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过程工程学报 ›› 2022, Vol. 22 ›› Issue (3): 347-356.DOI: 10.12034/j.issn.1009-606X.221100CSTR: 32067.14.jproeng.221100

• 研究论文 • 上一篇    下一篇

ARTP选育ε-聚赖氨酸高产菌株及其发酵条件优化

徐祖伟1,2， 季立豪1,2， 唐文秀1,2， 郭亮1,2， 陈修来1,2， 刘佳1,2， 刘立明1,2*
  

  1. 1. 江南大学食品科学与技术国家重点实验室，江苏 无锡 214122 2. 江南大学国际食品安全联合实验室，江苏 无锡 214122
  • 收稿日期:2021-03-24 修回日期:2021-05-21 出版日期:2022-03-28 发布日期:2022-03-28
  • 通讯作者: 徐祖伟 xzw125836@163.com
  • 作者简介:徐祖伟(1997-)，男，江苏省常州市人，硕士研究生，生物工程专业，E-mail: xzw125836@163.com；刘立明，通讯联系人，E-mail: mingll@jiangnan.edu.cn.
  • 基金资助:
    广东省重点领域研发计划项目;国家轻工技术与工程一流学科自主课题

Breeding of ε-poly-L-lysine high yield strain by ARTP and fermentation condition optimization

Zuwei XU1,2,  Lihao JI1,2,  Wenxiu TANG1,2,  Liang GUO1,2,  Xiulai CHEN1,2,  Jia LIU1,2,  Liming LIU1,2*   

  1. 1. State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu 214122, China 2. International Joint Laboratory on Food Safety, Jiangnan University, Wuxi, Jiangsu 214122, China
  • Received:2021-03-24 Revised:2021-05-21 Online:2022-03-28 Published:2022-03-28

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摘要： ε-聚赖氨酸(ε-PL)是由25~35个L-赖氨酸单体组成的一种天然聚合物，在食品、医学、农药等领域有很大的应用潜力。目前，微生物法生产ε-PL存在生产强度低、发酵周期长、工艺不稳定等问题。为此，本研究以S.albulus FMME-545为出发菌株，通过常温常压等离子诱变(ARTP)结合核糖体工程选育了一株具有利福霉素抗性的高产菌株S.albulus FMME-545RX，其ε-PL产量达到2.44 g/L，相较于出发菌株提升了105%。为了进一步提高ε-聚赖氨酸的产量，在5 L发酵罐中通过分批补料的方式对碳源的调控策略、pH调控方法、DO控制水平进行了系统的研究。结果表明，采用葡萄糖-蔗糖双碳源调控策略有助于提高菌体代谢强度；在发酵过程中添加柠檬酸钠能有效帮助菌体抵御酸性环境；产物合成所需的最适pH值和DO值分别为3.80和30%。经过192 h的分批补料发酵，ε-PL的产量、生产强度、单位细胞合成能力分别达到了53.0 g/L, 6.63 g/(L?d), 0.88 g/g，相比于原始菌株分别提高了130%, 131%, 118%。上述研究结果为ε-聚赖氨酸工业化生产提供了有益的借鉴。

关键词: ε-聚赖氨酸, 白色链霉菌, 核糖体工程, ARTP诱变, 发酵优化

Abstract: 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. To tackle these issues, S. albulus FMME-545RX 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, a series of fermentation optimization strategies, including carbon sources regulation, pH 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 pH 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.

Key words: ε-poly-L-lysine, Streptomyces albulus, ribosome engineering, ARTP mutagenesis, fermentation optimization