Construction Of A Sphingomonas Paucimobilis Mutant Producing Low-acyl Gellan Gum And Optimization Of Its Production Process

Gellan gum is a kind of extracellular soluble heteropolysaccharide produced by Sphingomonas paucimobilis.It has good rheological and gel properties,easy biodegradation,safety and non-toxicity,so it is widely used in food,daily chemical,pharmaceutical and other industrial fields,and can be used as thickener,emulsifier and stabilizer.The low-acyl gellan gum formed by deacetylation of natural high-acyl gellan gum has the advantages of low gel temperature,easy impurity separation and high gel strength.it is the most important commercial gel product at present.At present,the production methods of low-acyl gellan gum are scarce.In this thesis,the Red/ET recombinant system suitable for S.paucimobilis was constructed,which was used to knock out the acetyltransferase gene（nat）of S.paucimobilis ATCC31461 and construct the acetyl defective strain which could produce low-acyl gellan gum directly.Furthermore,the fermentation medium composition of acetyl deficient engineering strain S.paucimobilis LY126 was optimized by single factor test and response surface experiment,and the yield of low-acyl gellan gum was increased.The main contents of this thesis are as follows:（1）Five strains with different promoter expression plasmids were constructed.The strains containing different promoters were induced with corresponding inducers.The best inducible promoter controlling the expression of recombinase in S.paucimobilis was identified as the P_tac promoter through GFP fluorescence detection.In this study,Sp Rec ET,an endogenous protein with recombination function,was identified in S.paucimobilis LH128 through BLAST analysis by NCBI.To express the recombinase,an IPTG-induced plasmid(p BBR1-kan-P_tac-Sp Rec ET)was constructed and electrotransformed into S.paucimobilis.As a result,we successfully obtained S.paucimobilis Sp Rec ET.By optimizing the recombination conditions such as homologous arm length,DNA addition and induction time,the optimum homologous arm length of Red/ET recombinant system of S.paucimobilis was 100 bp,the optimum DNA addition was 1500 ng/m L,and the optimum induction time was 2 h.The Red/ET recombinant system of S.paucimobilis was successfully constructed.（2）The acetyltransferase gene（nat）of S.paucimobilis ATCC31461 was knocked out by the recombinant system of S.paucimobilis Red/ET,and the engineering strain S.paucimobilis LY126 with acetyl deficiency was obtained.Shake flask fermentation showed that the acetyl content of cold gel produced by acetyltransferase deletion strain S.paucimobilis LY126 was 1.66%,which was 32.79%lower than that of the original strain,the viscosity of fermentation liquid was 7520 m Pa·s,36.59%lower than that of the original strain,and the gum production rate was 1.53%.Increased by 8.65%compared with the original strain.After the acetyltransferase gene of S.paucimobilis ATCC31461 was knocked out by Red/ET recombinant system,the acetyl content of the gel produced by the strain was reduced successfully.（3）The fermentation medium composition of acetyl deficient engineering strain S.paucimobilis LY126 producing low-acyl gellan gum was optimized by single factor experiment.After optimization,the optimum carbon source was glucose,and the optimum addition amount of glucose was 50.0 g/L,and the optimum nitrogen source was soybean meal,and the optimum addition amount of soybean meal was 8.0 g/L.The optimum addition amount of K₂HPO₄ was 5.0 g/L,The optimum addition amount of KH₂PO₄ was 5.0g/L,and the optimum addition amount of Mg SO₄ was 0.9 g/L.The fermentation medium of low-acyl gellan gum was further optimized by response surface experiment,and the high yield medium composition of low-acyl gellan gum was optimized:glucose 50.0 g/L,soybean meal 8.0 g/L,K₂HPO₄ 5.0 g/L,KH₂PO₄ 5.0 g/L,Mg SO₄ 0.9 g/L.under these conditions,the gum production rate of low-acyl gellan gum was 2.38%,which was 55.40%higher than that before optimization.（4）Based on the optimized fermentation medium,the production experiment of acetyl defective engineering strain S.paucimobilis LY126 producing low-acyl gellan gum was carried out in 10 L fermentor.After 56 h fermentation in 10 L fermentor,the yield of low-acyl gellan gum was 2.41%,and the viscosity of fermentation liquid was 12143 m Pa·s.