Enhancing lipid production in plant cells through automated high-throughput genome engineering and phenotyping

Dong, Jia; Croslow, Seth W.; Lane, Stephan T.; Castro, Daniel C.; Blanford, Jantana; Zhou, Shuaizhen; Park, Kiyoul; Burgess, Steven; Root, Mike; Cahoon, Edgar B.; Shanklin, John; Sweedler, Jonathan V.; Zhao, Huimin; Hudson, Matthew E.

doi:10.1093/plcell/koaf026

Enhancing lipid production in plant cells through automated high-throughput genome engineering and phenotyping

Journal Article · Sun Feb 02 23:00:00 EST 2025 · The Plant Cell

DOI:https://doi.org/10.1093/plcell/koaf026· OSTI ID:2561350

^[1]; ^[1]; ^[1]; ^[1]; ^[2]; ^[1]; ^[3]; ^[1]; ^[1]; ^[3]; ^[2]; ^[1]; ^[1]; ^[1]

University of Illinois at Urbana-Champaign, IL (United States)
Brookhaven National Laboratory (BNL), Upton, NY (United States)
University of Nebraska–Lincoln, NE (United States)

Plant bioengineering is a time-consuming and labor-intensive process with no guarantee of achieving desired traits. Here, we present a fast, automated, scalable, high-throughput pipeline for plant bioengineering (FAST-PB) in maize (Zea mays) and Nicotiana benthamiana. FAST-PB enables genome editing and product characterization by integrating automated biofoundry engineering of callus and protoplast cells with single-cell matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). We first demonstrated that FAST-PB could streamline Golden Gate cloning, with the capacity to construct 96 vectors in parallel. Using FAST-PB in protoplasts, we found that PEG2050 increased transfection efficiency by over 45%. For proof-of-concept, we established a reporter-gene-free method for CRISPR editing and phenotyping via mutation of high chlorophyll fluorescence 136. We show that diverse lipids were enhanced up to 6-fold using CRISPR activation of lipid controlling genes. In callus cells, an automated transformation platform was employed to regenerate plants with enhanced lipid traits through introducing multigene cassettes. Lastly, FAST-PB enabled high-throughput single-cell lipid profiling by integrating MALDI-MS with the biofoundry, protoplast, and callus cells, differentiating engineered and unengineered cells using single-cell lipidomics. Furthermore, these innovations massively increase the throughput of synthetic biology, genome editing, and metabolic engineering and change what is possible using single-cell metabolomics in plants.

View Accepted Manuscript (DOE)

Research Organization:: Center for Advanced Bioenergy and Bioproducts Innovation (CABBI), Urbana, IL (United States)

Sponsoring Organization:: USDOE Office of Science (SC), Biological and Environmental Research (BER)

Grant/Contract Number:: SC0018420

OSTI ID:: 2561350

Journal Information:: The Plant Cell, Journal Name: The Plant Cell Journal Issue: 2 Vol. 37; ISSN 1040-4651

Publisher:: American Society of Plant Biologists (ASPB)Copyright Statement

Country of Publication:: United States

Language:: English

References (63)

