C,and fermentation at 200 rpm for 72 h.The yield reached12.39±0.48 g/L,which was 24.15%higher thanΔppc before optimization.To further adjusted carbon source metabolism and improve the efficiency of carbon source utilization,the enzyme gene,which is responsible for polysaccharide repeat unit assembly,β-1,4-glucuronyl transferase（gel K）was overexpression in knockout strainsΔpgi andΔppc and wild type strain S.elodea.The growth characteristics and polysaccharide yield of the recombinant strains S-1,Δpgi-1,Δppc-1were analyzed.The yield of gellan gum ofΔpgi-1 increased significantly,reaching 10.12±0.41 g/L,which was 39.2%higher thanΔpgi.Surfactants can not only act as stressors but also enhance the yield of target products by,for example,altering cell membrane permeability.In this study,one excellent strainΔppc-TW08 with high tolerance to Tween-80 and one excellent strainΔppc-Q15 with tolerance to Triton X-100 were obtained by trapezoidal acclimation.The effect of surfactant concentration on the yield of gellan gum was investigated.When triton X-100 was added at 5 g/L,Δppc-Q15 gellan gum production reached23.12±0.69 g/L,which was nearly 1 times higher thanΔppc.Combined with q PCR analysis of key gene expression and glucosyltransferase activity assay,the enchance yield may be related to the increased expression of UDP-glucose pyrophosphorylase gene and glucosyltransferase activity.