SummaryThe reduction of CO 2 emissions is a global effort which is not only supported by the society and politicians but also by the industry. Chemical producers worldwide follow the strategic goal to reduce CO 2 emissions by replacing existing fossil‐based production routes with sustainable alternatives. The smart use of CO and CO 2/H2 mixtures even allows to produce important chemical building blocks consuming the said gases as substrates in carboxydotrophic fermentations with acetogenic bacteria. However, existing industrial infrastructure and market demands impose constraints on microbes, bioprocesses and products that require careful consideration to ensure technical and economic success. The mini review provides scientific and industrial facets finally to enable the successful implementation of gas fermentation technologies in the industrial scale.

Kluyveromyces marxianus has a high potential for industrial production of aroma compounds, such as 2-phenylethanol, which is derived in a bioconversion from Lphenylalanine. 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 Lphenylalanine 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-phenylethanolproducing K. marxianus, the approach presented here provides important information on potential targets for genetic optimization of 2-phenylethanol-producing yeasts.

2-Phenylethanol (2-PE) is an important flavour and fragrance compound with a rose-like odour. Most of the world's annual production of several thousand tons is synthesised by chemical means but, due to increasing demand for natural flavours, alternative production methods are being sought. Harnessing the Ehrlich pathway of yeasts by bioconversion of L-phenylalanine to 2-PE could be an option, but in situ product removal is necessary due to product inhibition. This review describes the microbial production of 2-PE, and also summarizes the chemical syntheses and the market situation.

Yeasts can convert amino acids to flavor alcohols following the Ehrlich pathway, a reaction sequence comprising transamination, decarboxylation, and reduction. The alcohols can be further derivatized to the acetate esters by alcohol acetyl transferase. Using L: -methionine as sole nitrogen source and at high concentration, 3-(methylthio)-1-propanol (methionol) and 3-(methylthio)-propylacetate (3-MTPA) were produced with Saccharomyces cerevisiae. Methionol and 3-MTPA acted growth inhibiting at concentrations of >5 and >2 g L(-1), respectively. With the wild type strain S. cerevisiae CEN.PK113-7D, 3.5 g L(-1) methionol and trace amounts of 3-MTPA were achieved in a bioreactor. Overexpression of the alcohol acetyl transferase gene ATF1 under the control of a TDH3 (glyceraldehyde-3-phosphate dehydrogenase) promoter together with an optimization of the glucose feeding regime led to product concentrations of 2.2 g L(-1) 3-MTPA plus 2.5 g L(-1) methionol. These are the highest concentrations reported up to now for the biocatalytic synthesis of these flavor compounds which are applied in the production of savory aroma compositions such as meat, potato, and cheese flavorings.

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Die biotechnologische Produktion von natürlichen Aromastoffen scheitert häufig an zu niedrigen Produktkonzentrationen und -ausbeuten. Ein Grund dafür ist die starke Inhibition der hydrophoben Duft-und Aromastoffe. Die In-situ-Pervaporation, d. h. die Kopplung eines Pervaporations-Membranmoduls an einen aktiven Bioreaktor, stellt eine Möglichkeit dar, dieses Problem zu beheben. Sie ermöglicht eine kontinuierliche, effektive und selektive Abtrennung der Produkte. Im Folgenden wird beispielhaft die Biokonversion von L-Phenylalanin zu 2-Phenylethylacetat (2-PEA) und 2-Phenylethanol (2-PE) mit Williopsis saturnus dargestellt.In der durchgeführten klassischen Batch-Kultivierung konnten nur geringe Mengen 2-PE und 2-PEA gewonnen werden (s. Abb.). Durch die In-situ-Pervaporation konnte eine Produktabreicherung in der Kulturbrühe erreicht werden. Dies führte zu einer verringerten Inhibition und somit zu einer gesteigerten Aromastoffproduktion. So war im integrierten Bioprozess (IBP) mit einem Arbeitsvolumen von 2 L die 2-PEA-Ausbeute im Vergleich zum klassischen Batch 57 mal höher.Ein wesentlicher Parameter für die Effizienz des Verfahrens ist das Verhältnis zwischen Arbeitsvolumen und Membranfläche. Durch Halbierung dieses Verhältnisses konnte die Effizienz wesentlich erhöht und die Produktausbeuten weiter gesteigert werden. Die Ausbeute des 2-PEs vervierfachte sich im Vergleich zur klassischen Batch-Kultivierung, und die des 2-PEAs erhöhte sich um fast zwei Gröûenordnungen.Anhand dieser Ergebnisse wird offensichtlich, dass die Integration dieses Trennprozesses zur Produktabreicherung in einen produktinhibierten Bioprozess zu einer deutlichen Erhöhung der Produktausbeuten führt. Wesentlich für die Effizienz des Prozesses ist das Reaktorvolumen-Membranflächen-Verhältnis, da die Abreicherung durch den Stoffmengenfluss determiniert ist. So war die Produktausbeute (2-PE + 2-PEA) im IBP mit reduziertem Arbeitsvolumen im Vergleich zur klassischen Methode mehr als vierzehnmal höher. Abbildung.Vergleich der gebildeten Produktkonzentrationen in der klassischen Batch-Kultivierung im IBP mit 2 L Arbeitsvolumen und im IBP mit einem Arbeitsvolumen von 1 L.