Exploring the Fundamental Structures of Life: Non‐Targeted, Chemical Analysis of Single Cells and Subcellular Structures Neumann, Elizabeth K.; Do, Thanh D.; Comi, Troy J. Angewandte Chemie International Edition, Vol. 58, Issue 28 https://doi.org/10.1002/anie.201811951	journal	July 2019
Engineering Rhodosporidium toruloides for increased lipid production : Engineering Zhang, Shuyan; Skerker, Jeffrey M.; Rutter, Charles D. Biotechnology and Bioengineering, Vol. 113, Issue 5 https://doi.org/10.1002/bit.25864	journal	November 2015
A mass spectrometry‐based high‐throughput screening method for engineering fatty acid synthases with improved production of medium‐chain fatty acids Xue, Pu; Si, Tong; Mishra, Shekhar Biotechnology and Bioengineering, Vol. 117, Issue 7 https://doi.org/10.1002/bit.27343	journal	April 2020
Poly(ethylene glycol)‐block‐polyethylenimine copolymers as carriers for gene delivery: Effects of PEG molecular weight and PEGylation degree Zhang, Xuan; Pan, Shi‐Rong; Hu, Hai‐Mei Journal of Biomedical Materials Research Part A, Vol. 84A, Issue 3 https://doi.org/10.1002/jbm.a.31343	journal	July 2007
Probe‐based mass spectrometry approaches for single‐cell and single‐organelle measurements Castro, Daniel C.; Chan‐Andersen, Peter; Romanova, Elena V. Mass Spectrometry Reviews, Vol. 43, Issue 4 https://doi.org/10.1002/mas.21841	journal	April 2023
Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR and the 2−ΔΔCT Method Livak, Kenneth J.; Schmittgen, Thomas D. Methods, Vol. 25, Issue 4 https://doi.org/10.1006/meth.2001.1262	journal	December 2001
The small, versatilepPZP family ofAgrobacterium binary vectors for plant transformation Hajdukiewicz, Peter; Svab, Zora; Maliga, Pal Plant Molecular Biology, Vol. 25, Issue 6, p. 989-994 https://doi.org/10.1007/BF00014672	journal	September 1994
Plant extracellular vesicles Cui, Yong; Gao, Jiayang; He, Yilin Protoplasma, Vol. 257, Issue 1 https://doi.org/10.1007/s00709-019-01435-6	journal	August 2019
microMS: A Python Platform for Image-Guided Mass Spectrometry Profiling Comi, Troy J.; Neumann, Elizabeth K.; Do, Thanh D. Journal of The American Society for Mass Spectrometry, Vol. 28, Issue 9 https://doi.org/10.1007/s13361-017-1704-1	journal	June 2017
Rapid prototyping of microbial cell factories via genome-scale engineering Si, Tong; Xiao, Han; Zhao, Huimin Biotechnology Advances, Vol. 33, Issue 7 https://doi.org/10.1016/j.biotechadv.2014.11.007	journal	November 2015
Accelerating strain engineering in biofuel research via build and test automation of synthetic biology Zhang, Jianzhi; Chen, Yongcan; Fu, Lihao Current Opinion in Biotechnology, Vol. 67 https://doi.org/10.1016/j.copbio.2021.01.010	journal	February 2021
The limitless applications of single-cell metabolomics Guo, Shenghao; Zhang, Cissy; Le, Anne Current Opinion in Biotechnology, Vol. 71 https://doi.org/10.1016/j.copbio.2021.07.015	journal	October 2021
Understanding and manipulating plant lipid composition: Metabolic engineering leads the way Napier, Johnathan A.; Haslam, Richard P.; Beaudoin, Frederic Current Opinion in Plant Biology, Vol. 19 https://doi.org/10.1016/j.pbi.2014.04.001	journal	June 2014
Metabolic engineering for enhanced oil in biomass Vanhercke, Thomas; Dyer, John M.; Mullen, Robert T. Progress in Lipid Research, Vol. 74 https://doi.org/10.1016/j.plipres.2019.02.002	journal	April 2019
New insights into the role of lipids in plant hypoxia responses Xie, Li-Juan; Zhou, Ying; Chen, Qin-Fang Progress in Lipid Research, Vol. 81 https://doi.org/10.1016/j.plipres.2020.101072	journal	January 2021
