ACS Publications. Most Trusted. Most Cited. Most Read
My Activity

Figure 1Loading Img

Engineering an NADPH/NADP+ Redox Biosensor in Yeast

View Author Information
The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kongens Lyngby, 2800, Denmark
Joint BioEnergy Institute, Emeryville, California 94608, United States
§ Biological Systems & Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720, United States
∥ ⊥ Department of Chemical and Biomolecular Engineering and Department of Bioengineering, University of California, Berkeley, California 94709, United States
Cite this: ACS Synth. Biol. 2016, 5, 12, 1546–1556
Publication Date (Web):July 15, 2016
Copyright © 2016 American Chemical Society

    Article Views





    Read OnlinePDF (1 MB)
    Supporting Info (1)»


    Abstract Image

    Genetically encoded biosensors have emerged as powerful tools for timely and precise in vivo evaluation of cellular metabolism. In particular, biosensors that can couple intercellular cues with downstream signaling responses are currently attracting major attention within health science and biotechnology. Still, there is a need for bioprospecting and engineering of more biosensors to enable real-time monitoring of specific cellular states and controlling downstream actuation. In this study, we report the engineering and application of a transcription factor-based NADPH/NADP+ redox biosensor in the budding yeast Saccharomyces cerevisiae. Using the biosensor, we are able to monitor the cause of oxidative stress by chemical induction, and changes in NADPH/NADP+ ratios caused by genetic manipulations. Because of the regulatory potential of the biosensor, we also show that the biosensor can actuate upon NADPH deficiency by activation of NADPH regeneration. Finally, we couple the biosensor with an expression of dosage-sensitive genes (DSGs) and thereby create a novel tunable sensor-selector useful for synthetic selection of cells with higher NADPH/NADP+ ratios from mixed cell populations. We show that the combination of exploitation and rational engineering of native signaling components is applicable for diagnosis, regulation, and selection of cellular redox states.

    Supporting Information

    Jump To

    The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssynbio.6b00135.

    • Pasmids and yeast strains used in this study; additional figures supporting the text; constraint-based metabolic modeling (PDF)

    Terms & Conditions

    Most electronic Supporting Information files are available without a subscription to ACS Web Editions. Such files may be downloaded by article for research use (if there is a public use license linked to the relevant article, that license may permit other uses). Permission may be obtained from ACS for other uses through requests via the RightsLink permission system:

    Cited By

    This article is cited by 56 publications.

