3.1. Isolation and phylogenetic analysis of Meyerozymasp. 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 thede novo pathway (Fig. 1B), which surpassed that using metabolically engineered S. cerevisiae (96 mg/L) via de novo pathway (Shen et al., 2016). The 18S rDNA genes amplified from the genomic DNA of culture YLG18 showed 99% identity to Meyerozyma guilliermondii when blasted with the bacterial sequences in the GenBank database. Thus, this culture is designated as Meyerozyma sp. strain YLG18 (Table 2). M. guilliermondii is known to be an ascomycetous yeast, which is broadly used for the production of riboflavin, xylitol and industrial enzymes (Papon et al., 2013). Actually, M. guilliermondii has been reported to produce 2-PE through biological conversion of L-phe, and the highest 2-PE production could reach 1.61 g/L (Karolina, Katarzyna, Daria, & Jolanta, 2017), 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.
2-PE can increase the cell membrane fluidity and reduce the uptake of amino acids 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 (Seward, Willetts, Dinsdale, & Lloyd, 1996). Studies have reported that 2.0 g/L of 2-PE could completely inhibit the growth of S. cerevisiae W303-1A andS. cerevisiae Giv 2009 (Stark, Münch, Sonnleitner, Marison, & Stockar, 2002). 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 Fig. 1C, with the increase of exogenously added 2-PE, the inhibition effect on strain growth was obviously observed. For instance, with the supplementation of 3.5 g/L of 2-PE, OD600 reached 10, which is only half of that in the presence of 1.5 g/L 2-PE. 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 (Schrader, Etschmann, Sell, Hilmer, & Rabenhorst, 2004).