Single-cell metabolomics by mass spectrometry: Advances, challenges, and future applications Ali, Ahmed; Abouleila, Yasmine; Shimizu, Yoshihiro TrAC Trends in Analytical Chemistry, Vol. 120 https://doi.org/10.1016/j.trac.2019.02.033	journal	November 2019
Engineering biological systems using automated biofoundries Chao, Ran; Mishra, Shekhar; Si, Tong Metabolic Engineering, Vol. 42 https://doi.org/10.1016/j.ymben.2017.06.003	journal	July 2017
Using the Synergy between HPLC-MS and MALDI-MS Imaging to Explore the Lipidomics of Clear Cell Renal Cell Carcinoma Martín-Saiz, Lucía; Abad-García, Beatriz; Solano-Iturri, Jon D. Analytical Chemistry, Vol. 95, Issue 4 https://doi.org/10.1021/acs.analchem.2c03953	journal	January 2023
Metabolic Engineering: Methodologies and Applications Volk, Michael J.; Tran, Vinh G.; Tan, Shih-I Chemical Reviews, Vol. 123, Issue 9 https://doi.org/10.1021/acs.chemrev.2c00403	journal	December 2022
Bioprocessing, Recovery, and Mass Balance of Vegetative Lipids from Metabolically Engineered “Oilcane” Demonstrates Its Potential as an Alternative Feedstock for Drop-In Fuel Production Maitra, Shraddha; Viswanathan, Mothi Bharath; Park, Kiyoul ACS Sustainable Chemistry & Engineering, Vol. 10, Issue 50 https://doi.org/10.1021/acssuschemeng.2c05327	journal	December 2022
Spatially Resolved Mass Spectrometry at the Single Cell: Recent Innovations in Proteomics and Metabolomics Taylor, Michael J.; Lukowski, Jessica K.; Anderton, Christopher R. Journal of the American Society for Mass Spectrometry https://doi.org/10.1021/jasms.0c00439	journal	March 2021
A Look at Product Development with Genetically Modified Crops: Examples from Maize Mumm, Rita H. Journal of Agricultural and Food Chemistry, Vol. 61, Issue 35 https://doi.org/10.1021/jf400685y	journal	May 2013
Single-cell analysis reveals a stem-cell program in human metastatic breast cancer cells Lawson, Devon A.; Bhakta, Nirav R.; Kessenbrock, Kai Nature, Vol. 526, Issue 7571 https://doi.org/10.1038/nature15260	journal	September 2015
Automated multiplex genome-scale engineering in yeast Si, Tong; Chao, Ran; Min, Yuhao Nature Communications, Vol. 8, Issue 1 https://doi.org/10.1038/ncomms15187	journal	May 2017
Progress and prospects in plant genome editing Yin, Kangquan; Gao, Caixia; Qiu, Jin-Long Nature Plants, Vol. 3, Issue 8 https://doi.org/10.1038/nplants.2017.107	journal	July 2017
Building a global alliance of biofoundries Hillson, Nathan; Caddick, Mark; Cai, Yizhi Nature Communications, Vol. 10, Issue 1 https://doi.org/10.1038/s41467-019-10079-2	journal	May 2019
PlasmidMaker is a versatile, automated, and high throughput end-to-end platform for plasmid construction Enghiad, Behnam; Xue, Pu; Singh, Nilmani Nature Communications, Vol. 13, Issue 1 https://doi.org/10.1038/s41467-022-30355-y	journal	May 2022
Single-cell lipidomics enabled by dual-polarity ionization and ion mobility-mass spectrometry imaging Zhang, Hua; Liu, Yuan; Fields, Lauren Nature Communications, Vol. 14, Issue 1 https://doi.org/10.1038/s41467-023-40512-6	journal	August 2023
Lipids light up in plant membranes Mamode Cassim, Adiilah; Mongrand, Sébastien Nature Plants, Vol. 5, Issue 9 https://doi.org/10.1038/s41477-019-0494-9	journal	August 2019
High aspect ratio nanomaterials enable delivery of functional genetic material without DNA integration in mature plants Demirer, Gozde S.; Zhang, Huan; Matos, Juliana L. Nature Nanotechnology, Vol. 14, Issue 5 https://doi.org/10.1038/s41565-019-0382-5	journal	February 2019
A lipidome atlas in MS-DIAL 4 Tsugawa, Hiroshi; Ikeda, Kazutaka; Takahashi, Mikiko Nature Biotechnology, Vol. 38, Issue 10 https://doi.org/10.1038/s41587-020-0531-2	journal	June 2020
