A Redox Proteomics Approach for Decoding Lignin to Lipid Conversion by Rhodococci

Abstract

Background: Rhodococciare studied for their bacterial ligninolytic capabilities and proclivity to accumulate lipids. Lignin utilization is a resource intensive process requiring a variety of redox active enzymes and cofactors. Studying both protein abundance and regulation helps decode the metabolic rewiring that stymies lignin to lipid conversion in these bacteria. Herein, a redox proteomics approach was applied to investigate a fundamental driver of carbon catabolism and lipid anabolism: redox balance. Results: In this study, the importance of redox balance as it relates to nutrient availability is demonstrated from an unique angle by employing a modified bottom-up proteomics workflow to acquire a general relationship between protein abundance and protein redox states. In support of this, a previously demonstrated consortium of Rhodococcus strains was grown on glucose vs. lignin under nitrogen limitation, which is generally conducive to lipid accumulation. Global proteomics results affirm downregulation of enzymes involved in sugar catabolism and upregulation of those involved in lignin degradation and aromatics catabolism compared to glucose-fed cultures. Several enzymes in the lipid biosynthetic pathways were downregulated, whereas many involved in β-oxidation were upregulated. Interestingly, proteins involved in oxidative stress response were also upregulated perhaps in response to lignin degradation and aromatics catabolism, which require oxygen and reactive oxygen species. Enzymes displaying little-to-no change in abundance but differences in protein cysteine oxidation (i.e. redox state) were observed in various pathways for carbon utilization (e.g., β‑ketoadipate pathway), fatty acid and lipid metabolism, as well as nitrogen metabolism (e.g., purine scavenging/synthesis), suggesting potential redox-dependent regulation beyond protein expression. Conclusions: Efficient lipid production requires a steady carbon and energy flux while balancing fundamental requirements for enzyme production and cell maintenance. For lignin, we theorize that this balance is difficult to establish due to resource expenditure for enzyme production and oxidative stress response. This is supported by significant changes to protein abundances and protein cysteine oxidation in various pathways.