Breeding Of ε-poly-L-lysine High-yield Strain By Ribosome Engineering And Protoplast Fusion

ε-poly-L-lysine（ε-PL）is a homopolymer amino acid made of 25~35 L-lysine monomers through the dehydration condensation between ε-amino group and theα-carboxylic acid group,it is widely used as food preservatives and drug carriers in Europe,Central Asia and other regions because of its broad bacteriostasis,good thermal stability and high safety.Therefore,it is of great significance to the food industry in China to breed high-yield strain of ε-PL and realize low-cost,high-efficiency fermentation production ofε-PL,because China starts its research late and not realize the industrial production of1,000 tons.In this study,S.albulus AM which is ε-PL-producing strain was used as the initial strain.A high throughput platform system of 24-well microplates culture was established to replace flask fermentation screening.The ε-PL production of strain AM was improved by ribosome engineering and genome shuffling.Morphological study,gene analysis and fermentation performance of high-yield strains were studied to establishing an effective breeding method.The main research contents of this paper are as follows:1.Breeding of ε-poly-L-lysine high-yield strain by ribosome engineering.The significant correlation was found between 24-well microplates culture and shake flask fermentation of strain（P=0.022<0.05）.Streptomycin（Str）,gentamicin（Gen）and genomycin（G418）were identified from four antibiotics as effective antibiotics for strain AM.The positive mutation rate of single MIC streptomycin was the highest（33%）,and the effect of single MIC gentamicin on the synthesis ability of ε-PL was the strongest（36%）.It was found that the positive mutation rate of double resistance（Str+Gen）was the higher（17%）through the second round of ribosome engineering,and the yield of ε-PL reached1.16 g/L.On the basis of double resistance,the high Str-resistant strain S-53（1.41 g/L）and the high Gen-resistant strain G-154（1.58 g/L）were obtained by breeding multiple resistant mutants,which was 2.01-fold and 2.26-fold higher than the original strain.2.Breeding of ε-poly-L-lysine high-yield strain by protoplast fusion.The high Str resistant mutant S-53 and high Gen resistant mutant G-154 were used as parental strains.The optimal conditions for protoplast preparation of S-53 are as follows: Culture time 32 h,lysozyme concentration 5 mg/m L,enzymolysis time 90 min,enzymolysis temperature32℃.The inactivation conditions: 60℃ for 50 min,UV（20 W,10 cm）irradiation for 100 min.The optimal conditions for protoplast preparation of G-154 are as follows: Culture time 24 h,lysozyme concentration 7 mg/m L,enzymolysis time 120 min,enzymolysis temperature 32℃.The inactivation conditions: 60℃ for 60 min,UV（20 W,10 cm）irradiation for 100 min.The best condition of protoplast fusion is 40% PEG（6000）.Eight colony morphology different from original strain were selected from regeneration plate,and a high-yield ε-PL strain SG-162 was screened by two breeding methods,’parental inactivation protoplast fusion’ and ’protoplast fusion combined with gentamicin resistance screening’.The yield reach 2.51 g/L,which was 78% and 58% higher than the parent strain,and was 3.58 times higher than the original strain.3.Morphological study and gene analysis of high-yielding strains.Taking the high-yield strains SG-162 and AM as the research object,it was found that the colony morphology of strain SG-162 was significantly different from strain AM,and the spore growth rate and sporulation ability were weakened by comparing the differences between the two strains in colony morphology,spore growth and differentiation,mutation sites and fingerprint,etc.SG-162 can maintains dense and uniform mycelial pellets during shake flask fermentation.There was a site-specific substitution mutation in the rps L gene of SG-162 that the 34 th base G of S12 protein was mutated to T.By comparing the BOX-PCR,the high-yield strain SG-162 lacked a gene fragment of about 500 bp.4.Study on fermentation performance of high-yield strain SG-162.The results ofε-PL content detection in 6 kinds of fermentation medium showed that the optimum medium was optimized and the yield was 2.51 g/L after 96 h fermentation.After 168 h of fermentation,the final content of ε-PL reached 33.2 g/L,the dry weight of cell reached41.3 g/L,and the unit synthesis capacity of cell reached 0.8 g/g.