Image-guided MALDI mass spectrometry for high-throughput single-organelle characterization Castro, Daniel C.; Xie, Yuxuan Richard; Rubakhin, Stanislav S. Nature Methods, Vol. 18, Issue 10 https://doi.org/10.1038/s41592-021-01277-2	journal	September 2021
Single-cell metabolomics hits its stride Seydel, Caroline Nature Methods, Vol. 18, Issue 12 https://doi.org/10.1038/s41592-021-01333-x	journal	December 2021
Subcellular omics: a new frontier pushing the limits of resolution, complexity and throughput Eberwine, James; Kim, Junhyong; Anafi, Ron C. Nature Methods, Vol. 20, Issue 3 https://doi.org/10.1038/s41592-023-01788-0	journal	March 2023
Visualization of metabolites and microbes at high spatial resolution using MALDI mass spectrometry imaging and in situ fluorescence labeling Bourceau, Patric; Geier, Benedikt; Suerdieck, Vincent Nature Protocols, Vol. 18, Issue 10 https://doi.org/10.1038/s41596-023-00864-1	journal	September 2023
In vitro plant tissue culture as the fifth generation of bioenergy Norouzi, Omid; Hesami, Mohsen; Pepe, Marco Scientific Reports, Vol. 12, Issue 1 https://doi.org/10.1038/s41598-022-09066-3	journal	March 2022
Plant single-cell solutions for energy and the environment Cole, Benjamin; Bergmann, Dominique; Blaby-Haas, Crysten E. Communications Biology, Vol. 4, Issue 1 https://doi.org/10.1038/s42003-021-02477-4	journal	August 2021
The emerging role of nanotechnology in plant genetic engineering Squire, Henry J.; Tomatz, Sophia; Voke, Elizabeth Nature Reviews Bioengineering, Vol. 1, Issue 5 https://doi.org/10.1038/s44222-023-00037-5	journal	February 2023
Compartmentation of Triacylglycerol Accumulation in Plants Chapman, Kent D.; Ohlrogge, John B. Journal of Biological Chemistry, Vol. 287, Issue 4, p. 2288-2294 https://doi.org/10.1074/jbc.R111.290072	journal	November 2011
Inference of CRISPR Edits from Sanger Trace Data Conant, David; Hsiau, Tim; Rossi, Nicholas The CRISPR Journal, Vol. 5, Issue 1 https://doi.org/10.1089/crispr.2021.0113	journal	February 2022
Comprehensive investigation of pathway enrichment methods for functional interpretation of LC–MS global metabolomics data Lu, Yao; Pang, Zhiqiang; Xia, Jianguo Briefings in Bioinformatics, Vol. 24, Issue 1 https://doi.org/10.1093/bib/bbac553	journal	December 2022
A nuclear-encoded protein of prokaryotic origin is essential for the stability of photosystem II in Arabidopsis thaliana Meurer, Jörg; Plücken, Henning; Kowallik, Klaus V. The EMBO Journal, Vol. 17, Issue 18 https://doi.org/10.1093/emboj/17.18.5286	journal	September 1998
CRISPR-P: A Web Tool for Synthetic Single-Guide RNA Design of CRISPR-System in Plants Lei, Yang; Lu, Li; Liu, Hai-Yang Molecular Plant, Vol. 7, Issue 9 https://doi.org/10.1093/mp/ssu044	journal	September 2014
High-throughput methods for genome editing: the more the better Huang, Yong; Shang, Meiqi; Liu, Tingting Plant Physiology, Vol. 188, Issue 4 https://doi.org/10.1093/plphys/kiac017	journal	February 2022
Advances in single-cell metabolomics to unravel cellular heterogeneity in plant biology Pandian, Kanchana; Matsui, Minami; Hankemeier, Thomas Plant Physiology, Vol. 193, Issue 2 https://doi.org/10.1093/plphys/kiad357	journal	June 2023
GoldenBraid 2.0: A Comprehensive DNA Assembly Framework for Plant Synthetic Biology Sarrion-Perdigones, A.; Vazquez-Vilar, M.; Palaci, J. Plant Physiology, Vol. 162, Issue 3, p. 1618-1631 https://doi.org/10.1104/pp.113.217661	journal	May 2013
Sugar Potentiation of Fatty Acid and Triacylglycerol Accumulation Zhai, Zhiyang; Liu, Hui; Xu, Changcheng Plant Physiology, Vol. 175, Issue 2 https://doi.org/10.1104/pp.17.00828	journal	August 2017
High Efficiency Transformation of Cultured Tobacco Cells An, Gynheung Plant Physiology, Vol. 79, Issue 2 https://doi.org/10.1104/pp.79.2.568	journal	October 1985
