# Anaerobic Limonene Metabolism in a Methanogenic Enrichment Involves a Glycine Radical Enzyme

## TL;DR

A methanogenic enrichment culture degrades limonene using a novel glycine radical enzyme and involves a complex microbial community.

## Contribution

The discovery of a glycine radical enzyme for anaerobic limonene activation in methanogenic systems is novel.

## Key findings

- A glycine radical enzyme activates limonene by adding fumarate in methanogenic systems.
- The microbial community includes a predator species and fermenting bacteria recycling necromass.
- Genes for limonene degradation and syntrophic fermentation are highly transcribed in Syntrophobacteraceae.

## Abstract

Limonene is a natural monoterpene omnipresent in human environments. It enters wastewater and is also metabolised in methanogenic digesters. A stable limonene‐degrading methanogenic enrichment culture was investigated by metagenomic, metatranscriptomic and metaproteomic data sets to characterise the microbial community and identify the limonene degradation pathway. Thirty‐two metagenome‐assembled genomes revealed a complex community of bacteria and methanogenic archaea dominated by Candidatus Velamenicoccus archaeovorus as the top predator, contributing two‐thirds of the reads in the metagenome. The presence of several fermenting bacteria (Anaerolineaceae, Aminidesulfovibrio, Smithellaceae, Lentimicrobium) indicated the recycling of necromass in a microbial loop. Only one hydrocarbon‐activating enzyme system was expressed, a member of the alkyl‐ and arylsuccinate synthase family which is a glycine radical enzyme that adds fumarate to hydrocarbons. The limonenylsuccinate synthase gene encodes a modified substrate binding pocket with two smaller amino acids, suggesting an adaptation for the larger structure of limonene. The limonenylsuccinate synthase operon and a ring cleavage operon, as well as genes for the final syntrophic fermentation to acetate, hydrogen and formate were encoded in a Syntrophobacteraceae genome. Almost all genes for this degradation pathway were highly transcribed and expressed, demonstrating a catalytic role for glycine radical enzymes in methanogenic systems degrading limonene.

A methanogenic enrichment culture on the natural monoterpene limonene is characterised by a novel glycine radical enzyme for anaerobic hydrocarbon activation, the presence of a microbial loop for necromass utilisation and the presence of a predator of B
acteria and A
rchaea.