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

doi:10.13560/j.cnki.biotech.bull.1985.2023-0759

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

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.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. 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/Abstract

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

高凯月, 郭雨婷, 杜奕谋, 郑小梅, 马欣荣, 赵伟, 郑平, 孙际宾. 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[J]. 生物技术通报, 2023, 39(12): 71-80.

GAO Kai-yue, GUO Yu-ting, DU Yi-mou, ZHENG Xiao-mei, MA Xin-rong, ZHAO Wei, ZHENG Ping, SUN Ji-bin. A Quantitative Detection Approach for Glucose Uptake in Aspergillus niger: A Case Study of Glucose Transporter MstC[J]. Biotechnology Bulletin, 2023, 39(12): 71-80.

图/表 8

表1 本研究所用的菌株与质粒

Table 1 Strains and plasmids used in this study

Strains/Plasmids	Description	Source
Escherichia coli Trans1-T1	F- φ80（lacZ）, ΔM15, ΔlacX74, hsdR（rkmk+）, ΔrecA 1398, endA, tonA	TransGen.
Saccharomyces cerevisiae EBY.VW4000	CEN.PK2-1C, hxt1-17, stl1, agt1, ydl247w, yjr160c, gal2	[12]
S. cerevisiae EBY.MstC	EBY.VW4000 with pRS-MstC	This study
S. cerevisiae EBY.MstC^R188K	EBY.VW4000 with pRS-MstC^R188K	This study
A. niger D353.8	pyrG::hph, kusA::hph, Hyg^R	[13]
A. niger OE. MstC	D353.8 with the PgpdA:mstC expression cassette	This study
A. niger OE. MstC^R188K	D353.8 with the PgpdA:mstC^R188K expression cassette	This study
pRS426	Ppgk:gfp:Tpgk, Ura, Amp^R	[12]
pRS-MstC	pRS426 carring the mstC gene	This study
pRS-MstC^R188K	pRS426 carring the mstC^R188K gene	This study
pXMD7	PgpdA:TtrpC, pyrG, Amp^R	[14]
pOE-MstC	pOE carring PgpdA-mstC expression cassette	This study
pOE-MstC^R188K	pOE carring PgpdA-mstC^R188K expression cassette	This study

表2 本研究所用引物及其序列

Table 2 Primers and their sequences used in this study

Primer name	Primer sequence（5'-3'）	Source
MstC-F1	gcatactagtATGGGTGTCTCTAATATGATGTC	This study
MstC-R1	gcctaagcttCTCGCGGAGCTCAGTGGG	This study
MstC^R188K-F MstC^R188K-R	CTGCTCCCCGTCAGGTCAAGGGTGCCATGGTCAGTGCCTTC CACTGACCATGGCACCCTTGACCTGACGGGGAGCAGATTC	This study
MstC-F2	tcaccggatcccatatgttaattaaATGGGTGTCTCTAATATGATGTC	This study
MstC-R2	atctactcccgggtacgtaactagtCTACTCGCGGAGCTCAGTGGG	This study
pRS-F	TACGACTCACTATAGGGCGAA	This study
pRS-R	AAACAGAATTGTCCGAATCGT	This study

图1 不同2-NBDG孵育浓度对葡萄糖吸收荧光检测的影响 A-C为黑曲霉孢子与2-NBDG孵育不同浓度后的荧光成像检测；D-F为黑曲霉孢子与2-NBDG孵育不同浓度后的流式细胞检测。A与D为黑曲霉孢子在100 μmol/L 2-NBDG孵育后的荧光检测结果；B与E为黑曲霉孢子在150 μmol/L 2-NBDG孵育后的荧光检测结果；C与F为黑曲霉孢子在200 μmol/L 2-NBDG孵育后的荧光检测结果。FI表示所检测的105个孢子的平均荧光强度，Q2表示当荧光强度FITC-H大于103的孢子占总孢子数的比例，下同

Fig. 1 Effect of 2-NBDG concentration on the glucose uptake in A. niger A-C: The fluorescence imaging of A. niger spores incubated with different concentrations of 2-NBDG. D-F: Flow cytometric detection of A. niger spores incubated with different concentrations of 2-NBDG. A and D: 100 μmol/L 2-NBDG; B and E: 150 μmol/L 2-NBDG; C and F: 200 μmol/L 2-NBDG. FI refers to the average fluorescence intensity of 105 spores. Q2 refers to the proportion of spores with fluorescence intensity FITC-H（>103）to the total spore count, the same below

