| ε-poly-L-lysine(ε-PL)is a natural homo-polymer of microbial origin,consisting of 25～35 L-lysine monomers,which is mainly produced by aerobic microbial fermentation and secreted to extracellular accumulation.Due to its wide antimicrobial spectrum and high safety,ε-PL has been successfully used as a food preservative.Besides,as a safe and green biopolymer,it has also been extensively applied in biomedical,chemical,and many other fields.Unfortunately,the efficient microbial production of ε-PL has reached a bottleneck owing to the limitations of low productivity,long fermentation period and unstable fermentation process,which can not satisfy the demand of industrialized production and brought obstacles to its popularity.In this study,ribosome engineering and fermentation engineering were combined to control and optimize the combined mutagenesis and fermentation process of Streptomyces.albulus,which effectively improved the strain’s ability to produce ε-poly-L-lysine,and further analyzed the mechanism of ribosome engineering.The main research contents are as follows:1.Screening of ε-poly-L-lysine high-producing mutants based on ribosomal engineering.Firstly,a highthroughput screening strategy was determined: microplate culture technology combined with microplate analyzer for rapid detection.Second,S.albulus FMME-545 RX with high tolerance to rifamycin was firstly screened and obtained by atmospheric and room temperature plasmas(ARTP)mutagenesis combined with ribosomal engineering,which could produce 2.44 g/L of ε-PL,with an increase of 105% in comparison with that of the parent strain S.albulus FMME-545.Then,after the third round of ARTP mutagenesis,streptomycin resistance was introduced into the strain,and a double resistant strain of "rifamycin +streptomycin" S.albulus FMME-545RX-S was selected,and its shaking flask yield reached 2.88 g/L.After192 h of fermentation,the ε-PL yield of the strain reached 33.5 g/L,which increased by 50% compared with the original strain.2.Preliminary analysis of the high-yield mechanism of ribosome engineering.By comparing the growth performance of thallus and synthesis ability of products,colony morphology and transcription level of the primary strain and FMME-545RX-S,the analysis was carried out from the outside to the inside.1.By comparing the specific growth curve of bacteria and the specific synthesis curve of products,we found that ribosome engineering can significantly improve the growth performance of bacteria and the synthesis ability of products.2.Comparative analysis of colony morphology showed that the mutant strains had shorter morphological differentiation period and thicker mycelia,which was conducive to the synthesis of secondary metabolites.3.The difference analysis of transcription level showed that the central metabolic pathway and L-lysine production pathway of mutant were enhanced,which led to the further increase of ε-PL production.3.Improve ε-PL yield based on carbon supply strategy and fermentation conditions optimization.A series of fermentation optimization strategies,including carbon sources regulation,p H control and dissolved oxygen(DO)regulation,were employed in increasing the production of ε-PL.The final results demonstrated that the mixed carbon source of glucose and sucrose fermentation was helpful to improve the metabolic intensity of bacteria;the addition of sodium citrate in the fermentation process can effectively improve the ability of the bacteria to resist the acidic environment;the optimum p H and DO values for product synthesis were 3.80 and 30%,respectively.Finally,under the controlled fed-batch fermentation,the production,productivity,and dry cell weight(DCW)of ε-PL reached up to 53.0 g/L,6.63 g/(L?d),and 0.88 g/g,respectively,which were 130%,131%,and 118% higher than those of the parent strain S.albulus FMME-545.Taken together,this study shows great potential for industrial production of ε-PL and the strategies described here also pave the way to the production of other value-added chemicals. |
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