    1. Shixiu Cui, Xueqin Lv, Xianhao Xu, Taichi Chen, Hongzhi Zhang, Yanfeng Liu, Jianghua Li, Guocheng Du, Rodrigo Ledesma-Amaro, Long Liu. Multilayer Genetic Circuits for Dynamic Regulation of Metabolic Pathways. ACS Synthetic Biology 2021, 10 (7) , 1587-1597.
    2. David Romero-Suarez, Tune Wulff, Yixin Rong, Tadas Jakočiu̅nas, Satoshi Yuzawa, Jay D. Keasling, Michael K. Jensen. A Reporter System for Cytosolic Protein Aggregates in Yeast. ACS Synthetic Biology 2021, 10 (3) , 466-477.
    3. Ning Qin, Lingyun Li, Xu Ji, Xiaowei Li, Yiming Zhang, Christer Larsson, Yun Chen, Jens Nielsen, Zihe Liu. Rewiring Central Carbon Metabolism Ensures Increased Provision of Acetyl-CoA and NADPH Required for 3-OH-Propionic Acid Production. ACS Synthetic Biology 2020, 9 (12) , 3236-3244.
    4. Lion Konstantin Flachbart, Sascha Sokolowsky, Jan Marienhagen. Displaced by Deceivers: Prevention of Biosensor Cross-Talk Is Pivotal for Successful Biosensor-Based High-Throughput Screening Campaigns. ACS Synthetic Biology 2019, 8 (8) , 1847-1857.
    5. Yang Liu, Robert Landick, Srivatsan Raman. A Regulatory NADH/NAD+ Redox Biosensor for Bacteria. ACS Synthetic Biology 2019, 8 (2) , 264-273.
    6. Joshua T. Atkinson, Ian Campbell, George N. Bennett, and Jonathan J. Silberg . Cellular Assays for Ferredoxins: A Strategy for Understanding Electron Flow through Protein Carriers That Link Metabolic Pathways. Biochemistry 2016, 55 (51) , 7047-7064.
    7. Chufan Xiao, Yuyang Pan, Mingtao Huang. Advances in the dynamic control of metabolic pathways in Saccharomyces cerevisiae. Engineering Microbiology 2023, 3 (4) , 100103.
    8. Chufan Xiao, Songlyu Xue, Yuyang Pan, Xiufang Liu, Mingtao Huang. Overexpression of genes by stress-responsive promoters increases protein secretion in Saccharomyces cerevisiae. World Journal of Microbiology and Biotechnology 2023, 39 (8)
    9. Raquel Perruca Foncillas, Miguel Sanchis Sebastiá, Ola Wallberg, Magnus Carlquist, Marie F. Gorwa-Grauslund. Assessment of the TRX2p-yEGFP Biosensor to Monitor the Redox Response of an Industrial Xylose-Fermenting Saccharomyces cerevisiae Strain during Propagation and Fermentation. Journal of Fungi 2023, 9 (6) , 630.
    10. Shifan He, Zhanwei Zhang, Wenyu Lu. Natural promoters and promoter engineering strategies for metabolic regulation in Saccharomyces cerevisiae. Journal of Industrial Microbiology and Biotechnology 2023, 50 (1)
    11. Xia Liu, Lei Qin, Jie Yu, Wentao Sun, Jianhong Xu, Chun Li. Real-time monitoring of subcellular states with genetically encoded redox biosensor system (RBS) in yeast cell factories. Biosensors and Bioelectronics 2023, 222 , 114988.
    12. Ning Li, Lihong Li, Shiqin Yu, Jingwen Zhou. Dual-channel glycolysis balances cofactor supply for l-homoserine biosynthesis in Corynebacterium glutamicum. Bioresource Technology 2023, 369 , 128473.
    13. Indrajeet Yadav, Akhil Rautela, Shweta Rawat, Ajay Kumar Namdeo, Sanjay Kumar. Metabolic engineering of yeast for advanced biofuel production. 2023, 73-97.
    14. Maurizio Mormino, Ibai Lenitz, Verena Siewers, Yvonne Nygård. Identification of acetic acid sensitive strains through biosensor-based screening of a Saccharomyces cerevisiae CRISPRi library. Microbial Cell Factories 2022, 21 (1)
    15. Kendreze Holland, John Blazeck. High throughput mutagenesis and screening for yeast engineering. Journal of Biological Engineering 2022, 16 (1)
    16. Sumeng Wang, Wei Jiang, Xin Jin, Qingsheng Qi, Quanfeng Liang. Genetically encoded ATP and NAD(P)H biosensors: potential tools in metabolic engineering. Critical Reviews in Biotechnology 2022, 132 , 1-15.
    17. Hajar Yaakoub, Sara Mina, Alphonse Calenda, Jean-Philippe Bouchara, Nicolas Papon. Oxidative stress response pathways in fungi. Cellular and Molecular Life Sciences 2022, 79 (6)
    18. Raquel Perruca-Foncillas, Johan Davidsson, Magnus Carlquist, Marie F. Gorwa-Grauslund. Assessment of fluorescent protein candidates for multi-color flow cytometry analysis of Saccharomyces cerevisiae. Biotechnology Reports 2022, 34 , e00735.
    19. Eleni Theodosiou, Adrian Tüllinghoff, Jörg Toepel, Bruno Bühler. Exploitation of Hetero- and Phototrophic Metabolic Modules for Redox-Intensive Whole-Cell Biocatalysis. Frontiers in Bioengineering and Biotechnology 2022, 10
    20. Lisbeth Olsson, Peter Rugbjerg, Luca Torello Pianale, Cecilia Trivellin. Robustness: linking strain design to viable bioprocesses. Trends in Biotechnology 2022, 18
