Probing Lignin Deconstruction and Catabolism in Soil Pseudomonas Species

Authors:

Beth DiBiase¹, Nanquing Zhou¹, Rebecca A. Wilkes², Jacob Waldbauer³, Gregg T. Beckham², Allison Z. Werner², Neha P. Kamat¹, and Ludmilla Aristilde¹* (ludmilla.aristilde@northwestern.edu)

Institutions:

¹Northwestern University; ²National Renewable Energy Laboratory; and ³University of Chicago

Goals

The overall goal of this project is to elucidate the metabolic reaction networks within outer membrane vesicles (OMVs) secreted from soil Pseudomonas species. In particular, this project aims to evaluate how OMVs catabolize lignin-derived aromatics in Pseudomonas strains, and in turn to maximize aromatic catabolic activity via engineered or synthetic 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. Gram-negative soil Pseudomonas strains, which natively catabolize lignin-derived aromatics (LDAs), are commonly engineered for the conversion of LDAs to value-added compounds. It was recently shown that Pseudomonas putida secretes OMVs enriched with enzymes that catalyze LDA turnover (Salvachúa et al. 2020). However, the metabolic reaction networks of pseudomonad OMVs remain uncharacterized.

Towards characterizing the regulatory controls and bottlenecks of OMV-localized fluxes from LDAs, the team first characterized a method for OMV isolation and enumeration from Pseudomonas cultivations and applied this to compare OMV secretion rates across growth conditions and stages. For OMV isolation, affinity-based kits were compared to ultracentrifugation (UC). Nanoparticle tracking analysis (NTA) was used both to count and measure the size of OMVs. Affinity-based isolation had improved throughput and lower processing time, but lowered particle yields. The isolation method did not have an effect on OMV size distribution, and OMV secretion was determined at different growth stages during growth on hydroxycinnamic acid, p-coumarate, or nutrient-rich medium. Proteomics analysis is underway to evaluate whether isolation strategies result in selective enrichment of certain OMV populations. The objective is to identify optimal sampling strategy for P. putida cultivations on p-coumarate.

Regarding OMV function, researchers hypothesized that the differential abundance of enzymes packaged into OMVs from P. putida fed on different LDAs will give rise to OMVs with different reaction networks. To test this hypothesis, the OMV metabolic functionality is being characterized for a variety of LDAs using OMVs produced by cultivation in lignin-rich media prepared with alkaline pretreated lignin liquor. After addition of an LDA and cofactors (i.e., ATP and NAD(P)H) were added to purified OMVs, preliminary metabolic profiling was conducted. Metabolic intermediates such as 4-hydroxybenzoate and protocatechuate were identified, demonstrating OMVs are actively catabolizing the LDA substrate. High-resolution kinetic profiling and kinetic ¹³C-labeling experiments with several LDAs will be performed next to both obtain direct evidence of and to quantify the metabolic functionality of OMV-localized reaction networks.

Lastly, quantification of metabolic functionalities in the OMVs may reveal metabolic bottlenecks within the pathways for LDA catabolism. Overcoming these bottlenecks would be of interest in engineering improved biocatalysts for lignin valorization. However, genetic tools for OMV biogenesis and cargo packaging are not currently available in P. putida. To this end, a library of genetic mutants has been screened to identify mutations that induce vesiculation but do not significantly impact growth on LDAs. Current work is focused on developing a SpyCatcher-SpyTag system for targeting protein cargo into OMVs. Protocols for proteomics analysis of OMV preparations from wild-type and mutant strains are under refinement and will enable assessment of aberrant protein cargo sorting in hypervesiculating mutants. The project aims to enable OMV deployment as a standardized synthetic biology tool in pseudomonads.

References

Salvachúa, D., et al. 2020. “Outer Membrane Vesicles Catabolize Lignin-Derived Aromatic Compounds in Pseudomonas putida KT2440,” Proceedings of the National Academy of Sciences of the United States of America 117, 9302–10.

Funding Information

This research was supported by the DOE Office of Science, Biological and Environmental Research (BER) Program, grant no. DE-SC0022181.