3.2. Determination of influencing factors for 2-PE production byMeyerozyma 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 (Mu, Hu, Liu, Zhao, & Xu, 2014). 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 (Fig. 2B). The highest 1.55 g/L of 2-PE was obtained at 30ºC with molar conversion of 70.86%. Studies have shown M. guilliermondii preferred 37ºC for ethanol production, however, slightly lower 2-PE production of 1.3 g/L occurred, and an OD600 of 14.32 was achieved at 37ºC, indicating that elevated temperatures unfavored 2-PE production and microbial growth for strain YLG 18. Similar results were also obtained when otherM. guilliermondii strain, such as M. guilliermondii WUT22 was used for bioconversion of L-phe to 2-PE (Diniz, Rodrigues, Fietto, Passos, & Silveira, 2013; Ferreira et al., 2015).
During the bioconversion process, 30 g/L of glucose can be rapidly consumed during 48 h, however, 3.65 g/L of L-phe was still left over with initial concentration of 5 g/L (data not shown). Hence, different initial glucose concentrations were assessed for their effects on 2-PE production. As seen in Fig. 2C, when initial glucose concentration was 40.0 g/L, the highest 2.17 g/L of 2-PE and 77.6% of molar conversion were achieved, respectively. The molar conversion would decrease with the increase of glucose concentration. When glucose concentration was above 40 g/L, OD600 dropped from 38.15 to 31.96 with the increase 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 OD600 of 35.72 were achieved, which was higher than that using S. cerevisiae Ye9- 612 E (0.85 g/L) (Eshkol, Sendovski, Bahalul, Kashi, & Fishman, 2015; Stark et al., 2003). 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 study, 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 (Azevedo et al., 2018).