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).