Enhanced 2-phenylethanol production by newly isolated Meyerozyma sp. strain YLG18 and characterization of its synthetic pathways

2-Phenylethanol (2-PE) is an aromatic alcohol which has been widely used in cosmetics, perfume and food industries owning to its delicate rose scent. The newly isolated yeast Meyerozyma guilliermondii YLG18 was able to tolerate high exogenous 2-PE and produce 2-PE with two different pathways. A unique Meyerozyma sp. strain YLG18 was obtained in this study, which was capable of tolerating 4.0 g/L exogenous 2-PE. Response surface methodology (RSM) was implemented to improve the maximum 2-PE production. At optimized conditions: temperature, 24.7℃; initial glucose, 63.54 g/L; initial L-phe, 10.70 g/L, 2-PE production was increased to 2.55 g/L, leading to 104% increase compared to the pre-optimized one. In situ product recovery (ISPR) could further help improve the final 2-PE production to 3.20 g/L with fatty acid methyl ester as the extractant, representing the highest 2-PE production by using Meyerozyma sp.. Furthermore, genes involved in 2-PE synthesis were identified and their expression levels between Shikimate pathway and Ehrlich pathway were compared. Based on the genomic and transcriptional analysis, a penta-functional enzyme AroM in Shikimate pathway and an aspartate aminotransferase (AAT) with the potential to convert phenylalanine into phenylpyruvate in Ehrlich pathway were identified. These findings broaden add to the pool of known 2-PE generating microbes and genes. Moreover, this study describes a potential, new 2-PE producer that lays foundation for the industrial-scale production of 2-PE and its

Background 2-Phenylethanol (2-PE) is an aromatic alcohol, which can be naturally found in essential oils of many plants, such as hyacinths, jasmine and lilies. 2-PE has been widely used in cosmetics, perfume and food industries owning to its delicate rose scent [1]. Furthermore, 2-PE is also an important raw material for the derivatives synthesis, among which phenylethyl acetate is a valuable fragrance compound, and p-hydroxyphenylethanol is widely used in pharmaceutical and fine chemicals industries [2,3]. Although 2-PE can be extracted from flowers and plants, the extremely low concentration hinders it application [4]. Alternatively, 2-PE can be chemically synthesized, however, its quality is greatly affected because of the harsh condition and toxic reagents used [5]. Currently, the price of 2-PE produced through natural routes, such as extraction from rose petals or bio-converted from renewable resources is approximately USD 1,000/kg. However, it is only USD 5.0/kg for chemically synthesized 2-PE from benzene and styrene [6]. Therefore, bio-synthesis of 2-PE has become an appealing option owning to its environmentally friendly property and mild conditions.
In nature, many wild-type microorganisms have been identified and characterized to be capable of producing 2-PE, most of which are from eukaryotes, including Saccharomyces sp., Kluyveromyces marxianus, Yarrowia lipolytica, Aspergillus oryzae, and Pichia sp., etc [2,7]. In addition, some prokaryotes have also been reported to produce 2-PE, such as Microbacterium foliorum, Proteus vulgaris, and Psychrobacter sp.. 2-PE can be either converted from L-phenylalanine (L-phe) through three steps catalysis of Ehrlich pathway or produced from glucose through Shikimate pathway. As multiple steps are needed for 2-PE production through Shikimate pathway, lower concentration usually occurred when 2-PE was directly synthesized from glucose. Accordingly, Ehrlich pathway is thought to be more promising for 2-PE production. It should be noticed that the lipophilic 2-PE could make the lipid membrane structure a preferential binding target, resulting in the collapse of transmembrane gradients and consequently the loss of cell viability [8], hence, strain development including mutation, selection, or genetic modification has been comprehensively adopted to improve the final 2-PE production. For instance, the newly isolated K. marxianus CCT 7735 could generate 3.44 g/L of 2-PE through Ehrlich pathway under optimized conditions [9]. S. cerevisiae BY4741, which overexpressed ARO10 and contained an aro8Δ deletion could produce 96 mg/L of 2-PE from glucose [10]. S. cerevisiae SPO810, in which ARO8 and ARO10 were coexpressed could finally produce 2.61 g/L of 2-PE with fed batch fermentation [3].
In the present study, novel 2-PE-generating microbes were first isolated and characterized. To further improve 2-PE production, statistical design of experimental strategy and in situ extraction strategy were used to improve final 2-PE titer. Genes involved in 2-PE synthesis were also identified and characterized by transcriptome analysis.

