Physiological Responses Of Streptomyces Albulus M-Z18 To Acidic PH For High ε-polyl-lysine Production

ε-Poly-L-lysine(ε-PL) is a homopolymer of 25-35 L-lysine residues with amide linkage between ε-amino and α-carboxyl groups. It is biodegradable, water-soluble, heat-stable and exhibits a wide antimicrobial spectrum, including yeast, fungi, Gram-positive and Gram-negative bacteria, as well as antiphage activity. Moreover, ε-PL also shows excellent behavior in high safety. Therefore, ε-PL has been used widely as a natural food preservative in many countries, including Japan, Korea and the United States as well as China.In this dissertation, a high ε-PL producing strain, S. albulus M-Z18, was used as the study object. An economical mudium was developed by artificial neural network(ANN) with agro-industrial by-products as alternative organic nitrogen sources. Then, microbial physiology and RNA-sequencing(RNA-Seq) approaches were used to disclose the response mechanism of S. albulus M-Z18 to acidic p H. Based on these, an industrial fermentation strategy was established employing an environmental stress of acidic p H shock. Finally, the physiological changes induced by the acidic p H shock were investigated in fed-batch fermentation. The detailed results were depicted as below:1) Agro-industrial by-products, i.e. fish meal coupled with corn steep liquor, were employed as alternative organic nitrogen sources for industrial ε-PL production. An economical medium was then developed in shake-flask fermentation by ANN. Amino acids analyses showed that the improved medium was rich in glutamate, arginine, lysine and aspartate, which not only elevated acid tolerance capability of the mycelia but also enhanced cell growth and ε-PL production. As a result, ε-PL production and productivity reached 35.24 g·L-1 and 4.85 g·L-1·d-1 at 176 h of fed-batch fermentation in a 5 L fermenter, which were 17.0% and 15.1% higher than those of the original medium.2) The investigation of physiological response mechanism of S. albulus M-Z18 to acidic p H was based on cell wall, cell membrane and micro-environment analyses. Scanning electron microscope analysis showed that cell wall maintained intact under different environmental p H(5.0, 4.0, 3.0), and the vesicular protuberance of mycelia at p H 4.0 may be responsible for the production or secretion of ε-PL. Besides, cell membrane also maintained its selective permeability at acidic environment, which would contribute to decrease the effuse of intracellular materials and the influx of extracellular toxicant. Analysis of the content of membrane fatty acids showed that acidic p H led to the increased ratio of unsaturated fatty acids to saturated ones and the decreased carbon chain length, which could increase the membrane fluidity but decrease its stability. Meanwhile, in response to acidic p H, S. albulus M-Z18 could maintain p H i homeostasis at about 7.7 by the increase of intracellular H+-ATPase activity, and concentrations of ATP and amino acids(arginine, glutamate, aspartate, serine and glycine). In addition, the physiological responses of cells to acidic p H were also benefit for ε-PL production.3) A comparative transcriptomic analysis based on RNA-sequencing(RNA-Seq) was conducted to investigate the transcription differences of global genes at different environmental p H(5.0, 4.0, 3.0), which helped to further disclose the transcriptional response mechanism of S. albulus M-Z18 to acidic p H. Acidic p H could invoke a global change in cells. The genes related to transcription regulation, stress response protein, translocator, cell wall and cell membrane, secondary metabolite production, DNA and RNA, as well as ribosome played improtant roles in the acidic p H response. As a result, the acidic p H response mechanism in S. albulus M-Z18 was preliminary illustrated based on the physiological and transcriptional studies. We deduced that ε-PL production is also a response manner of S. albulus M-Z18 to acidic p H.4) Environmental stress is an important approach for the promotion of secondary metabolites production by Streptomyces. In this study, the effect of acidic p H shock on enhancing ε-PL production by S. albulus M-Z18 was investigated in a 5 L fermenter. Based on the evaluation of acidic p H on mycelia metabolic activity and shock parameters optimization, an integrated p H-shock strategy was developed as follows: pre-acid-shock adaption at p H 5.0 to alleviate the damage that caused by the followed p H shock, and then acidic p H shock at 3.0 for 12 h(including p H decline from 4.0 to 3.0) to positively regulate mycelia metabolic activity, finally restoring p H to 4.0 to provide optimal condition for ε-PL production. After 192 h of fed-batch fermentation, the maximum ε-PL production and productivity reached 54.70 g·L-1 and 6.84 g·L-1·d-1, respectively, which were 52.50% higher than those of control without p H shock. These results demonstrated that acidic p H shock is an efficient approach for improving ε-PL production.5) The introduction of acidic p H shock had successfully solved the common deficiency existed in ε-PL production, viz. the distinct decline of ε-PL productivity in the feeding phase of the fed-batch fermentation. To unravel the underlying mechanism, we comparatively studied the physiological changes of S. albulus M-Z18 during fed-batch fermentations with the p H shock strategy(PS) and p H non-shock strategy(PNS). Morphology investigation showed that pellet-shape change was negligible throughout both fermentations. In addition, the distribution of pellet size rarely changed in the PS, whereas pellet size and number decreased substantially with time in the PNS. This was consistent with the performances of ε-PL productivity in both strategies, demonstrating that morphology could be used as a predictor of ε-PL productivity during fed-batch fermentation. Furthermore, a second growth phase happened in the PS after p H shock, followed by the re-appearance of live mycelia in the dead core of the pellets. Meanwhile, mycelia respiration and key enzymes in the central metabolic and ε-PL biosynthetic pathways were overall strengthened until the end of the fed-batch fermentation. As a result, the physiological changes induced by the acidic p H shock have synergistically and permanently contributed to the stimulation of ε-PL productivity. However, this second growth phase and re-appearance of live mycelia were absent in the PNS. These results indicated that the introduction of a short-term suppression on mycelia physiological metabolism would guarantee the long-term high ε-PL productivity.