生物技术通报 ›› 2023, Vol. 39 ›› Issue (12): 71-80.doi: 10.13560/j.cnki.biotech.bull.1985.2023-0759

• 技术与方法 • 上一篇    下一篇

黑曲霉葡萄糖吸收定量检测的方法建立及其在MstC功能研究中的应用

高凯月1,2,3(), 郭雨婷1,2,3, 杜奕谋1,2,3, 郑小梅2,3,4(), 马欣荣1(), 赵伟5, 郑平2,3,4, 孙际宾2,3,4   

  1. 1.天津科技大学生物工程学院,天津 300457
    2.中国科学院天津工业生物技术研究所,天津 300308
    3.国家合成生物技术创新中心,天津 300308
    4.中国科学院大学,北京 100049
    5.山东福洋生物科技股份有限公司,德州 253100
  • 收稿日期:2023-08-09 出版日期:2023-12-26 发布日期:2024-01-11
  • 通讯作者: 郑小梅,女,博士,副研究员,研究方向:真菌系统与合成生物学;E-mail: zheng_xm@tib.cas.cn
    马欣荣,女,博士,教授,研究方向:RNA介导的基因沉默机制;E-mail: xinrong.ma@tust.edu.cn
  • 作者简介:高凯月,女,硕士研究生,研究方向:真菌合成生物学;E-mail: gaoky@tib.cas.cn
    第一联系人:

    郭雨婷为本文共同第一作者

  • 基金资助:
    国家自然科学基金项目(32070082);国家自然科学基金项目(31961133021);天津市合成生物技术创新能力提升行动项目(TSBICIP-PTJS-003);天津市合成生物技术创新能力提升行动项目(TSBI-CIP-IJCP-003);山东省重点研发计划(2022SFC0101)

A Quantitative Detection Approach for Glucose Uptake in Aspergillus niger: A Case Study of Glucose Transporter MstC

GAO Kai-yue1,2,3(), GUO Yu-ting1,2,3, DU Yi-mou1,2,3, ZHENG Xiao-mei2,3,4(), MA Xin-rong1(), ZHAO Wei5, ZHENG Ping2,3,4, SUN Ji-bin2,3,4   

  1. 1. College of Biotechnology, Tianjin University of Science & Technology, Tianjin 300457
    2. Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences Tianjin, 300308
    3. National Technology Innovation Center of Synthetic Biology, Tianjin 300308
    4. University of Chinese Academy of Sciences, Beijing 100049
    5. Shandong Fuyang Biological Technology Co., Ltd., Dezhou 253100
  • Received:2023-08-09 Published:2023-12-26 Online:2024-01-11

摘要:

黑曲霉是有机酸与酶制剂重要的工业生产菌株,具有强大的水解酶系与葡萄糖转运系统,可快速响应胞外碳源变化,摄取环境中葡萄糖供给细胞生长和产物合成。作为重要的碳源底物与关键的信号分子,葡萄糖的摄取吸收直接影响黑曲霉细胞生长与发酵性能。针对目前丝状真菌缺乏简便葡萄糖吸收能力定量表征方法的问题,本文以2-(N-(7-硝基苯并-2-氧杂-1,3-二唑-4-氨基)-2-脱氧葡萄糖(2-NBDG)作为葡萄糖吸收探针,通过将预培养的黑曲霉孢子与2-NBDG孵育后,利用荧光显微成像与流式细胞分析,建立了丝状真菌的葡萄糖吸收的定量检测方法。结果发现,2-NBDG 的最佳使用浓度为150 μmol/L,最佳孵育时间为4 h。进一步利用该定量分析方法,发现低亲和力葡萄糖转运蛋白MstC的过表达使黑曲霉葡萄糖吸收提高1.44倍,同时在前期研究的基础上,利用多序列比对分析,设计了MstC的突变体R188K,通过检测发现该点突变可直接导致MstC葡萄糖转运活性的丧失,这表明Arg188是影响MstC葡萄糖转运能力的关键氨基酸位点。本文葡萄糖摄取定量检测方法的建立及其在MstC功能研究上的应用,不仅加深了丝状真菌葡萄糖吸收的定量认识,也为葡萄糖转运系统的改造优化提供了技术支撑。

关键词: 黑曲霉, 葡萄糖吸收, 2-NBDG, 葡萄糖转运蛋白, MstC, 荧光成像, 流式细胞检测

Abstract:

Aspergillus niger is an important strain for industrial production of organic acids and enzymes, possessing a powerful hydrolytic enzyme system and glucose transport system, which enable to copy with extracellular carbon sources variance and uptake glucose to support cell growth and industrial production. As an important carbon source and an essential signaling molecule, glucose and its uptake has considerable influence on cell growth and fermentation performance. Regarding to issue of lacking simple methods for quantitative characterization of glucose absorptive capacity in filamentous fungi, we developed a quantitative detection pipeline for glucose uptake capacity assay for filamentous fungi, using a non-metabolizable, fluorescent glucose analog, 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose(2-NBDG)as a fluorescent probe, then incubating pre-cultured A. niger spores with 2-NBDG, and using with fluorescence microscopy and flow cytometry. It showed that the optimized 2-NBDG work concentration and incubation time were 150 μmol/L and 4 h, respectively. Using this quantitative analysis method, it was found that the overexpression of low affinity glucose transporter MstC increased glucose uptake by 1.44 times. Meanwhile, on the basis of previous studies, mutant R188K of MstC was designed, and the point mutation directly caused the loss of glucose transport activity of MstC, which indicates that Arg188 is a key amino acid site affecting the glucose transport capacity of MstC. The establishment of this quantitative glucose uptake detection method and its application to the study of MstC function not only deepens the quantitative understanding of glucose absorption in filamentous fungi, but also provides technical support for the transformation and optimization of glucose transport system. This study provides a feasible quantitative approach to investigate the glucose uptake of filamentous fungi, and also revealed the physiological function of key glucose transporter MstC, which paves the way for glucose uptake engineering in this industrially important fungal cell factory.

Key words: Aspergillus niger, glucose uptake, 2-NBDG, glucose transporter, MstC, fluorescence imaging, flow cytometry