Results and discussion
Isolation and phylogenetic analysis of Meyerozyma sp. strain YLG18 In this study, L-phe was used as the sole nitrogen source for the isolation of 2-PE generating strains (Fig. 1A). After more than 5 consecutive transfers in the medium spiked with 5 g/L of L-phe and 40 g/L of glucose, one colony named YLG18 gave the highest 2-PE production and molar conversion. Furthermore, YLG18 can even produce 100 mg/L of 2-PE in synthetic medium without supplementation of L-phe via the de novo pathway (Fig. 1B), which surpassed that using metabolically engineered S. cerevisiae (96 mg/L) via de novo pathway [10]. The 18S rDNA genes amplified from the genomic DNA of culture YLG18 showed 99% identity to Meyerozyma guilliermondii when blasted with the microbio sequences in the GenBank database.
guilliermondii is known to be an ascomycetous yeast, which is broadly used for the production of riboflavin, xylitol and industrial enzymes [11]. Actually, M.
guilliermondii has been reported to produce 2-PE through biological conversion of Lphe, and the highest 2-PE production could reach 1.61 g/L [12], however, the underlying mechanism for 2-PE production has not been clearly elaborated. Therefore, this newly isolated wild-type 2-PE-producing Meyerozyma sp. strain YLG18 may further broaden our knowledge and add to the pool of known 2-PE generating microbes. and glucose, leading to the low microbial growth and 2-PE production. The increased permeability of membrane would accelerate the transmembrane diffusion of ions and small molecular metabolites, and disrupt the transmembrane proton potential [13]. Studies have reported that 2.0 g/L of 2-PE could completely inhibit the growth of S. cerevisiae W303-1A and S. cerevisiae Giv 2009 [14]. Therefore, to further determine the 2-PE tolerance potential of strain YLG18, the 2-PE tolerance of strain YLG18 was investigated. Different concentrations of exogenous 2-PE ranging from 1.5 g/L to 4.0 g/L were added into the fermentation medium initially. As seen from When the concentration of exogenous 2-PE reached 4.0 g/L, strain growth was almost completely inhibited. Nevertheless, the higher 2-PE tolerance level of strain YLG18 compared to current reported 2-PE producers indicated that it may be promising candidate for high 2-PE production [15].
Determination of influencing factors for 2-PE production by Meyerozyma sp. strain

