Anaerobic Degradation of Syringic Acid by an Adapted Strain of Rhodopseudomonas palustris

Abstract

While lignin represents a major fraction of the carbon in plant biomass, biological strategies to convert the components of this heterogeneous polymer into products of industrial and biotechnological value are lacking. Syringic acid (3,5-dimethoxy-4-hydroxybenzoic acid) is a by-product of lignin degradation, appearing in lignocellulosic hydrolysates, deconstructed lignin streams, and other agricultural products. Rhodopseudomonas palustris CGA009 is a known degrader of phenolic compounds under photoheterotrophic conditions via the benzoyl coenzyme A (CoA) degradation (BAD) pathway. However, R. palustris CGA009 is reported to be unable to metabolize meta-methoxylated phenolics, such as syringic acid. We isolated a strain of R. palustris (strain SA008.1.07), adapted from CGA009, which can grow on syringic acid under photoheterotrophic conditions, utilizing it as a sole source of organic carbon and reducing power. An SA008.1.07 mutant with an inactive benzoyl-CoA reductase structural gene was able to grow on syringic acid, demonstrating that the metabolism of this aromatic compound is not through the BAD pathway. Comparative gene expression analyses of SA008.1.07 implicated the involvement of products of the vanARB operon (rpa3619, rpa3620, rpa3621), which has been described as catalyzing aerobic aromatic ring demethylation in other bacteria, in anaerobic syringic acid degradation. In addition, experiments with a vanARB deletion mutant demonstrated the involvement of the vanARB operon in anaerobic syringic acid degradation. These observations provide new insights into the anaerobic degradation of meta-methoxylated and other aromatics by R. palustris. IMPORTANCE Lignin is the most abundant aromatic polymer on Earth and a resource that could eventually substitute for fossil fuels as a source of aromatic compounds for industrial and biotechnological applications. Engineering microorganisms for the production of aromatic-based biochemicals requires detailed knowledge of the metabolic pathways for the degradation of aromatics that are present in lignin. Our isolation and analysis of a Rhodopseudomonas palustris strain capable of syringic acid degradation reveal a previously unknown metabolic route for aromatic degradation in R. palustris. This study highlights several key features of this pathway and sets the stage for a more complete understanding of the microbial metabolic repertoire required to metabolize aromatic compounds from lignin and other renewable sources.

Kinetic modeling of anaerobic degradation of plant-derived aromatic mixtures by Rhodopseudomonas palustris

“…In the experiments of Austin et al ( 2015 ), vanillic acid accumulated as an extracellular product, but it is not known whether its accumulation represents the complete inability of R. palustris to degrade vanillic acid or a slow and incomplete degradation of this product. Experiments with vanillic acid as the sole carbon source show that this substrate does not support anaerobic growth of R. palustris (Harwood and Gibson 1988 ; Oshlag et al 2020 ), and thus, if vanillic acid was degraded, we hypothesize that it occurred by co-metabolism.…”

“…Syringic acid is not a substrate that supports anaerobic growth of wild type R. palustris . However, we recently adapted an R. palustris strain able to photoheterotrophically grow on this substrate as the sole organic carbon source (Oshlag et al 2020 ). This adaptation took several rounds of selection that were not used in the experiments of Austin et al ( 2015 ), in which syringic acid degradation was not apparent, and therefore, we hypothesize that syringic acid was not significantly co-metabolized in the experiments of Austin et al ( 2015 ).…”

“…The existing literature on anaerobic degradation of aromatics by R, palustris shows that aromatic acids with substitutions other than a hydroxyl at the para position to the carboxylic group cannot serve as sole organic carbon sources for photoheterotrophic growth of R. palustris (Harwood and Gibson 1988 ). There is a recent report of an R. palustris strain evolved to degrade syringic acid, which does not use the benzoyl-CoA pathway (Oshlag et al 2020 ). In addition, even though there is a limited number of aromatic compounds that support photoheterotrophic growth as sole organic carbon sources, R. palustris can metabolize a broad set of aromatic compounds when multiple aromatics are present (Austin et al 2015 ; Gall et al 2013 ).…”

Functional pathway redundancy in the metabolism of plant-derived phenolics byNovosphingobium aromaticivorans

“…Rhodopseudomonas palustris (11)(12)(13) have been extensively studied for their ability to transform the plant-derived phenolic compounds often present in deconstructed plant biomass.…”

“…This funneling of phenolic mixtures to single compounds has been demonstrated with engineered strains of Pseudomonas putida (6, 7), Rhodococcus jostii (8), and Novosphingobium aromaticivorans (9). In addition, other microbes, such as the yeast Rhodosporidium toruloides (10) and the photoheterotrophic bacterium Rhodopseudomonas palustris (11-13) have been extensively studied for their ability to transform the plant-derived phenolic compounds often present in deconstructed plant biomass.…”