Inhibition of Rumen Methanogens by a Novel Archaeal Lytic Enzyme Displayed on Tailored Bionanoparticles

Abstract

Methane is a potent greenhouse gas, 25 times more efficient at trapping heat than carbon dioxide. Ruminant methane emissions contribute almost 30% to anthropogenic sources of global atmospheric methane levels and a reduction in methane emissions would significantly contribute to slowing global temperature rises. Here we demonstrate the use of a lytic enyzme, PeiR, from a methanogen virus that infects Methanobrevibacter ruminantium M1 as an effective agent inhibiting a range of rumen methanogen strains in pure culture. We determined the substrate specificity of soluble PeiR and demonstrated that the enzyme is capable of hydrolysing the pseudomurein cell walls of methanogens. Subsequently, peiR was fused to the polyhydroxyalkanoate (PHA) synthase gene phaC and displayed on the surface of PHA bionanoparticles (BNPs) expressed in Eschericia coli via one-step biosynthesis. These tailored BNPs were capable of lysing not only the original methanogen host strain, but a wide range of other rumen methanogen strains in vitro. Methane production was reduced by up to 97% for 5 days post-inoculation in the in vitro assay. We propose that tailored BNPs carrying anti-methanogen enzymes represent a new class of methane inhibitors. Tailored BNPs can be rapidly developed and may be able to modulate the methanogen community in vivo with the aim to lower ruminant methane emissions without impacting animal productivity.

Rumen Virus Populations: Technological Advances Enhancing Current Understanding

“…Sequence analysis of rumen sample RNA (metatranscriptomics) has also been used to identify RNA virus populations (Hitch et al, 2019). In addition, alternative technologies are being applied for the isolation of virus particles (for example, chromatography) and enabling the expression of novel viral proteins in vitro (Figura et al, 2011;Krajacic et al, 2017;Altermann et al, 2018). The emerging field of proteomics, is also providing fresh insights into the roles viruses and the proteins produced by viruses, may play within the rumen microbial ecosystem (Solden et al, 2018).…”

“…Recently the in vitro expression of peiR was reported (Altermann et al, 2018). In this study, initial experiments were conducted to produce this protein using traditional gene cloning, transformation, protein expression in a commerciallyavailable E. coli expression strain (Schofield et al, 2015), with the final purified protein employed in lytic activity assays.…”

“…To further improve the stability and enable practical delivery of the enzyme, an alternative protein expression strategy was adopted. This approach involved the peiR gene being fused to the polyhydroxyalkanoate (PHA) synthase gene which was then expressed in E. coli to create PHA bionanoparticles with the lytic enzyme being displayed on the outer surface of the bionanoparticle (Altermann et al, 2018). Using this combination of techniques, sufficient quantities were produced to allow testing for cell-wall degrading effects against several strains of Methanobrevibacter, which are usually the most highly abundant methanogen genera found in the rumen (St-Pierre and Wright, 2012), and other methanogenic genera (Methanobacterium, Methanosphaera, Methanospirillum, and Methanonosarcina).…”

“…Chromatography is a traditional chemistry technique for the separation of mixtures, and has the potential be applied for the purification of viruses from rumen samples. Currently the use of chromatography in rumen research has been limited to metabolomic and proteomic studies with only a few publications combining chromatography and virus protein research (Schofield et al, 2015;Altermann et al, 2018). Given the proteinaceous nature of virus structures, particularly phages, implementing protein chromatography methods to separate or isolate whole phages directly should be possible.…”

“…Mechanisms for the delivery and release of viral material from the host often involves enzymes (lysins/holins, peptidases, depolymerases, tail spike proteins), which can be repurposed as enzybiotics (Murphy et al, 1995;Andres et al, 2012;São-José, 2018). Indeed, enzybiotics have been tested in animals (O'Flaherty et al, 2009) and targeted nanoparticles displaying enzybiotics have been demonstrated to reduce methanogenesis by rumen-associated archaea (Altermann et al, 2018). Virus-derived enzybiotics are appealing, due to their scalability and ease of use within existing guidelines, enzybiotics could represent the future direction of phage research.…”