Yarrowia lipolytica as a potential producer of 2-phenylethanol from L-phenylalanine biotransformation A. Braga 1,* , A. Oliveira 1 , B. Freitas 1 , E. Nagy 2 , D. Q. Nguyen 2 , I. Belo 1 1 CEB - Centre of Biological Engineering, University of Minho, Campus de Gualtar, 4710–057 Braga, Portugal 2 Research Centre for Bioengineering and Process Engineering, Szent István University, Ménesi út 45, H-1118 Budapest Hungary. * abraga@deb.uminho.pt 2-Phenylethanol (2-PE) is an aromatic alcohol with a delicate fragrance of rose petals. The non-conventional yeast Yarrowia lipolytica is extensively explored for flavor compounds production, but the production of 2-PE has been very poorly described. This study investigated the potential of different Y. lipolytica strains (W29, CBS2075, CH 1/5 and CH 3/4) for 2-PE production. It was confirmed that all strains were able to produce 2-PE by L- phenylalanine (L-Phe) bioconversion, but were inhibited by 2-PE concentrations above 2 g L -1 . The strain Y. lipolytica CH 1/5 was selected for further studies since it produced the highest 2-PE titer (2.2 g L -1 ). Afterwards, the effect of L-Phe concentration and carbon source (glucose and crude glycerol) on 2-PE production was studied, and it was observed that increasing L-Phe concentration decreases the aroma production, and that the highest titer was obtained with glycerol. This study demonstrates the promising production of 2-PE using Y. lipolytica as biotechnological platform for flavors production. Introduction 2-Phenylethanol (2-PE) is an aromatic alcohol with a fresh rose scent, and is the second most widely used flavor after vanillin [1]. It is commonly used in the cosmetics and perfumes industries [1], with a world market volume of nearly 10 000 t, mainly produced by chemical synthesis. Nevertheless, the use of the chemically synthesised flavor compounds is restricted to some applications and natural flavor compounds are preferred by consumers [2]. However, its natural production includes the extraction from plants and this process involves several steps of down-stream operations, which makes the market price of natural 2-PE more expensive ($1000/kg) than the chemically synthesised ($5/kg) [3]. Hence, great interest has been arising for the biotechnological production of 2-PE. Several microorganisms have been described as possessing the ability to synthesise 2- PE [1]. 2-PE can be synthesized in yeast through shikimate and Ehrlich pathways (Graphical abstract). Either way, the main bottleneck for yeast fermentation of 2-PE is its toxicity [1] due to the fact that concentrations between 2 and 3 g L -1 inhibit the cellular growth [1] and the results obtained so far do not seem viable for industrial scale-up. Among several microorganisms able to produce 2-PE, the yeast Y. lipolytica appears to be promising due to its interesting characteristics, such as the Crabtree negative trait and absence of ethanol production, however this process has been fairly described. Material and Methods The strains used in this work were Y. lipolytica W29 (ATCC 20460), CBS2075, CH 1/5 and CH 3/4 (isolated from chesee). Y. lipolytica strains were cultivated for 16-17 hours in of YPD medium (glucose 20 g L -1 , peptone 20 g L -1 , yeast extract 10 g L -1 ) at 200 rpm and 27 ºC, and further used to inoculate the bioconversion experiments with an initial OD600 of 0.5. Bioconversion of L-Phe to 2-PE was carried out in cultivation medium containing per liter of deionized water: glucose or glycerol 40 g, KH2PO4 15 g, MgSO4.7H2O 0.5 g, YNB without amino acids 0.02 g, thiamine 3 mg, pH 6.5, supplemented with L-Phe 4 g or 6 g incubated at 27 ºC and 200 rpm. Glucose and glycerol were quantified by high- performance liquid chromatography. The 2-PE and L-Phe quantification was obtained using a SHIMADZU UHPLC system equipped with a diode array detector (SPD-M20A) at a fixed wavelength of 215 nm. LC separation was carried out with a YMC ODS-Aq (250 mm × 4.6 mm) reverse phase column at 25 °C. For elution, water (solvent A) and acetonitrile (solvent B) were applied as the mobile phases at a flow rate of 1 mL min -1 . A gradient was used, where the amount of solvent A was increased stepwise: 0 min – 100% A, 10 min – 100% A, 16.7 min - 70% A, 26.7 min – 70% A, 33.3 min – 100% A; 41.7 min – 100% A. Results and Discussion The main challenge for microbial production of 2-PE is its cytotoxicity [1]. To study the tolerance of different Y. lipolytica strains toward 2-PE, cells were cultivated in solid medium containing various concentrations of 2-PE. As shown in Fig. 1 the growth of all strains was completely repressed at a 2-PE concentration of 2 g L -1 . Comparing the performance of the four Y. lipolytica strains herein studied it was possible to observe that W29 and CBS2075 strains were more tolerant to 2-PE. Figure 1. Inhibitory effect of 2-PE for the Y. lipolytica W29, CH 1/5, CH 3/4 and CBS2075 strains. Cells were incubated in YPD medium for 16 h and diluted to an OD600 of 0.6. Cells (3 L) at a dilution of 10 0 , 10 -1 , 10 -2 , 10 -3 and 10 -4 were spotted on solid media (glucose 40 g L -1 , agar 20 g L -1 and (NH4)2SO4 2 g L -1 ) containing different concentrations of 2-PE (0 (control), 1, 2 and 3, g L -1 ). Cells were incubated at 30 ºC during 48h. [O-BB07] CHEMPOR 2018 41