YLG18
To further improve the final 2-PE titer, various strategies including fermentation condition optimization and process integration have been developed. During fermentation process, carbon source has been proved as an important factor influencing cell growth, L-phe consumption and 2-PE molar conversion. Therefore, different carbon sources including glycerol, glucose, xylose, rapeseed oil and NaAc were chosen for 2-PE production ( Fig. 2A). After 96 h fermentation in mineral salts medium, the highest 1.25 g/L of 2-PE with glucose as the carbon source occurred with 51.3% molar conversion, which is 38.89%, 56.25%, 316.67% and 150% higher than that using xylose, glycerol, rapeseed oil and NaAc as substrate, respectively. It should be noticed that although glycerol is more reduced than glucose and provide more NADH, which is critical for the last reduction step in Ehrlich pathway, it only gave 0.8 g/L of 2-PE. Also, previous studies have shown that lower pH is relatively favorable for cell growth, but unfavorable for 2-PE production [16]. However, when sodium acetate was used as carbon source to increase pH, 2-PE production was only 0.5 g/L.
The effect of temperature ranging from 25 to 37ºC was also evaluated in synthetic medium containing 30.0 g/L of glucose ( concentration. Especially, 2-PE production kinetics is paralleled to that of microbial growth, suggesting that 2-PE production by strain YLG18 was closely related to cell growth. As L-phe was used as the nitrogen source for both microbial growth and 2-PE production, different amounts of L-phe were also evaluated for the improvement of 2-PE production. It can be seen from Fig. 2D that 2-PE production kinetics was basically consistent with the yeast growth. When L-phe concentration was 7 g/L, 2.22 g/L of 2-PE and a maximum OD 600 of 35.72 were achieved, which was higher than that using S. cerevisiae Ye9-612 E (0.85 g/L) [19,20]. However, the molar conversion with 7.0 g/L of 2-PE was only 79%, which could be because the molar conversion was negatively correlated with the initial L-phe concentration. This finding proved that high L-phe concentration in a certain range is beneficial for 2-PE production, but would reduce the conversion yield, which was also verified by other studies, in which L-phe concentrations above 4.0 g/L did not lead to the increase of 2-PE titer by using K. marxianus CCT 7735 [9].
Optimization of fermentation conditions for enhanced 2-PE production from L-phe Based on the above results using the method of "one time one factor", further statistical experimental design methodology was applied to evaluate the interaction and determine the optimal level of three influencing factors including temperature, initial glucose and initial L-phe levels. For the response surface analysis, 17 experiments with triplicates were conducted according to the RSM design, where Y is 2-PE production (g/L), X 1 denotes temperature (℃), X 2 denotes initial glucose level (g/L) and X 3 denotes initial L-phe level (g/L) ( Table 2). According to the response values obtained from the experimental results, a second order regression equation was generated for the response surface, as follows:  (1)) gave a high R 2 value of 0.9710 (Table 3), which indicated aptness of the model [21]. The R 2 value is between 0 and 1. When R 2 is close to 1.0, the model will better predict the response [22,23]. Notably, the model F value of 26.06 and values of probability (P) > F (0.0001) indicated that the model terms were significant [24]. The three-dimensional response surface plot is generally used to demonstrate relationships between the response and experimental levels of each variable (Fig. 3). The highest point on the contour profiles in Fig. 3 indicates the optimal parameter values of the highest 2-PE production. As shown, there was obvious interaction between each pair of variables, and the interaction between the selected three variables was significant. The optimal 2-PE production value predicted from the response surface model was 2.37 g/L, when the temperature was controlled at about 24.7℃ and initial glucose and L-phe concentrations were 63.5 and 10.7 g/L, respectively. The following validation experiment in 3.0 L bioreactor was carried out under the optimized conditions with 63.54 g/L of initial glucose, 10.70 of L-phe g/L at 24.7℃.
The 2-PE production was finally improved up to 2.55 g/L after 96 h, which is 7.8% higher than the predicted level, representing the highest 2-PE production from Lphe by using M. guilliermondii [ 12]. Hence, the models developed were considered to be accurate and reliable for predicting 2-PE production from L-phe by using strain YLG18.
Biosynthesis of 2-PE by using Meyerozyma sp. strain YLG18 with ISPR technology As mentioned, 2-PE is toxic to microbial cells, as it can make the lipid membrane structure a preferential binding target, resulting in the collapse of transmembrane gradients and the loss of cell viability [8]. To further increase the final 2-PE production and productivity, more robust strains or novel extraction technology should be developed. In situ product recovery (ISPR) techniques can simultaneously remove 2-PE from the fermentation broth while it is produced. Thereby, 2-PE concentration could be maintained below the inhibitory level, and microbes are able to continuously produce 2-PE. Suitable extractants including fatty acid methyl ester (FAME), oleic acid and ethyl acetate were first identified. As seen from Fig. 4A, the partition coefficients of these organic solvents were similar for 2-PE. However, different organic solvents had different impacts on the dissolved oxygen level, which would affect microbial growth and lead to significant difference in the final 2-PE titer [25]. Among these organic solvents, FAME gave the highest 2.53 g/L of 2-PE.
The volume ratio of medium and extractant phases will also affect the distribution and mass transfer of products. Low ratio of FAME could not extract 2-PE effectively, affecting the bioconversion rate. High ratio of FAME may increase toxicity to cells.
Results showed that the highest 2.48 g/L of 2-PE was obtained when FAME : water ratio was maintained at 1:1 (Fig. 4B). It should be noticed that when the ratio of FAME to water was 1:2, the production of 2-PE was 2.45 g/L, which is close to the highest one. Accordingly, the optimal ratio of 1:2 was chosen for following experiments.
Generally, microbes would show a log phase after inoculated into the fresh medium.
The presence of FAME in the initial stage may prolong this period and affect the transformation activity of yeasts. Therefore, a proper delay of FAME addition may help to improve the metabolic activity of yeasts and ultimately increase 2-PE production. From Fig. 4C, it can be seen that the earlier extractants were added, the more obvious the strain growth inhibition was. Compared with the experimental results of groups 4, 5 and 6, when FAME was supplemented when strain growth reached exponential phase, the death rate would be decreased significantly. In addition, it can be seen from Fig. 4D that when the strain death rate decreases, the 2-PE production in the group 4 was also the highest, and it can reach 3.20 g/L, which was improved by 25.49% compared to single-phase biotransformation. To confirm the reproducibility and accuracy of differential gene expression identified through the Illumina analysis, genes related to phenylalanine biosynthesis and phenylalanine metabolism were selected for qRT-PCR analysis to determine the FPKM value. As can be seen from Fig. 5 [3,26]. However, AAT has rarely been reported for 2-PE production.
Shrawder et al. [27] have proved that AAT from porcine heart had phenylalanine transaminase activity, and Cárdenas-Fernández et al. [28] have successfully synthesized L-phenylalanine with immobilized AAT. Therefore, AAT may be a potential enzyme for 2-PE synthesis, and Meyerozyma sp. strain YLG18 may also serve as a potential candidate for industrial 2-PE production from L-phe. Future studies are needed to elaborate AroM and AAT function, and the tolerance mechanism of strain YLG18.

Conclusion
This study presents how a newly identified M. guilliermondii strain YLG18 can be used as a potential candidate for high 2-PE production from L-phe. After determination of influencing factors, process optimization using RSM and reducing toxicity of 2-PE with ISPR techniques, 3.20 g/L of 2-PE can be produced from 63.54 g/L of glucose and 10.70 g/L of L-phe. The high 2-PE production by using strain YLG18 indicates that it may show great potential for 2-PE production.
Candidate genes related to 2-PE biosynthesis and metabolism can be used as target genes for marker-assisted selection through genetic engineering to further improve final 2-PE titer in future studies.

Materials and methods
Isolation and molecular identification of strain YLG18 Soil samples from Xuanwu Lake, Nanjing, China were used as inocula to screen   [30]. The values reported represent the average of 3 biological replicates [31]. Table 4 Primers used for qRT-PCR analysis. Predicted function Gene name  Primers used for qPCR  K13830  pentafunctional  AROM polypeptide  aroM  F  R  GCGGAATCGAACA  TTTGTCGAGCA  TTGAGGGTTTTCAG  CGGCCAAATA  K01736  chorismate  synthase  aroC  F  R  ATTCAGAGTGACCA  CCTATGGTGAG  CACAAGCATACCAA  TTGGAGATCCC  K01626 3  Proposed metabolic pathway for 2-PE production within strain YLG18 and comparison of expr