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.
Wittmann
Christoph
C
Biochemical Engineering, Saarland University, Saarbruecken, Germany. c.wittmann@mx.uni-saarland.de
Hans
Michael
M
Bluemke
Wilfried
W
D001709:Biotechnology / Q000379:methods; D002247:Carbon Isotopes / Q000378:metabolism; D003470:Culture Media; D008401:Gas Chromatography-Mass Spectrometry; D015966:Gene Expression Regulation, Fungal; D005947:Glucose / Q000378:metabolism; D007716:Kluyveromyces / Q000378:metabolism*; D010648:Phenylacetates / Q000378:metabolism*; D010649:Phenylalanine / Q000378:metabolism; D010626:Phenylethyl Alcohol / Q000378:metabolism*
D002247:Carbon Isotopes; D003470:Culture Media; D010648:Phenylacetates; D010649:Phenylalanine; C025136:phenylacetic acid; D005947:Glucose; D010626:Phenylethyl Alcohol