Biocatalytic synthesis of L-methionine via enzymatic carbon dioxide fixation

In the era of a steadily growing world population and ongoing accumulation of greenhouse gases, in particular carbon dioxide, providing food while protecting the environment constitutes an increasing challenge.

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The paper in Nature Catalysis is here:

In animal breeding, essential amino acids are added to the feedstock in order to increase the efficiency of weight gain. In this context, L-methionine (L-Met) is one of the most important food additives, comprising a world-wide production volume of more than 1 million metric tons per year. However, the use of the extremely toxic reactant hydrogen cyanide (HCN) during conventional chemical synthesis of this amino acid makes this route challenging with regard to process safety.

Would it not be nice to produce L-Met in a safer way – without HCN – while utilising the abundant industrial side product CO2?

In frame of a call for research proposals, our cooperation partner Evonik Industries was looking for an alternative, HCN-free route for the synthesis of L-Met starting from the bulk product methional. Albeit this call was primarily addressed to scientists in the area of preparative chemistry, we proposed an enzymatic approach that involved reversal of the catabolic Ehrlich pathway and, in particular, the biocatalytic fixation of gaseous CO2.

First and critical step in our strategy was the carboxylation of the aliphatic aldehyde methional employing a decarboxylase in the reverse mode. This was followed by conversion of the resulting α-ketoacid (4-methylthio-2-oxobutanoate, MTOB) into L-Met via a coupled stereospecific amination reaction catalysed by an aminotransferase or an amino acid dehydrogenase.

Use of a decarboxylase for CO2 fixation appeared challenging as in biochemistry simple decarboxylation reactions are generally regarded irreversible under physiological conditions. There was one previous publication that had claimed the enzymatic incorporation of CO2 into acetaldehyde using a concentrated carbonate solution but not gaseous CO2. However, such strongly alkaline conditions are deleterious for enzyme as well as substrate and product stability, and both we and other labs had difficulties in reproducing this study.

Thus, in the beginning of our endeavour there were several crucial questions: Is it possible to reverse a decarboxylase, especially under the slightly acidic pH that develops under a CO2 atmosphere? Which is a suitable enzyme and what is its Michaelis-Menten constant for CO2? Finally, what is the chemical equilibrium constant for the incorporation of CO2 into methional (without the use of an explicit biochemical energy source such as ATP), and which CO2 pressure is needed to drive the biocatalytic reaction?

In our topical paper in Nature Catalysis 1, 555–561 (2018) [DOI 10.1038/s41929-018-0107-4] we provide the answers: The decarboxylase KdcA from Lactococcus lactis can catalyse its reverse reaction, namely the carboxylation of methional, with a relatively low KM for CO2 of merely 3.3 bar (110 mM) and at pH 6–7. Hence, 2 bar gaseous CO2 applied in a small glass pressure vessel were sufficient to synthesise L-Met when coupling the chemically unfavoured carboxylation (1/554 M-1, as quantified in our study using the Haldane relationship) to an energetically preferred transamination reaction, either catalysed by the aminotransferase YbdL from Escherichia coli or the amino acid (leucine) dehydrogenase from Lysinibacillus sphaericus.

The same strategy was successfully applied to produce the amino acids L-leucine and L-isoleucine from the appropriate aldehyde substrates. While our experiments were performed in vitro, it can easily be envisaged to combine the pair of enzymes – possibly including a regeneration system for the cosubstrate of the amination reaction – into a bacterial whole cell biocatalyst. Thus, our work opens a novel (and HCN-free) route to a variety of amino acids and valuable building blocks for pharmaceutical chemistry in an environmental friendly manner.

Written by Julia Martin and Arne Skerra

Arne Skerra

Professor, Technical University of Munich