Refactoring Metabolism to Control the Persistence of Genetically Engineered Microorganisms in the Environment
Joshua Elmore1* (firstname.lastname@example.org), Adam Guss2, Ryan McClure1, Gara Dexter2, Henri Baldino1, Jay Huenemann2, Ryan Francis1, George Peabody V2, Jessica Martinez-Baird2, Lauren Riley2, Tuesday Simmons3, Devin Colemann-Derr3, Andrew Wilson1, Elise Van Fossen1, Ritu Shrestha1, Andrew Frank1, Valentine Trotter4, Brenton Poirier1, Young-Mo Kim1, Adam Deutschbauer4, Yasuhiro Oda5, William Nelson1, Yuliya Farris1, Aaron Ogden1, Caroline Harwood5, William Alexander3, and Robert Egbert1
1Pacific Northwest National Laboratory (PNNL); 2Oak Ridge National Laboratory; 3University of California–Berkeley; 4Lawrence Berkeley National Laboratory; and 5University of Washington
The Persistence Control Science Focus Area (PerCon SFA) at PNNL is focused on developing fundamental understanding of factors governing the persistence of engineered microbial functions in rhizosphere environments. From this understanding, the team will establish design principles to control the environmental niche of native rhizosphere microbes for the model bioenergy crop sorghum through data-driven genome reduction and engineered metabolic addiction to plant root exudates. These principles will lead to secure plant-microbe biosystems that promote secure, stress-tolerant, and highly productive biomass crops.
Persistence control is an engineering approach in which survival of genetically modified microorganisms is restricted to a target environmental niche. Refactoring the catabolic repertoire of these organisms such that they have reduced fitness outside of the target niche and enhanced fitness within the target niche is one of several powerful tools for achieving persistence control (Fig. 1). Within the PerCon SFA, researchers are refactoring the catabolism of plant growth–promoting rhizobacteria (PGPR) to use crop-specific root exudate compounds and not use abundant, wide-spread soil compounds (e.g., lignocellulose, chitin, and non-crop-specific root exudates) as carbon sources. This has the potential to enable responsible application of beneficial genetically modified PGPR in the environment, while preventing their uncontrolled spread outside of the crop rhizosphere. Through the use of Sorghum bicolor (sorghum) as a model crop, and with random barcode transposon mutagenesis (RB-TnSeq), researchers have identified genes whose removal from the model sorghum rhizobacteria Pseudomonas facilor TBS28 will abolish its use of common carbon sources. Sorgoleone (a lipophilic benzoquinone) and MHPP (a phenylpropanoid methyl ester) are two compounds entirely or almost entirely unique to sorghum root exudates, respectively. This makes them excellent model nutrients for expansion of the TBS28 catabolic repertoire, but the catabolic pathways for their consumption are unknown. Researchers isolated three bacteria, including a new species, that utilize sorgoleone and found that the model rhizobacteria Pseudomonas fluorescens SBW25 utilizes MHPP. Using RB-TnSeq and RNAseq individually, dozens of genes were found that were potentially involved in sorgoleone and MHPP catabolism. When used together, the number of candidate genes were reduced by an order of magnitude, enabling detection of key enzymes in each catabolic pathway. A core set of four highly conserved genes are essential for use of sorgoleone, serve as a biomarker for sorgoleone catabolic function, and are enriched in members of the sorghum rhizosphere. With a combination of genetic characterization and biochemical enzyme assays, the team identified two enzymes responsible for funneling MHPP into a common aromatic catabolism pathway. This includes a novel family of esterases that demethylates MHPP and other plant-derived phenylpropanoid methyl esters. To enable facile and efficient site-specific integration of non-native catabolic pathways and other heterologous genetic programs into bacterial genomes, researchers developed serine recombinase-assisted genome engineering (SAGE; Elmore et al. 2023). For this project, the team is actively using a multiplexed version of SAGE to evaluate genetic parts for rational control of gene expression in diverse bacteria, complement gene deletions for pathway discovery, introduce machinery for CRISPR interference, and engineer PGPR that can consume MHPP and sorgoleone as carbon sources.
Figure 1. Refactoring the metabolism of bacteria for persistence control.
Elmore, J. R. et al. “The SAGE Genetic Toolkit Enables Highly Efficient, Iterative Site-Specific Genome Engineering in Bacteria,” Science Advances. In press.
This research was supported by the U.S. Department of Energy’s (DOE) Biological and Environmental Research (BER) Program as part of BER’s Genomic Science program (GSP) and is a contribution of the Pacific Northwest National Laboratory (PNNL) Secure Biosystems Design SFA “Persistence Control of Engineered Functions in Complex Soil Microbiomes.” A portion of this work was performed in the William R. Wiley Environmental Molecular Sciences Laboratory (EMSL), a national scientific user facility sponsored by BER and located at PNNL. PNNL is a multiprogram national laboratory operated by Battelle for the DOE under Contract DE-AC05-76RL01830.