ε-Poly-L-lysine (ε-PL) is a kind of food preservatives mainly produced by Streptomyces albulus, which possesses excellent antimicrobial activity and has broad industrial applications. In this study, the pH and dissolved oxygen (DO) levels in the fed-batch fermentation by an ε-PL high-producing strain S. albulus WG-608 were systemically optimized, and a novel pH-DO regulation strategy was subsequently established. Specifically, the fermentation process was divided into two stages: stage I, DO concentration and pH level were respectively kept at 40% and 4.0, for the accumulation of biomass and ε-PL; stage II, DO and pH levels were maintained at 20% and 4.3, to improve the specific formation rate and ε-PL production. After 240 h of fed-batch fermentation, the ε-PL production and average specific formation rate of WG-608 reached (68.77±2.53) g/L and (2.33±0.08) d-1, which were 28.23% and 12.02% higher than those of the control strategy. The total glucose consumption rate increased, especially the substrate consumption rate increased from 315.5 g/L to 528.8 g/L in the stage II, which increased by 67.6%. And the glucose conversion rate increased by 6.7%. Changes in key enzyme activities, respiratory chain activity, and intracellular nucleotide levels were detected and analyzed to further investigate the metabolic differences between the pH-DO regulation strategy and control strategy. The result showed that the enhanced glucose consumption rate, key enzyme activity of central carbon metabolism (include glucose-6-phosphate dehydrogenase, phosphoenolpyruvate carboxylase, citrate synthase, aspartokinase and ε-poly-L-lysine synthase), respiratory chain activity, L-lysine biosynthesis pathway are responsible for the improved ε-PL production by pH-DO regulation strategy. Overall, the novel pH-DO regulation strategy is a convenient and effective approach for promoting ε-PL biosynthesis by strengthening carbon and energy metabolism. As mentioned above, the new strategy increases the demand for energy, and the demand in the cell may be in short supply. To further increase the yield of ε-PL in S. albulus, balancing the intracelluar cofactors by metabolic engineering may become the next step research. This developed method has shed new light on the ε-PL production by other Streptomyces strains.
WU Mengping
,
WANG Liang
,
ZHANG Jianghua
,
ZHANG Hongjian
,
CHEN Xusheng
. Enhancing the epsilon-poly-L-lysine biosynthesis of Streptomyces albulus by a pH-DO combined regulation strategy[J]. Food and Fermentation Industries, 2023
, 49(5)
: 9
-17
.
DOI: 10.13995/j.cnki.11-1802/ts.031500
[1] SHIMA S, SAKAI H.Polylysine produced by Streptomyces[J].Agricultural and Biological Chemistry, 1977, 41(9):1 807-1 809.
[2] WANG L, ZHANG C Y, ZHANG J H, et al.Epsilon-poly-L-lysine:Recent advances in biomanufacturing and applications[J].Frontiers in Bioengineering and Biotechnology, 2021, 9:748976.
[3] HUANG M W, CHEN H Q, TANG X, et al.Two-stage pH control combined with oxygen-enriched air strategies for the highly efficient production of EPA by Mortierella alpina CCFM698 with fed-batch fermentation[J].Bioprocess and Biosystems Engineering, 2020, 43(9):1 725-1 733.
[4] KAHAR P, IWATA T, HIRAKI J, et al.Enhancement of epsilon-polylysine production by Streptomyces albulus strain 410 using pH control[J].Journal of Bioscience and Bioengineering, 2001, 91(2):190-194.
[5] REN X D, CHEN X S, ZENG X, et al.Acidic pH shock induced overproduction of ε-poly-L-lysine in fed-batch fermentation by Streptomyces sp.M-Z18 from agro-industrial by-products[J].Bioprocess and Biosystems Engineering, 2015, 38(6):1 113-1 125.
[6] XU Z X, BO F F, XIA J, et al.Effects of oxygen-vectors on the synthesis of epsilon-poly-lysine and the metabolic characterization of Streptomyces albulus PD-1[J].Biochemical Engineering Journal, 2015, 94:58-64.
[7] XU Z X, CAO C H, SUN Z Z, et al.Construction of a genetic system for Streptomyces albulus PD-1 and improving poly(epsilon-L-lysine) production through expression of Vitreoscilla hemoglobin[J].Journal of Microbiology and Biotechnology, 2015, 25(11):1 819-1 826.
[8] 吴光耀, 陈旭升, 王靓, 等.核糖体工程技术选育ɛ-聚赖氨酸高产菌株[J].微生物学通报, 2016, 43(12):2 744-2 751.
WU G Y, CHEN X S, WANG L, et al.Screening of high-yield ε-poly-L-lysine producing strains through ribosome engineering[J].Microbiology China, 2016, 43(12):2 744-2 751.
[9] 谢志鹏, 徐志南, 郑建明, 等.靛酚蓝反应测定发酵液中的氨态氮[J].浙江大学学报(工学版), 2005, 39(3):437-439;444.
