Study On The Promotion Of ε-polylysine Production Of Streptomyces Albulus By Adaptive Evolution And Co-culture

ε-poly-L-lysine(ε-PL)is a polypeptide polymer containing 25-35 L-lysine monomers.It has some advantages of strong bacteriostasis,wide bacteriostatic spectrum,strong water solubility,good thermal stability,biodegradability and high safety.It can be hot-processed at the same time as food,and has been approved by many countries as a food preservative for food preservation.However,due to the late start of ε-PL industry in China,the low production of ε-PL limits the development ofε-PL preservatives,so improving the yield of ε-PL-producing bacteria is the current research focus.Streptomyces albulus CICC 11022 is a ε-PL producing strain.It can be used as the starting strain of this study.In this thesis,the adaptive evolution experiment of S.albulus CICC 11022 was carried out by using antibiotic and low p H pressure,and a strain S.albulus C214 with high ε-PL production was successfully obtained.Then,the mutant genes and intracellular differential metabolites were analyzed by genome re-sequencing and non-targeted metabonomics to explore the mechanism of drug resistance,acid tolerance and high ε-PL production of S.albulus C214.Finally,Bacillus subtilis 168 was screened to increase the ε-PL yield of S.albulus C214 again in the co-culture system.The main results are as follows:1.S.albulus CICC 11022 was adaptively evolved by continuous application of multiple antibiotics and low p H pressure.A high-yield strain S.albulus C214 was screened many times,and the yield of ε-PL was 1.57 g/L,which was 153.23% higher than that of S.albulus CICC 11022(0.62 g/L).After 6 generations of subculture,the yield of ε-PL remained at 1.57±0.01 g/L.The fermentation kinetic parameters of the original strain and the high yield strain were investigated.It was found that the growth and production performance of the high yield strain S.albulus C214 were significantly higher than those of the original strain under the same culture conditions.2.At the level of gene and metabolites,with S.albulus CICC 11022 as control,genome resequencing and intracellular differential metabolites detection of S.albulus C214 were performed respectively.And the key mutant genes and differential metabolites were analyzed.The results showed that there were significant differences and positive mutations in genes and metabolites related to amino acid metabolism,energy metabolism,molecular transcriptional regulation and cell membrane function.S.albulus C214 has a stronger regulatory mechanism,which can make it survive under the pressure of antibiotics and low p H pressure and produce a large number of active secondary metabolites ε-PL.3.There are new findings on ε-PL synthesis and resistance response of S.albulus.In the synthesis of ε-PL,it was found that there were genes encoding serine hydrolase,L-antibiotic dehydrase and mycocerosic acid synthas in S.albulus C214 and positive mutations occurred.The genes encoding serine hydrolase and L-antibiotic dehydrase may play important roles in the degree of polymerization and spatial structure of ε-PL.which is mentioned for the first time in the mechanism of polymerization of L-lysine toε-PL.This discovery has a significance to the study of the synthesis mechanism ofε-PL.The gene encoding mycocerosic acid synthas has been found for the first time in Streptomyces,which is resistant to antibiotic stress and previously existed only in Mycobacterium,Corynebacterium and Nocardia.4.By using the co-culture technique,the high-yield strain S.albulus C214 was co-cultured with 54 strains which may have co-culture characteristics,and 9 strains of primary screening bacteria with good performance were obtained.The re-screening experiment of primary screening bacteria was carried out under different gradient inoculum.It was found that Saccharomyces cerevisiae FD7-1 and B.subtilis 168 could stably promote the ability of S.albulus C214 to produce ε-PL.After the final screening,B.subtilis 168 was selected as a symbiotic bacteria to co-culture with S.albulus C214.The co-culture conditions were optimized.After 96 h of shake flask fermentation,theε-PL yield of S.albulus C214 was 3.30 g/L,which was 33.60% higher than that of pure culture(2.47 g/L).This study proved that co-culture technology is feasible to promote the production of ε-PL by S.albulus,which is also the first time that S.albulus have been studied in the field of co-culture.