| ε-poly-L-lysine(ε-PL) is a homo-polymer composed of 25 to 35 residues of L-lysine(L-Lys) with linkages between α-carboxyl group and ε-amino group,which is mainly produced by aerobic microbial fermentation and extracellularly accumulated. With wide antimicrobial spectrum and high safety, ε-PL has been successfully used as a food preservative in Japan, the United States, South Korea and China at present. Besides, as a safe and green biopolymer, it has also been extensively applied in the medical industry. Therefore, ε-PL is a novel biotechnology product with widespread application and tremendous vendibility.The present study sought to examine the physiological mechanism of the improved ε-PL production in culture using glucose and glycerol as a mixed carbon source by investigating a high ε-PL producing strain, Streptomyces albulus M-Z18(S. albulus M-Z18). For this purpose, analysis of differences in cell physiological parameters, genes transcription and metabolic fluxes was carried out to disclose the mechanism of the enhanced ε-PL production in the mixed carbon source; Besides, the physiological basis of glucose-glycerol simultaneous consumption was discussed by genes transcription analysis and exogenous addition of auxiliary carbon sources; Finally, from the prospective of oxidative stress, we systematically analyzed the decrease of ε-PL productivity at the middle and later periods of the fed-batch fermentation with the mixed carbon source. The major conclusions were attached as follows:(1) By monitoring the activity of key enzymes in ε-PL production, it was found that in the mixed carbon source, the activity of 6-phosphogluconate dehydrogenase(G6PDH) at 31 h were 1.6- and 1.9-fold higher than that in glucose and glycerol, respectively, thus leading to enhancements of both L-Lys and cell growth. In addition, the activity of pyruvate kinase(PK) in embden-meyerhof-parnas(EMP) pathway and citrate synthase(CS) during tricarboxylic acid(TCA) cycle in the mixed carbon source were 2-fold higher than that in single carbon sources, which was beneficial to the production of NADH; Similarly, phosphoenolpyruvate carboxylase(PEPC), pyruvate carboxylase(PYC) and aspartate kinase(ASK) in the anaplerotic pathway and diaminopimelate(DAP) pathway, whose activity was increased as well, promoted the L-lysine biosynthesis; Moreover, the activity of ε-PL synthase(Pls) in the mixed carbon source was 2-fold higher than that in glucose, which directly enhanced the ε-PL production. Moreover, analysis of exhaust gas composition and intracellular energy cofactors levels disclosed that the mixed carbon source resulted in higher cell respiration activity, which led to increased ability of NADH and ATP biosynthesis. Therefore, the glucose-glycerol mixed carbon source enhanced the ε-PL production mainly through promoting the activity of key enzymes in ε-PL biosynthesis and cell respiration.(2) The enhancement of ε-PL production in the mixed carbon source was accompanied with rapid cell growth. However, whether the enhanced ε-PL production was attributed to the mixed carbon source or the accompanied rapid cell growth still remained unknown. Theeffect of dilution rate(0.02, 0.04, 0.05, 0.06, 0.08 h-1) on ε-PL production was studied through chemostat culture, which indicated a positive correlation between the specific ε-PL formation rate and specific cell growth rate at dilution rate of 0.02-0.06 h-1. Through the comparison of cell physiological status among different dilution rates, it was found that rapid-growing cells(0.06 h-1) showed higher activity of CS and PEPC, higher levels of intracellular L-aspartate(L-Asp) and L-Lys, higher efficiency of NADH oxidation, higher level of energy charge and antioxidant ability, which consequently provided more ATP and L-Lys for ε-PL biosynthesis; Based on comparing carbon fluxes among cultures using different carbon sources with the same dilution rate(0.05 h-1), it was found that the fluxes were improved in the mixed carbon source in terms of EMP, anaplerotic, DAP pathways and TCA cycle, thus providing sufficient precursor L-Lys for ε-PL biosynthesis. Hence, the enhanced ε-PL production was attributed to both the superior carbon source and the rapid-growing cells.(3) A comparative transcriptome study of cells cultured with single glucose, glycerol and the mixed carbon source was performed to reveal the physiological mechanism of the enhanced ε-PL production in the mixed carbon source. It was found that above 1.5-fold up-regulated genes transcription was observed in the mixed carbon source in terms of biosynthetic pathways of ribose 5-phosphate, basic group, cell wall, DNA replication as well as the ribosome production, which accelerated the cell growth. Furthermore, the mixed carbon source enhanced the transcription of genes involved in TCA cycle, anaplerotic metabolic pathway and DAP pathway, promoting precursor L-lysine biosynthesis and relevant carbon skeletons provision. In addition, the mixed carbon source significantly enhanced the transcription of genes involved in oxidoreductase, cytochrome, F-type H+-transporting ATPase and several cofactors related to the cell respiration(50% of which showed 2-fold up-regulated transcription than single carbon sources), increasing the ATP production. However, no significant difference was observed in the transcription of pls and pld among the three different carbon sources. Therefore, the mixed carbon source enhanced the ε-PL production mainly through the improvement of precursor and energy re-generation.(4) Based on the transcriptome technology, the transcription of genes related to carbon source utilization was compared and analyzed among cultures using glucose, glycerol and the mixed carbon source. It was found that the glucose uptake did not suppress the transcription of glycerol uptake facilitator protein and glycerol kinase, which was regarded as a precondition for simultaneous consumption of glucose and glycerol by S. albulus M-Z18; Analysis of genes transcription in the central carbon metabolism demonstrated that the cells in single glucose or glycerol showed potential demand for another substrate, while the mixed carbon source could satisfy this potential demand; The existence of such demand was further confirmed by auxiliary carbon source addition; The simultaneous consumption of glucose and glycerol could provide abundant precursors and energy for the production of both ε-PL and biomass. Therefore, the simultaneousconsumption of glucose and glycerol by S. albulus M-Z18 was based on the absence of mutual suppression between glucose and glycerol utilization. Moreover, the mixed carbon source also met the potential demand of cells for more carbon skeletons.(5) Through systematical analysis of fermentation parameters in fed-batch fermentation with glucose, glycerol and the mixed carbon source, it was found that the cell metabolic activity declined rapidly after 36 h; Through monitoring cells physiological status, sharp decline of cell viability, activity of G6 PDH, intracellular ATP and L-Lys levels were observed after 36 h; By examining reactive oxygen species(ROS) and oxidative damage in the whole fed-batch fermentation, it revealed that in the fed-batch fermentation with the mixed carbon source, intensive cell respiration at the early period rapidly generated ROS and caused oxidative damage, resulting in decreased activity of electron transport chain(ETS) and insufficient ATP provision; Transcriptome analysis indicated that the oxidative stress in the mixed carbon source induced higher transcription of genes related to antioxidant enzymes and damage repairing(above 1.5-fold up-regulation); However, activity measurement of antioxidant enzymes manifested much lower activity of antioxidant enzymes in the mixed carbon source, which could not protect cell against ROS. Therefore, intensive cell respiration in the mixed carbon source produced abundant ROS, which impaired the cell physiology and hence caused the sharp decline of cell metabolic ability at the middle and later periods of the fed-batch fermentation. |
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