Determining which of the halogenated small molecules from Asparagopsis is responsible for the observed activity in inhibiting methane production in cattle and sheep

Summary

This project is undertaken as basic R&D toward developing a commercially viable feed additive for sheep and cattle with acceptable methane emission reduction (MER) for the red meat industry. It is aimed at examining quantitatively the MER effect of halogenated compounds from Asparagopsis seaweeds and their nonhalogenated analogues as inhibitors of the Factor F430 containing enzymes in two methanogenic archaea: Methanothermobacter marburgensis (MM) and Methanobrevibacter ruminantium (MR). MM is a model organism that has been well studied and is well understood in terms of methanogenic pathways. MR is one of the principle methanogens in cattle rumen, responsible to ruminant methane emissions.

In stage I of the project, an in vitro assay using live organisms as well as an enzyme assay, under strictly controlled anaerobic conditions, using purified methyl CoM reductase enzyme complex (MCR) from MM, were established to test potentially active compounds. A total of 18 compounds were assayed with in vitro assays and 3 known compounds assayed with the enzyme MCR assay. It was confirmed that 3-NOP and 2-BES inhibit MCR but that bromoform is a weak inhibitor of the enzyme but a strong inhibitor in vitro, suggesting it inhibits methane production via a different mechanism.

The development of a defined feed additive for the mitigation of methane emissions from ruminant animals will have potential economic impacts on the red meat industry in terms of increased production and helping to meet the industry’s net zero carbon emission by 2030 target (CN30). The long-term goal is to develop a cost-effective and commercially viable solution based on additives with well-defined mode-of-action.

Objectives

Optimally grown Methanobacter marburgensis from culture
Isolated and purified active MCR-red1 enzyme
Synthesised and purified methyl-coenzyme M, coenzyme B, and 5 potential inhibitors, which have already been designed and that are believed to be potential inhibitors of MCR based on the evidence that halogenated Asparagopsis alkanes inhibit MCR, not B12, enzymes)
Developed MCR-red1 methane production bioassay and initial testing of inhibitors
Provided recommendations to MLA for further R and D.

Key findings

Methanothermobacter marburgensis and Methanobrevibacter ruminantium were successfully grown under strictly anaerobic conditions continuously over 1 year;
MCRred1 was isolated and purified from Methanothermobacter marburgensis and shown to generate methane directly from Methyl-coenzyme M and Coenzyme B;
Methyl-coenzyme M and Coenzyme B were successfully synthesised at 0.3 g scale.
In vitro assay for MCR inhibition was successfully established using head space analysis with positive controls and in vitro assays for MER was established for both Methanothermobacter marburgensis (MM) and Methanobrevibacter ruminantium (MR).
18 potential inhibitors were tested, including 10 methyl-CoM mimics, 3 CoB mimics and 5 Asparagopsis compounds.
IC50 values were calculated on known methanogenesis inhibitors 2-BES, 3-NOP and bromoform.

Benefits to industry

A scalable and field-worthy compound as a feed additive, with defined mode of action (MOA) and reliable MER effects, would be highly valued by the red meat industry in maintaining its commercial viability and increasing production. In addition, it would enable the industry to pursue the Red Meat 2030 Vision of “doubling the value of red meat sales as the trusted source of the highest quality protein” while achieving its Carbon Neutral by 2030 target (CN30). The rapid reduction of methane emissions will also disproportionally slow global warming compared to reductions in CO2 emissions.

MLA action

Further development and adoption of this approach will be critical to maintaining the competitiveness of the Australian Red Meat industry and securing the long-term sustainability by meeting the emission reduction requirements within the CN30 framework.

Future research

With the groundwork set for finding and developing nature-mimicking compounds for defined MER effects, we strongly recommend proceeding with the second stage of this project:
Aim 1) Finding new chemical entities (NCEs), by designing and testing rationally nature-mimicking compounds, as potential feed additives with target-specific activity against the MCR enzyme;
Aim 2) Testing in vitro combinations of inhibitors for synergistic effects of MER with the potential of finding new targetable aspects of methanogenesis.