Plant Callus: Mechanisms of Induction and Repression Ikeuchi, Momoko; Sugimoto, Keiko; Iwase, Akira The Plant Cell, Vol. 25, Issue 9 https://doi.org/10.1105/tpc.113.116053	journal	September 2013
Arabidopsis SEIPIN Proteins Modulate Triacylglycerol Accumulation and Influence Lipid Droplet Proliferation Cai, Yingqi; Goodman, Joel M.; Pyc, Michal The Plant Cell, Vol. 27, Issue 9 https://doi.org/10.1105/tpc.15.00588	journal	September 2015
A Multipurpose Toolkit to Enable Advanced Genome Engineering in Plants Čermák, Tomáš; Curtin, Shaun J.; Gil-Humanes, Javier The Plant Cell, Vol. 29, Issue 6 https://doi.org/10.1105/tpc.16.00922	journal	May 2017
Phosphorylation of WRINKLED1 by KIN10 Results in Its Proteasomal Degradation, Providing a Link between Energy Homeostasis and Lipid Biosynthesis Zhai, Zhiyang; Liu, Hui; Shanklin, John The Plant Cell, Vol. 29, Issue 4 https://doi.org/10.1105/tpc.17.00019	journal	March 2017
Trehalose 6-Phosphate Positively Regulates Fatty Acid Synthesis by Stabilizing WRINKLED1 Zhai, Zhiyang; Keereetaweep, Jantana; Liu, Hui The Plant Cell, Vol. 30, Issue 10 https://doi.org/10.1105/tpc.18.00521	journal	September 2018
Cocobean Tissue Culture: Lipid Composition and Fatty Acid Metabolism Tsai, C. H.; Wen, M. C.; Kinsella, J. E. Journal of Food Science, Vol. 47, Issue 3 https://doi.org/10.1111/j.1365-2621.1982.tb12711.x	journal	May 1982
A Revised Medium for Rapid Growth and Bio Assays with Tobacco Tissue Cultures Murashige, Toshio; Skoog, Folke Physiologia Plantarum, Vol. 15, Issue 3, p. 473-497 https://doi.org/10.1111/j.1399-3054.1962.tb08052.x	journal	July 1962
Metabolic engineering of biomass for high energy density: oilseed-like triacylglycerol yields from plant leaves Vanhercke, Thomas; El Tahchy, Anna; Liu, Qing Plant Biotechnology Journal, Vol. 12, Issue 2 https://doi.org/10.1111/pbi.12131	journal	October 2013
Roles of lipids as signaling molecules and mitigators during stress response in plants Okazaki, Yozo; Saito, Kazuki The Plant Journal, Vol. 79, Issue 4 https://doi.org/10.1111/tpj.12556	journal	June 2014
t‐SNAREs bind the Rhg1 α‐SNAP and mediate soybean cyst nematode resistance Dong, Jia; Zielinski, Raymond E.; Hudson, Matthew E. The Plant Journal, Vol. 104, Issue 2 https://doi.org/10.1111/tpj.14923	journal	August 2020
Accelerating a paradigm shift: The Common Fund Single Cell Analysis Program Roy, Ananda L.; Conroy, Richard; Smith, Jessica Science Advances, Vol. 4, Issue 8 https://doi.org/10.1126/sciadv.aat8573	journal	August 2018
Targeted genome modifications in soybean with CRISPR/Cas9 Jacobs, Thomas B.; LaFayette, Peter R.; Schmitz, Robert J. BMC Biotechnology, Vol. 15, Issue 1 https://doi.org/10.1186/s12896-015-0131-2	journal	March 2015
Extracellular Vesicles from Plants: Current Knowledge and Open Questions Urzì, Ornella; Raimondo, Stefania; Alessandro, Riccardo International Journal of Molecular Sciences, Vol. 22, Issue 10 https://doi.org/10.3390/ijms22105366	journal	May 2021
Ectopic Expression of OLEOSIN 1 and Inactivation of GBSS1 Have a Synergistic Effect on Oil Accumulation in Plant Leaves Zhai, Zhiyang; Liu, Hui; Shanklin, John Plants, Vol. 10, Issue 3 https://doi.org/10.3390/plants10030513	journal	March 2021
CRISPR/dCas9-Based Systems: Mechanisms and Applications in Plant Sciences Karlson, Chou Khai Soong; Mohd-Noor, Siti Nurfadhlina; Nolte, Nadja Plants, Vol. 10, Issue 10 https://doi.org/10.3390/plants10102055	journal	September 2021

Similar Records

Related Subjects

59 BASIC BIOLOGICAL SCIENCES

Enhancing lipid production in plant cells through automated high-throughput genome engineering and phenotyping

Citation Formats

References (63)

Similar Records

Related Subjects