Pyrazine Biosynthesis in Corynebacterium glutamicum

The volatile compounds released by Corynebacterium glutamicum were collected by use of the CLSA technique (closed-loop stripping apparatus) and analysed by GC-MS. The headspace extracts contained several acyloins and pyrazines that were identified by their synthesis or comparison to commercial standards. Feeding experiments with [H-2(7)]acetoin resulted in the incorporation of labelling into trimethylpyrazine and tetramethylpyrazine. Several deletion mutants targeting genes of the primary metabolism were constructed to elucidate the biosynthetic pathway to pyrazines in detail. A deletion mutant of the ketol-acid reducto-isomerase was not able to convert the acetoin precursor (S)-2-acetolactate into the pathway intermediate (R)-2,3-dihydroxy-3-methylbutanoate to the branched amino acids. This mutant requires valine, leucine, and isoleucine for growth and produces significantly higher amounts and more different compounds of the acyloin and pyrazine classes. Gene deletion of the acetolactate synthase (AS) resulted in a mutant that is not able to convert pyruvate into (S)-2-acetolactate. This mutant also requires branched amino acids and produces only very small amounts of pyrazines likely from valine via the valine biosynthetic pathway operating in reverse order. A Delta AS Delta KR double mutant was constructed that does not produce any pyrazines at all. These results open up a detailed biosynthetic model for the formation of alkylated pyrazines via acyloins.