Study On ε-poly-L-lysine Biosynthesis And Metabolic Regulation

ε-Poly-L-lysine (ε-PL), produced by Streptomyces strains, is a homopolymer of L-lysinewith a pomerization degree of25-35. Due to its highly safety, ε-PL is being receivedincreasing interest as a natural food preservation. In this paper, the product isolated fromStreptomyces ahygroscopicus GIM8fermentation broth was identified and its molecularweight was determined for assessing as a suitable food preservative. With the main aim ofimproving ε-PL production, study emphasis was paid on ε-PL biosynthesis and metabolicregulation. The main results obtained were summarized in the following.The product isolated from Streptomyces ahygroscopicus GIM8was confirmed as ε-PLafter TLC, ultraviolet spectrum, infrared spectrum (IR) and nuclear magnetic resonance(NMR) analysis. Tricine-SDS-PAGE demonstrated that the molecular weight of ε-PLproduced by this strain was about3300kDa, and this polymer has a pomerization degree ofranging from28to34obtained from MALDI-TOF-MS spectrum. On the basis of thesefindings, S. ahygroscopicus is a strain capable of producing ε-PL and the product is suitablefor using in the food industry as a food preservative.In a neutral buffer, large amounts of ε-PL adsorbed onto the cells, and declined with adecrease in pH value. When the pH was at4.0, which pH is required for ε-PL biosynthesis,there was minimal adsorption of ε-PL on the cells. The adsorption was also confirmed byfluorescence microscopy through labeling ε-PL with FITC. Treatment of the cells with ε-PL atpH7.0, increased membrane permeability, cell membrane damage, and decreased cell activitywere observed. However, ε-PL inhibited the intracellular materials leakage in the pH4.0buffer even though minimal adsorption of ε-PL onto the cells occurred, and decreased cellactivity considerably. From these findings, it was reasonably postulated that ε-PL could enterthe cells at pH4.0, thereby exerting its function. The entrance of ε-PL into the cells may bedue to the increase in membrane permeability and ε-PL conformation change caused by lowpH.As ε-PL could significantly decrease metabolic activity of the cells, it is necessary toseparate ε-PL from fermentation broth as it was produced by the cells. For development of insitu removal of ε-PL, adsorption experiments were performed. Among Amberlite IRC-76,Amberlite IRC-50, and Amberlite IR-120, D152was chosen using adsorption capactity anddesoprtion rate as bases. Inclusion of D152in shaken cultures grown in the production phase,the production of ε-PL was increased to2.86g/L from0.81g/L. In a5L fermentor, ε-PLconcentration as high as23.4g/L, with an increase of522%relative to the control (3.76g/L), was obtained by affixing two bags of D152resin to the probes and baffles of the fermentor,indicating in situ product removal an efficient technique for the production of ε-PL.To significantly improve cell density and overcome the inhibitory effect of ε-PL, acombination of cell immobilization and in situ adsorption was developed to test in improvingfermentation efficiency in shaken flasks. Among loofah sponge, sugarcane bagasse andsynthetic sponge, loofah sponge-immobilzed S. ahygroscopicus GIM8behaved the best on thebasis of ε-PL productivity and cell growth. Using loofah sponge as cell carrier forimmobilization and D152resin as an adsorbent for in situ adsorption of ε-PL, a final ε-PL titreof3.64g/L was achieved, signficantly higher than those obtained by the single technique(immobilization,0.54g/L; in situ product removal,2.73g/L). Furthermore, the immobilizedcells could be repeatedly used three times, with a total ε-PL amount of8.05g/L at flask level.Using BacLight Live/Dead as a viability dye, laser scanning confocal microsocpy(LSCM) data demonstrated that most of the cells was active from0to12h. At18h andwhereafter, typical BacLight images could not be observed. This may be related to the changein membrane permeability and DNA conformation. With CTC as a dye based on metabolicactivity, it was found that cell activity increased from0to18h, and thereafter it began todecline. At48h, when ε-PL biosynthesis ceased, the cells exhibited little metabolic activity.With a colorimetric procedure, lower metabolic activity was observed after6h inoculation,and increased gradually to18h. However, a sharp decline was observed between18to24hcultivation. In the later cultivation, the decline trend continued, and at48h only15.9%ofinitial activity remained. From the evolutionary of ε-PL formation and cell activity, it could beconcluded that the ε-PL biosynthesis was closely related to metabolic activity of the cells.Based on the correlation between ε-PL biosynthesis and metabolic activity, enhancingcell activity is very necessary for efficient ε-PL production. Therefore, malt, beef extract, andyeast extract were tested in improving ε-PL production in the flask culture of S.ahygroscopicus GIM8. The results indicated that yeast extract stimulated ε-PL biosynthesisthe most. Further studies indicated that enhancement of cell activity and production capacityof the cells was observed. By intermittently feeding yeast extract to fed-batch cultures in30Lfermenters grown in the production phase, both the ε-PL production time and biomass hadsignificant improvement as compared with those of the control cultures. ε-PL concentration ashigh as28.2g/L was achieved—73%higher than the concentration of the control culture(16.3g/L).Considering that lysine is a precursor for the biosynthesis of ε-PL, its effect on ε-PLproduction in a fermentation medium was examined. The results demonstrated that L-lysine added in the production phase mainly served as a precursor for ε-PL biosynthesis, leading togreater ε-PL production. At an optimum level of3mM L-lysine, a ε-PL yield of1.16g/L wasattained, with a41.4%increment relative to the control (0.78g/L). Interestingly, ε-PLproduction was also enhanced considerably when D-lysine at3mM was supplemented intothe initial fermentation medium in flasks, even higher than that of L-lysine initial addition (3mM). The mechanism by which D-lysine improves ε-PL biosynthesis is that its utilization ledto greater biomass and, consequently, greater ε-PL production.When S. ahygroscopicus GIM8was cultivated in the defined medium containing L-lysine,several key metabolites of lysine metabolism including5-aminovalerate, pipecolate, andL-2-aminoadipate were identified in the cells by the LC-MS analysis; whereas onlyL-2-aminoadipate was detected after D-lysine metabolism. Apparently, L-lysine and D-lysinehave different catabolism pathways in the cells. On the other hand, cadaverine, a toxiccompound, was not formed in the cells after metabolism of both L-lysine and D-lysine. Theresults are expected to aid the understanding of ε-PL biosynthesis, and serve as reference forthe formulation of an alternative approach to improve ε-PL productivity using L-lysine as anadditional substrate in fermentation medium.The results of this paper provide insights on ε-PL biosynthesis and could serve forfermentation control and optimization in industrial fermentation processes.