    21. Luca Torello Pianale, Peter Rugbjerg, Lisbeth Olsson. Real-Time Monitoring of the Yeast Intracellular State During Bioprocesses With a Toolbox of Biosensors. Frontiers in Microbiology 2022, 12
    22. Kun Zhu, Jing Kong, Baixiang Zhao, Lanxin Rong, Shiqi Liu, Zhihui Lu, Cuiying Zhang, Dongguang Xiao, Krithi Pushpanathan, Jee Loon Foo, Adison Wong, Aiqun Yu. Metabolic engineering of microbes for monoterpenoid production. Biotechnology Advances 2021, 53 , 107837.
    23. Yuki Yoshikawa, Ryo Nasuno, Hiroshi Takagi. An NADPH‐independent mechanism enhances oxidative and nitrosative stress tolerance in yeast cells lacking glucose‐6‐phosphate dehydrogenase activity. Yeast 2021, 38 (7) , 414-423.
    24. Xiaofan Feng, Mario Marchisio. Saccharomyces cerevisiae Promoter Engineering before and during the Synthetic Biology Era. Biology 2021, 10 (6) , 504.
    25. Louis C Dacquay, David R McMillen. Improving the design of an oxidative stress sensing biosensor in yeast. FEMS Yeast Research 2021, 21 (4)
    26. Viviënne Mol, Martyn Bennett, Benjamín J. Sánchez, Beata K. Lisowska, Markus J. Herrgård, Alex Toftgaard Nielsen, David J. Leak, Nikolaus Sonnenschein. Genome-scale metabolic modeling of P. thermoglucosidasius NCIMB 11955 reveals metabolic bottlenecks in anaerobic metabolism. Metabolic Engineering 2021, 65 , 123-134.
    27. Liang Song, Jun-Yan Shi, Shou-Fu Duan, Da-Yong Han, Kuan Li, Ri-Peng Zhang, Peng-Yu He, Pei-Jie Han, Qi-Ming Wang, Feng-Yan Bai. Improved redox homeostasis owing to the up-regulation of one-carbon metabolism and related pathways is crucial for yeast heterosis at high temperature. Genome Research 2021,
    28. Yiming Zhang, Shuobo Shi. Transcription Factor-Based Biosensor for Dynamic Control in Yeast for Natural Product Synthesis. Frontiers in Bioengineering and Biotechnology 2021, 9
    29. Qiang Ding, Wenwen Diao, Cong Gao, Xiulai Chen, Liming Liu. Microbial cell engineering to improve cellular synthetic capacity. Biotechnology Advances 2020, 45 , 107649.
    30. Yiming Zhang, Mo Su, Ning Qin, Jens Nielsen, Zihe Liu. Expressing a cytosolic pyruvate dehydrogenase complex to increase free fatty acid production in Saccharomyces cerevisiae. Microbial Cell Factories 2020, 19 (1)
    31. Muskan Bhatia, Jyotika Thakur, Shradha Suyal, Ruchika Oniel, Rahul Chakraborty, Shalini Pradhan, Monika Sharma, Shantanu Sengupta, Sunil Laxman, Shyam Kumar Masakapalli, Anand Kumar Bachhawat. Allosteric inhibition of MTHFR prevents futile SAM cycling and maintains nucleotide pools in one-carbon metabolism. Journal of Biological Chemistry 2020, 295 (47) , 16037-16057.
    32. Lei Qin, Shuxin Dong, Jie Yu, Xiaoyu Ning, Ke Xu, Sen-Jia Zhang, Li Xu, Bing-Zhi Li, Jun Li, Ying-Jin Yuan, Chun Li. Stress-driven dynamic regulation of multiple tolerance genes improves robustness and productive capacity of Saccharomyces cerevisiae in industrial lignocellulose fermentation. Metabolic Engineering 2020, 61 , 160-170.
    33. Vasil D'Ambrosio, Eleonora Dore, Roberto Di Blasi, Marcel van den Broek, Suresh Sudarsan, Jolanda ter Horst, Francesca Ambri, Morten O.A. Sommer, Peter Rugbjerg, Jay D. Keasling, Robert Mans, Michael K. Jensen. Regulatory control circuits for stabilizing long-term anabolic product formation in yeast. Metabolic Engineering 2020, 61 , 369-380.
    34. Hongting Tang, Yanling Wu, Jiliang Deng, Nanzhu Chen, Zhaohui Zheng, Yongjun Wei, Xiaozhou Luo, Jay D. Keasling. Promoter Architecture and Promoter Engineering in Saccharomyces cerevisiae. Metabolites 2020, 10 (8) , 320.
    35. Helene Martin-Yken. Yeast-Based Biosensors: Current Applications and New Developments. Biosensors 2020, 10 (5) , 51.
    36. Jason M. Held. Redox Systems Biology: Harnessing the Sentinels of the Cysteine Redoxome. Antioxidants & Redox Signaling 2020, 32 (10) , 659-676.
    37. Xia Wan, Monireh Marsafari, Peng Xu. Engineering metabolite-responsive transcriptional factors to sense small molecules in eukaryotes: current state and perspectives. Microbial Cell Factories 2019, 18 (1)
    38. Chenxi Qiu, Haotian Zhai, Jin Hou. Biosensors design in yeast and applications in metabolic engineering. FEMS Yeast Research 2019, 19 (8)
    39. Kwasi Kyere-Yeboah, Jessica Denteh, Kun Liu, Penglin Ye, E-Bin Gao. Monitoring Nicotinamide Adenine Dinucleotide and its phosphorylated redox metabolism using genetically encoded fluorescent biosensors. Sensing and Bio-Sensing Research 2019, 26 , 100307.
