Bioproduction of the aroma compound 2‐Phenylethanol in a solid–liquid two‐phase partitioning bioreactor system by Kluyveromyces marxianus

Abstract

The rose-like aroma compound 2-phenylethanol (2-PE) is an important fragrance and flavor ingredient. Several yeast strains are able to convert l-phenylalanine (l-phe) to 2-PE among which Kluyveromyces marxianus has shown promising results. The limitation of this process is the low product concentration and productivity primarily due to end product inhibition. This study explored the possibility and benefits of using a solid-liquid Two-Phase Partition Bioreactor (TPPB) system as an in situ product removal technique. The system applies polymer beads as the sequestering immiscible phase to partition 2-PE and reduce the aqueous 2-PE concentration to non-inhibitory levels. Among six polymers screened for extracting 2-PE, Hytrel 8206 performed best with a partition coefficient of 79. The desired product stored in the polymer was ultimately extracted using methanol. A 3 L working volume solid-liquid batch mode TPPB using 500 g Hytrel as the sequestering phase generated a final overall 2-PE concentration of 13.7 g/L, the highest reported in the current literature. This was based on a polymer phase concentration of 88.74 g/L and aqueous phase concentration of 1.2 g/L. Even better results were achieved via contact with more polymers (approximately 900 g) with the aqueous phase applying a semi-continuous reactor configuration. In this system, a final 2-PE concentration (overall) of 20.4 g/L was achieved with 1.4 g/L in the aqueous and 97 g/L in the polymer phase. The overall productivities of these two reactor systems were 0.38 and 0.43 g/L h, respectively. This is the first report in the literature of the use of a polymer sequestering phase to enhance the bioproduction of 2-PE, and exceeds the performance of two-liquid phase systems in terms of productivity as well as ease of operation (no emulsions) and ultimate product recovery.

Bioproduction of benzaldehyde in a solid–liquid two-phase partitioning bioreactor using Pichia pastoris

“…Over the course of the biotransformation period, 1 g polymer samples were periodically collected and tested for benzyl alcohol and benzaldehyde concentrations using the methanol extraction technique described by Gao and Daugulis (2009), in which the polymer beads are extracted repeatedly with methanol and the concentrations of target molecules determined from mass balances and polymer density.…”

“…These studies have shown initially promising results with an increase in total benzaldehyde concentration achieved compared to that of single phase systems. However, limitations associated with the use of organic solvents, including potential toxicity to the biocatalyst as well as the negative effect on the flavour/fragrance compound due to fugitive aromas, have led to the use of commodity polymers, which are biocompatible, non-bioavailable as the sequestering phase, chemically inert, and inexpensive (Gao and Daugulis 2009;Khan and Daugulis 2010;Morrish and Daugulis 2008;Prpich and Daugulis 2007). In this work, the biotransformation of benzyl alcohol to benzaldehyde using P. pastoris was investigated using rational polymer selection for sequestering phase selection in a solid-liquid TPPB.…”

Elevated production of the aromatic fragrance molecule, 2‐phenylethanol, using Metschnikowia pulcherrima through both de novo and ex novo conversion in batch and continuous modes

“…This is higher than 2PE production from other yeast systems presented in the literature using the adsorbants Hytrel, polypropylene glycol 1200, or polystyrene resin 14, 15…”

Identification of auxotrophic mutants of the yeast Kluyveromyces marxianus by non‐homologous end joining‐mediated integrative transformation with genes from Saccharomyces cerevisiae

“…In contrast to S. cerevisiae and K. lactis, which are widely used for molecular biology and industrial applications (Mattiazzi et al, 2012;Schaffrath and Breunig, 2000;Schwimmer et al, 2006;Smith and Snyder, 2006;van Leeuwen et al, 2012), K. marxianus has received relatively little attention, although there are an increasing number of reports describing the potential application of K. marxianus for the production of ethanol, biomass, endogenous enzymes and aromatic compounds (Bansal et al, 2008;Fonseca et al, 2008;Gao and Daugulis, 2009;Limtong et al, 2007;Nonklang et al, 2008Nonklang et al, , 2009Rodrussamee et al, 2011;Wittmann et al, 2002) and for protein structural analysis (Watanabe et al, 2012;Yamaguchi et al, 2012aYamaguchi et al, , 2012b. Thus, in the present study, we generated and characterized 79 auxotrophic mutants and identified 35 complementing genes using a novel integrative transformation approach for the development of genetic tools in K. marxianus.…”