Screeing Of ε-poly-L-lysine High-yield Strain Based On Ribosome Engineering Technology And Analysis Of Its High-yield Mechanism

ε-Poly-L-lysine（ε-PL）is a cationic polypeptide made of 25-35 L-lysine residues linkedtogether via amide bonds between theε-amino group and theα-carboxylic acid group fromStreptomyces and Bacillus.It has been widely used as natural food preservatives and freshness in Japan,South Korea,United States,China and other countries because of wide range of use of pH,good thermal stability,its broad bacteriostasis,strong water solubility and high safety.Meanwhile,as a cationic biopolymer,ε-PL is also widely used in drug carriers and biomaterials.Therefore,it is very important to breed high-yield strain ofε-PL and explore its high-yield mechanism to realize low-cost,high-efficiency fermentation production ofε-PL.In this paper,a high throughput platform for S.albulus culture andε-PL detection was established.Theε-PL production of S.albulus M-Z18 was improved by continuous introduction of streptomycin resistance and genome shuffling.Then,the differences between mutant and the parent strain were systematically analyzed from the physical and chemical level.Meanwhile,the changes in protein level were analyzed with the help of comparative proteomics technology,which was verified by metabolic engineering.Finally,with the help of comparative genomics technology,the changes in genome level between the mutant and the starting strain were deeply analyzed,and the high-yielding metabolic map ofε-PL was preliminarily constructed.Major results achieved with this research are highlighted below:（1）A high throughput platform for S.albulus culture andε-PL detection was established.Taking the titer ofε-PL A₄₆₅ as an index,the best culture system of 24/48-well microplates,the evaluation ofε-PL yield by using microplates culture instead of shaking flask culture,and the comparison between microplates culture and shaking flask culture were investigated.The titer of 24-well microplates and shake flask had a good correlation,and the correlation coefficient R reached 0.812.The 24/48-well microplates can be determined by microplate reader.The results showed that there was a good correlation between spectrophotometer and microplate reader for the titer of shake flask,and the titer of shake flask and 24-well microplates fermentation.The correlation coefficients R were 0.924 and 0.868,respectively.The results show that the high-throughput platform andε-PL detection was easy to operate and have large processing capacity,saving raw materials,fast and efficient.Compared with the traditional method,it saves a lot of reagents,shortens the screening cycle of mutants,and can significantly improve the screening efficiency ofε-PL high-yield mutations（2）Highε-PL-producing strains were obtained by continuous introduction of streptomycin resistance and genome shuffling from S.albulus M-Z18.Four effective antibiotics were identified from 8 antibiotics with high mutation to streptomyces,streptomycin was the best.So,streptomycin was selected as the resistance marker of repeated mutagenesis.The highest-yield mutant S.albulus SS-62 was generated after three rounds of screening,and achievedε-PL production of 3.04 g·L^-1 in shake-flask fermentation,which was 1.79-fold higher than the parent strain.On this basis,the highest-yield mutants S.albulus SG-86 and S.albulus GR-42 were obtained by genome shuffling and genome shuffling combined with ribosome engineering.The production ofε-PL reached 4.16 g·L^-1 and 4.01 g·L^-1,respectively,which increased by 145%and 136%compared with the original strain.（3）The differences between S.albulus M-Z18 and S.albulus SS-62 were systematically analyzed from the physical and chemical level.Firstly,the differences in morphology,differentiation,streptomycin resistance,fermentation performance and mutation sites among them were compared.In addition,the mutations of rpsL and rsmG genes were found in SS-62.The metabolic flow distribution,ATP synthesis and key enzyme activities of the two strains were compared.It was found that the metabolic flow of the mutant strain was redistributed.The flux to the precursor ofε-PL,the ATP synthesis and key enzyme activity were all increased.（4）The changes of S.albulus M-Z18 and S.albulus SS-62 in protein level were analyzed with the help of iTRAQ quantitative proteomics technology.A total of 401 differentially expressed proteins were identified in S.albulus SS-62.These differentially expressed proteins are mainly related to carbohydrate metabolism,amino acid metabolism and energy metabolism.It was indicated that SS-62 could not only strengthen theε-PL precursor metabolism and energy metabolism but also tune the pathways related to transcriptional regulation and translation,suggesting a better intracellular metabolic environment for the synthesis ofε-PL.Based onthe results of comparative proteomics analysis,overexpression production ofε-PL synthetase and ribosome recycling factor increased the batch fermentation ofε-PL andε-PL/DCW by43.3%and 9.7%,respectively.（5）With the help of comparative genomics technology,the changes in genome level between S.albulus M-Z18 and S.albulus SG-86 were deeply analyzed.The results showed that the genome of SG-86 was 2.1 M less than that of M-Z18.There were a lot of gene deletions,even metabolic pathways deletions on SG-86.Genome shuffling removed many metabolic pathways unrelated to the synthesis ofε-PL in SG-86,resulting in the reduction of genome of SG-86.The reduction of metabolic byproducts synthesis made more metabolic flow to the synthesis ofε-PL.In addition,insert and deletion mutation induced global response of cells.Some genes related to membrane proteins,transporters,transcriptional regulation and synthesis of secondary metabolites have mutated.Finally,based on comparative genomics,the metabolic map of highε-PL-producing strain was preliminarily established.