图2 不同2-NBDG孵育时间对葡萄糖吸收荧光检测的影响 A-C为黑曲霉孢子与2-NBDG孵育不同时间后的荧光成像检测；D-F为黑曲霉孢子与2-NBDG孵育不同时间后的流式细胞检测。A与D为黑曲霉孢子与2-NBDG孵育0 h后的荧光检测结果；B与E为黑曲霉孢子与2-NBDG孵育 2 h后的荧光检测结果；C与F与2-NBDG孵育4 h后的荧光检测结果

Fig. 2 Effect of 2-NBDG incubation time on the glucose uptake fluorescence detection in A. niger A-C: The fluorescence imaging of spores incubated 2-NBDG for different incubation time. D-F: Flow cytometric detection of spores incubated 2-NBDG for different incubation time. A and D: 0 h; B and E: 2 h; C and F: 4 h

图3 酵母重组菌株EBY.MstC在不同葡萄糖浓度下的生长情况

Fig. 3 Effect of glucose concentration on the cell growth of recombinant E. coli strain EBY.MstC

图4 黑曲霉MstC过表达菌株的葡萄糖吸收能力检测 A-B为黑曲霉孢子与150 μmol/L 2-NBDG孵育4 h后的荧光成像检测；C-D为黑曲霉孢子与150 μmol/L 2-NBDG孵育4 h后的流式细胞检测。A与C为出发菌株D353.8荧光检测结果；B与D为MstC过表达菌株OE.MstC的荧光检测结果

Fig. 4 Glucose uptake capacity detection based on MstC-overexpressed strain A. niger A-B: The fluorescence imaging of spores incubated 150 μmol/L 2-NBDG for 4 h. C-D: Flow cytometric detection of spores incubated 150 μmol/L 2-NBDG for 4 h.A and C: The parent strain D353.8; B and D: MstC overexpression strain OE.MstC

图5 葡萄糖转运蛋白MstC的序列结构分析与葡萄糖转运能力检测 A为葡萄糖转运蛋白MstC的序列结构分析；B为酵母重组菌株EBY.MstC与EBY.MstCR188K在不同葡萄糖浓度下的生长情况。在多序列比对中，AnMstC与AnMstA分别代表来自黑曲霉的内源葡萄糖转运蛋白MstC与MstA，AndMstE代表来自构巢曲霉的葡萄糖转运蛋白MstE，NcGlt1、NcHgt1与NcHgt2分别代表来自粗燥脉孢霉葡萄糖转运蛋白Glt1、Hgt1与Hgt2

Fig. 5 Structure analysis and glucose transport capacity detection of MstC A is the sequence structure analysis of glucose transporter protein MstC, and B is the growth of yeast recombinant strains EBY.MstC and EBY.MstCR188K at different glucose concentrations. In multiple sequence alignment, AnMstC and AnMstA represent endogenous glucose transporters MstC and MstA from A. niger, AndMstE represents glucose transporter MstE from A. niger, and NcGlt1, NcHgt1, and NcHgt2 represent glucose transporters Glt1, Hgt1, and Hgt2 from A. niger, respectively

图6 黑曲霉MstC突变体过表达菌株OE.MstCR188K的葡萄糖吸收检测 A-B为黑曲霉孢子与150 μmol/L 2-NBDG孵育4 h后的荧光成像检测；C-D为黑曲霉孢子与150 μmol/L 2-NBDG孵育4 h后的流式细胞检测。A与C为出发菌株D353.8荧光检测结果；B与D为MstC突变体过表达菌株OE.MstCR188K的荧光检测结果

Fig. 6 Glucose uptake capacity detection of A. niger MstC point mutant over-expressed strain OE.MstCR188K A-B: The fluorescence imaging of spores incubated 150 μmol/L 2-NBDG for 4 h. C-D: Flow cytometric detection of spores incubated 150 μmol/L 2-NBDG for 4 h. A, C: The parent strain D353.8; B, D: MstC point mutant overexpression strain MstCR188K

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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

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

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