TY - JOUR
T1 - Enhancing lipid production in plant cells through automated high-throughput genome engineering and phenotyping
AU - Dong, Jia
AU - Croslow, Seth W
AU - Lane, Stephan T
AU - Castro, Daniel C
AU - Blanford, Jantana
AU - Zhou, Shuaizhen
AU - Park, Kiyoul
AU - Burgess, Steven
AU - Root, Mike
AU - Cahoon, Edgar
AU - Shanklin, John
AU - Sweedler, Jonathan V
AU - Zhao, Huimin
AU - Hudson, Matthew E
N1 - We acknowledge the Center for Advanced Bioenergy and Bioproducts Innovation (CABBI) (United States Department of Energy, Office of Science, Office of Biological and Environmental Research under Award Number DE-SC0018420). Any opinions, findings, and conclusions or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the views of the United States Department of Energy. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
To overcome this limitation, we sought to integrate an automated biofoundry with single-cell metabolomics to expedite the engineering of plant genomes and characterization of cellular effects, which has never been done before. Biofoundries are specialized workstations that integrate robotics, high-throughput instrumentation, computer-aided design, and informatics to speed up iterative biological design-build-test-Learn cycles in a scalable manner (; ; ). These workstations are highly reproducible, and optimize resource utilization by reducing human time and labor costs, increasing experimental throughput, and enabling researchers to devote more time toward experimental design as well as analysis and interpretation of results (). The Illinois Biological Foundry for Advanced Biomanufacturing (iBioFAB) has demonstrated success in automating synthetic biology processes such as plasmid assembly (), yeast genome editing (), and antimicrobial discovery (). This success paves the way for the development of high-throughput plant genome editing technologies. Beyond the iBioFAB, it is important to note that biofoundry development has been strongly emphasized worldwide, including initiatives in the United States, South Korea, China, and others. The U.S. federal government has prioritized the acceleration of design-build-test-learn capabilities through initiatives such as Executive Order 14,081 and subsequent funding opportunities from the National Science Foundation (NSF 24-556 and NSF 23-585). Despite these advances, most biofoundry initiatives have focused on microbial, mammalian, and DNA-based systems, and progress in automating plant biotechnology has been limited ().
PY - 2025/2
Y1 - 2025/2
N2 - 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. 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.
AB - 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. 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.
UR - https://www.scopus.com/pages/publications/85218897897
UR - https://www.scopus.com/pages/publications/85218897897#tab=citedBy
U2 - 10.1093/plcell/koaf026
DO - 10.1093/plcell/koaf026
M3 - Article
C2 - 39899469
SN - 1040-4651
VL - 37
JO - The Plant cell
JF - The Plant cell
IS - 2
M1 - koaf026
ER -