    40. Xiaolin Shen, Jia Wang, Chenyi Li, Qipeng Yuan, Yajun Yan. Dynamic gene expression engineering as a tool in pathway engineering. Current Opinion in Biotechnology 2019, 59 , 122-129.
    41. Viridiana Olin-Sandoval, Jason Shu Lim Yu, Leonor Miller-Fleming, Mohammad Tauqeer Alam, Stephan Kamrad, Clara Correia-Melo, Robert Haas, Joanna Segal, David Alejandro Peña Navarro, Lucia Herrera-Dominguez, Oscar Méndez-Lucio, Jakob Vowinckel, Michael Mülleder, Markus Ralser. Lysine harvesting is an antioxidant strategy and triggers underground polyamine metabolism. Nature 2019, 572 (7768) , 249-253.
    42. Felix Moser, Eléonore Tham, Lina M. González, Timothy K. Lu, Christopher A. Voigt. Light‐Controlled, High‐Resolution Patterning of Living Engineered Bacteria Onto Textiles, Ceramics, and Plastic. Advanced Functional Materials 2019, 29 (30)
    43. Chandran Sathesh-Prabu, Kwang Soo Shin, Geun Hwa Kwak, Sang-Kyu Jung, Sung Kuk Lee. Microbial Production of Fatty Acid via Metabolic Engineering and Synthetic Biology. Biotechnology and Bioprocess Engineering 2019, 24 (1) , 23-40.
    44. Peng Xu. Production of chemicals using dynamic control of metabolic fluxes. Current Opinion in Biotechnology 2018, 53 , 12-19.
    45. Alexander Carpenter, Ian Paulsen, Thomas Williams. Blueprints for Biosensors: Design, Limitations, and Applications. Genes 2018, 9 (8) , 375.
    46. Shuobo Shi, Ee Lui Ang, Huimin Zhao. In vivo biosensors: mechanisms, development, and applications. Journal of Industrial Microbiology and Biotechnology 2018, 45 (7) , 491-516.
    47. Jiaheng Liu, Huiling Li, Guangrong Zhao, Qinggele Caiyin, Jianjun Qiao. Redox cofactor engineering in industrial microorganisms: strategies, recent applications and future directions. Journal of Industrial Microbiology and Biotechnology 2018, 45 (5) , 313-327.
    48. Tom Delmulle, Sofie L. De Maeseneire, Marjan De Mey. Challenges in the microbial production of flavonoids. Phytochemistry Reviews 2018, 17 (2) , 229-247.
    49. Xue‐Feng Chen, Xiao‐Xia Xia, Sang Yup Lee, Zhi‐Gang Qian. Engineering tunable biosensors for monitoring putrescine in Escherichia coli. Biotechnology and Bioengineering 2018, 115 (4) , 1014-1027.
    50. Benjamin M. Woolston, Timothy Roth, Ishwar Kohale, David R. Liu, Gregory Stephanopoulos. Development of a formaldehyde biosensor with application to synthetic methylotrophy. Biotechnology and Bioengineering 2018, 115 (1) , 206-215.
    51. Shuobo Shi, Yook Wah Choi, Huimin Zhao, Meng How Tan, Ee Lui Ang. Discovery and engineering of a 1-butanol biosensor in Saccharomyces cerevisiae. Bioresource Technology 2017, 245 , 1343-1351.
    52. Fumio Matsuda, Yoshihiro Toya, Hiroshi Shimizu. Learning from quantitative data to understand central carbon metabolism. Biotechnology Advances 2017, 35 (8) , 971-980.
    53. Jyun-Liang Lin, James M. Wagner, Hal S. Alper. Enabling tools for high-throughput detection of metabolites: Metabolic engineering and directed evolution applications. Biotechnology Advances 2017, 35 (8) , 950-970.
    54. Linda Dekker, Karen M Polizzi. Sense and sensitivity in bioprocessing — detecting cellular metabolites with biosensors. Current Opinion in Chemical Biology 2017, 40 , 31-36.
    55. Anne Sofie Lærke Hansen, Rebecca M. Lennen, Nikolaus Sonnenschein, Markus J Herrgård. Systems biology solutions for biochemical production challenges. Current Opinion in Biotechnology 2017, 45 , 85-91.
    56. Thomas C. Williams, Xin Xu, Martin Ostrowski, Isak S. Pretorius, Ian T. Paulsen. Positive-feedback, ratiometric biosensor expression improves high-throughput metabolite-producer screening efficiency in yeast. Synthetic Biology 2017, 2 (1)

    Pair your accounts.

    Export articles to Mendeley

    Get article recommendations from ACS based on references in your Mendeley library.

    Pair your accounts.

    Export articles to Mendeley

    Get article recommendations from ACS based on references in your Mendeley library.

    You’ve supercharged your research process with ACS and Mendeley!

    STEP 1:
    Click to create an ACS ID

    Please note: If you switch to a different device, you may be asked to login again with only your ACS ID.

    Please note: If you switch to a different device, you may be asked to login again with only your ACS ID.

    Please note: If you switch to a different device, you may be asked to login again with only your ACS ID.

    Your Mendeley pairing has expired. Please reconnect