Metabolic physiology of aroma‐producing Kluyveromyces marxianus

Kluyveromyces marxianus has a high potential for industrial production of aroma compounds, such as 2‐phenylethanol, which is derived in a bioconversion from L ‐phenylalanine. In the present work the product yield of K. marxianus in batch cultivation was estimated as 0.65 mol 2‐phenylethanol/mol L ‐phenylalanine and thus significantly below the theoretical optimum of 1 mol/mol. By a comprehensive approach of stoichiometric balancing and GC–MS analysis of various substrates and products of K. marxianus a detailed insight into its metabolism was gained. For this purpose ring‐labelled ( 13 C 6 ) L ‐phenylalanine and naturally labelled glucose were applied as substrates in tracer studies in batch culture. The produced aroma compounds 2‐phenylethanol and 2‐phenylethylacetate stem exclusively from the supplied L ‐phenylalanine, whereas glucose was not converted into these products because of efficient feed‐back inhibition of prephenate dehydratase in the L ‐phenylalanine biosynthetic pathway. It could be further shown that the supplied L ‐phenylalanine completely covers the anabolic cellular demand for this amino acid. Quantification of 13 CO 2 in the exhaust gas provided clear evidence for catabolic breakdown of L ‐phenylalanine during cultivation. Metabolic balancing around the pool of free intracellular L ‐phenylalanine revealed a significant loss of L ‐phenylalanine into catabolic and anabolic pathways. While 73.3% of L ‐phenylalanine was converted into 2‐phenylethanol or 2‐phenylethylacetate, 22.4% was catabolized through the cinnamate pathway and 4.3% was directed towards protein biosynthesis. Catabolic breakdown of L ‐phenylalanine via hydroxylation to L ‐tyrosine could be excluded. In addition to an insight into metabolic functioning and regulation of 2‐phenylethanol‐producing K. marxianus , the approach presented here provides important information on potential targets for genetic optimization of 2‐phenylethanol‐producing yeasts. Copyright © 2002 John Wiley & Sons, Ltd.