XIE Z P, XU Z N, ZHENG J M, et al.Determination of ammonium nitrogen in fermentationbroth through indophenol blue reaction[J].Journal of Zhejiang University (Engineering Science), 2005, 39(3):437-439;444.
[10] ITZHAKI R F.Colorimetric method for estimating polylysine and polyarginine[J].Analytical Biochemistry, 1972, 50(2):569-574.
[11] 李芹, 赵俊杰, 刘永娟, 等.链霉素抗性选育ε-聚赖氨酸高产菌株[J].现代食品科技, 2020, 36(3):150-158;195.
LI Q, ZHAO J J, LIU Y J, et al.Breeding of high ε-poly-L-lysine-producing strain by streptomycin resistance[J].Modern Food Science and Technology, 2020, 36(3):150-158;195.
[12] 王靓. ε-聚赖氨酸产生菌的育种、发酵及高产机制的初步生理解析[D].无锡:江南大学, 2018.
WANG L.Study on breeding and fermentation of ε-poly-L-lysine producing strains and preliminary physiological analysis of the improved production[D].Wuxi:Jiangnan University, 2018.
[13] TANG Z Y, XIAO C Y, ZHUANG Y P, et al.Improved oxytetracycline production in Streptomyces rimosus M4018 by metabolic engineering of the G6PDH gene in the pentose phosphate pathway[J].Enzyme and Microbial Technology, 2011, 49(1):17-24.
[14] BRAMWELL H, NIMMO H G, HUNTER I S, et al.Phosphoenolpyruvate carboxylase from Streptomyces coelicolor A3(2):Purification of the enzyme, cloning of the ppc gene and over-expression of the protein in a streptomycete[J].The Biochemical Journal, 1993, 293(1):131-136.
[15] MURAKAMI M, KOUYAMA T.Crystal structures of two isozymes of citrate synthase from Sulfolobus tokodaii strain 7[J].Biochemistry Research International, 2016, 2016:7560919.
[16] YAMANAKA K, KITO N, IMOKAWA Y, et al.Mechanism of epsilon-poly-L-lysine production and accumulation revealed by identification and analysis of an epsilon-poly-L-lysine-degrading enzyme[J].Applied and Environmental Microbiology, 2010, 76(17):5 669-5 675.
[17] 曾昕. 小白链霉菌同步代谢葡萄糖和甘油合成ε-聚赖氨酸的生理机制研究[D].无锡:江南大学, 2016.
ZENG X.Physiological analysis of the ε-poly-L-lysine biosynthesis in Streptomyces albulus used glucose and glycerol as mixed carbon source[D].Wuxi:Jiangnan university, 2016.
[18] 陈旭升. Streptomyces sp.M-Z18发酵生产ε-聚赖氨酸的碳源供给策略与过程调控研究[D].无锡:江南大学, 2011.
CHEN X S.Enhancement of ε-poly-L-lysine production through carbon source supply strategy optimization and fermentation process regulation by Streptomyces sp.M-Z18[D].Wuxi:Jiangnan University, 2011.
[19] XU Z X, FENG X H, SUN Z Z, et al.Economic process to co-produce poly(ε-L-lysine) and poly(L-diaminopropionic acid) by a pH and dissolved oxygen control strategy[J].Bioresource Technology, 2015, 187:70-76.
[20] YAMANAKA K, MARUYAMA C, TAKAGI H, et al.Epsilon-poly-L-lysine dispersity is controlled by a highly unusual nonribosomal peptide synthetase[J].Nature Chemical Biology, 2008, 4(12):766-772.
[21] WANG C Y, REN X D, YU C, et al.Physiological and transcriptional responses of Streptomyces albulus to acid stress in the biosynthesis of ε-poly-L-lysine[J].Frontiers in Microbiology, 2020, 11:1379.
[22] LYU P J, QIANG S, LIU L, et al.Dissolved-oxygen feedback control fermentation for enhancing β-carotene in engineered Yarrowia lipolytica[J].Scientific Reports, 2020, 10(1):17114.
[23] IMLAY J A.The molecular mechanisms and physiological consequences of oxidative stress:Lessons from a model bacterium[J].Nature Reviews Microbiology, 2013, 11(7):443-454.
[24] ZHU Y Q, LIU Y F, LI J H, et al.An optimal glucose feeding strategy integrated with step-wise regulation of the dissolved oxygen level improves N-acetylglucosamine production in recombinant Bacillus subtilis[J].Bioresource Technology, 2015, 177:387-392.
[25] XU J Z, RUAN H Z, CHEN X L, et al.Equilibrium of the intracellular redox state for improving cell growth and L-lysine yield of Corynebacterium glutamicum by optimal cofactor swapping[J].Microbial Cell Factories, 2019, 18(1):1-12.
