Investigation of fungal methionine synthesizing enzymes in the context of solid-state fermentation of peas

Methionine is an indispensable amino acid, which means that humans must ingest adequate amounts of it through their diets. The daily recommended intake for an adult is 10.4 mg per kg bodyweight. Methionine is part of proteins, but also fulfills roles as a free species, for example in cellular methylation processes. Animal-derived protein is a good source of methionine, but the production of meat and dairy causes greenhouse gas emissions and is resource-demanding. The production of plant foods is generally more climate-friendly, but the amino acid composition of their protein is often not ideal for humans. Pea protein, for example, contains low levels of methionine. Fungi can produce methionine de novo from precursors by the action of their enzymes. Solid-state fermentation of peas with filamentous fungi could, thus, be a way to produce new foods that contain more methionine than unfermented peas. By understanding the enzymes involved, it could be possible to choose optimal fungal strains for such fermentations.

In this thesis, we studied enzymes that catalyze methionine synthesis by transferring a methyl group to homocysteine. In fungi, cobalamin-independent methionine synthase is one such enzyme. We found that within fungi, these sequences are relatively well conserved. To assess kinetic differences between these enzymes, we characterized homologues of Aspergillus sojae, Rhizopus delemar, and Rhizopus microsporus. It was found that these enzymes had a low kcat. Aspergillus sojae and Rhizopus microsporus were found to have a k_cat of 3.35 min^-1 and 3.02 min^-1, respectively. The Rhizopus delemar homologue was even slower with a k_cat of 1.18 min^-1. With K_M values of 6.18 μM and 6.79 μM, the affinity for the methyl donor substrates is similar for Aspergillus sojae and Rhizopus microsporus homologues respectively. Rhizopus delemar MetE showed a K_M of 0.82 μM. For these studies, we used a colorimetric assay protocol that had been published, but that we optimized to work in 96 well microtiter plates.

To understand the role of these enzymes in solid-state fermentation systems, peas were fermented with Aspergillus oryzae IBT 21458 and Rhizopus oryzae IBT 7729. Both strains consumed available free methionine present in the peas between 0 h and 24 h. They then build up levels of free methionine between 24 h and 72 h. In Aspergillus oryzae, the levels are 79 % of the initial levels, while in Rhizopus oryzae the levels are 29 % of the initial levels. Cobalamin-independent methionine synthase rate in Aspergillus oryzae fermentation product was found to be circa 2.5 times higher than in Rhizopus oryzae fermentation product.

Despite sharing no sequence similarity, cobalamin-independent methionine synthase and cobalamin-dependent methionine synthase catalyze similar reactions and fulfill the same metabolic function. Fungi were believed to only encode cobalamin-independent methionine synthase until recently bioinformatic evidence was found for the presence of cobalamin-dependent methionine synthase in non-dikarya fungi including Rhizopus. We expect these enzymes to contribute to methionine production in fungi. We tested the fungal response to cobalamin, which was assessed via proteomics. In the absence of cobalamin, cobalamin-dependent methionine synthase was not detected in the proteome of Rhizopus oryzae IBT 7729. However, when grown in 2.5 μM or 10 μM of cobalamin, expression of the cobalamin-dependent methionine synthase is induced. The expression of one of the two copies of cobalamin-independent methionine synthase was found to be reduced in the presence of 10 μM of cobalamin. The induction of this enzyme in solid-state fermentation with Rhizopus could offer a way to enhance methionine production.

Homocysteine methyltransferase is another enzyme whose product is methionine. In some species, some homologues of this enzyme class can utilize S-methyl-methionine, which leads to the stoichiometric formation of two methionine per cycle. When looking into the diversity of publicly available sequences, we found that some species of Aspergillus and Penicillium encode two distantly related homologues of homocysteine methyltransferase. We speculate that one of these homologues is specialized on the utilization of S-methyl-methionine, which could make strains encoding this enzyme suitable candidates for future solid-state fungal fermentation efforts that have the goal to produce food products with enhanced methionine levels.

In summary, we have generated new insights about three enzymes involved in the biosynthesis of methionine in filamentous fungi.