Investigating Cellular Network and Outer-Membrane Vesicles for the Metabolism of Lignin-Derived Aromatics in Soil Pseudomonas Species
Authors:
Nanqing Zhou1* ([email protected]), Rebecca A. Wilkes2, Jacob Waldbauer3, Neha P. Kamat1, Allison Z. Werner2, Gregg T. Beckham2, Ludmilla Aristilde1
Institutions:
1Northwestern University; 2National Renewable Energy Laboratory; 3University of Chicago
Goals
The overall goal of this project is to elucidate the relationship between the cellular metabolic network and the metabolic reactions in outer membrane vesicles secreted by soil Pseudomonas species. In particular, the research team aims to evaluate the catabolism of lignin-derived aromatics in Pseudomonas strains toward maximizing aromatic catabolic activity via engineered or synthetic cellular and vesicle systems. The results from this work will enhance understanding of carbon cycling by soil bacteria and have implications in the use of engineered pseudomonads for lignin valorization to value-added compounds to support the bioeconomy.
Abstract
Valorization of lignin is an important component of a sustainable bioeconomy. Soil Pseudomonas strains, which natively catabolize lignin-derived aromatics (LDAs), are commonly engineered for the conversion of LDAs to value-added compounds. It was shown that Pseudomonas putida secretes outer membrane vesicles (OMVs) enriched with enzymes that catalyze LDA turnover (Salvachúa et al. 2020). However, the metabolic reaction networks of pseudomonad OMVs and their relationships to intracellular metabolism remain uncharacterized.
To reveal how OMVs potentially facilitate P. putida utilizing LDAs, potential bottlenecks of cells catabolizing different LDAs were identified by measuring the intracellular metabolite levels in cells fed with ferulate (FER), p-coumarate (COU), vanillate (VAN), or 4-hydroxybenzoate (4HB) as the sole carbon source. When P. putida was fed with FER and COU, intermediates accumulated in the peripheral pathways involved in the conversion of FER to VAN and COU to 4HB, suggesting the presence of bottlenecks in these pathways. Specifically, in FER-fed P. putida, vanillin was 20-fold higher than its upstream metabolite feruloyl-CoA and 4-fold higher than its downstream metabolite VAN; in COU-fed cells, 4HB accumulated 3-fold higher than its upstream metabolite 4-hydroxybenzoaldehyde and its downstream metabolite protochatechuate (PCA) was undetectable. When VAN was the carbon source, the PCA level was 25-fold smaller than in P. putida fed with 4HB. Based on quantification of intracellular metabolite levels, bottlenecks were identified at four metabolic nodes in the peripheral pathways for different LDA catabolism. The research team aims to overcome these bottlenecks by (1) overexpressing key enzymes involved in the bottlenecks and (2) synthesizing vesicles encapsulating key metabolites and delivering them directly to cells.
Evaluation of the metabolic capabilities of OMVs versus cells can provide insights into the spatial organization of catabolic pathways, providing further insights into potential bottlenecks in the LDA catabolic pathways. To overcome these bottlenecks, genetic tools for the manipulation of OMV biogenesis and enzyme packaging are needed. The current work aims to develop genetic tools in P. putida both to induce vesiculation and to target specific enzymes into the OMVs, thus providing additional approaches for engineering LDA bioconversion. To identify genetic targets that influence vesiculation, nine knockout mutants were screened for a hypervesiculation phenotype. Out of these mutants, only two knockouts, both involved in establishing linkages between the outer membrane and peptidoglycan layers, were found to induce biogenesis. Interestingly, high production of OMVs (i.e., 4-fold greater than wildtype) was found to coincide with higher cell membrane permeability and increased cell stress, whereas a moderate increase in OMVs (i.e., 1.5-fold greater than wildtype) did not impact cell performance.
Additionally, a SpyCatcher-SpyTag system was utilized to selectively target specific protein cargo into OMVs, which was demonstrated by an increase in extracellular enzyme activity. These advancements represent strides toward harnessing OMVs as a valuable synthetic biology tool.
References
Salvachúa, D., et al. “Outer membrane vesicles catabolize lignin-derived aromatic compounds in Pseudomonas putida KT2440,” Proceedings of the National Academy of Sciences 117, 9302–10.
Funding Information
This research was supported by the DOE Office of Science, BER Program, grant no. DE-SC0022181.