Engineering plant protein turnover rate to increase biomass yield

• Applicant: Dr. Ulschan Bathe
• Subject Area: Plant Biochemistry and Biophysics ; Biochemistry
• Term: from 2021 to 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 455236359

To ensure adequate nutrition for all people, overall production of the major food crops must rise drastically. However, conventional breeding will not suffice to reach this goal. Metabolic engineering is a new tool to decrease carbon loss by lowering respiration rates, thereby saving carbon that can subsequently be channeled into biomass. Notably, as much as 60% of photosynthetically fixed carbon in crop plants is lost by respiration. Growth and maintenance respiration each constitute 50% of total respiration, which means that up to 30% of the carbon fixed by crop plants can never be stored in biomass because it is used to support maintenance processes. Roughly half of maintenance respiration supports protein turnover which is of high cost in plants. This is especially the case for enzymes that are abundant but short-lived. Therefore, engineering protein turnover is a suitable target to reduce respiratory costs.Individual candidates to reduce protein turnover rates are present in all plants and can serve as a starting point to engineer maintenance respiration. Five Arabidopsis genes were chosen from available datasets, and will be subjected to continuous directed evolution. To do so, I will delete the corresponding gene from the platform genomes of Escherichia coli and yeast using the Keio collection in combination with P1 phage transduction, and the Euroscarf collection combined with CRISPR/Cas9 mediated genome editing, respectively. The selected candidate genes will then be evolved using synthetic biology tools: EvolvR in E. coli and OrthoRep in yeast, both allow high mutation rates. By shutting down expression in such systems suddenly and completely, when followed by a period to allow growth, it will allow selection for the cells with the longest‐lived target enzyme because these cells will continue growing for the greatest length of time. Evolved genes will then be sequenced to determine which amino acids have mutated. In addition, these genes will be expressed in E. coli, purified and characterized in terms of kinetic parameters. The five most successfully evolved enzymes from either the yeast or E. coli platform will be further subjected to structure determination by homology modeling and X-ray crystallography (performed by project partners). We aim to identify structural features that have been evolved.To test whether evolved enzymes can contribute to gain in biomass, the two most successfully evolved enzymes will be ported into Arabidopsis. For this, CRISPR/Cas9-mediated gene editing will be used to make the amino acid changes in the native Arabidopsis gene that match the changes introduced by directed evolution. Total above-ground biomass and seed yields will be measured and compared to control plants. Evolved enzymes in Arabidopsis mutants will also be analyzed by project partners in terms of metabolic flux and turnover rates which will provide fundamental knowledge needed to engineer energy-efficient crops.

• DFG Programme: WBP Fellowship
• International Connection: USA
• Host: Professor Andrew D. Hanson, Ph.D.