## 3 Results and Discussion
### Optimization of Culture Conditions by Single Factor Design  
Although producing 3-Met by yeasts from sugars via de novo biosynthesis has been rare, previous reports have emphasized that its biosynthesis by yeasts requires sugars [2; 3]. Therefore, the effect of glucose concentration on 3-Met production was evaluated: it increased initially as the glucose concentration increased and then decreased at higher concentrations. The yield of 3-Met was highest when the concentration range was 30-60 g/L (Supplementary Figure S2a), which agreed with previous reports [19]. When glucose is used as a carbon source, it provides energy for yeast growth and its concentration influences the rate of growth and the metabolic pathway and intensity. At a low glucose concentration, the biosynthesis of 3-Met was affected by a slow growth rate and fewer cells [54]. Although previous reports have shown that a higher glucose concentration was conducive to accumulating 3-Met, an excessive concentration can change the metabolic pathway because the oxygen concentration was insufficient due to rapid cell growth [11; 55]. Some studies have also shown that more ethanol and other higher alcohols may be produced at an excessive glucose concentration, which would be toxic to cells in combination with 3-Met [43]. Yeast extracts contain many vitamins and microelements, which can be used for yeast growth and affect the accumulation of yeast metabolites [8; 43; 56]. In the present study, 3-Met production was lower when yeast extract was absent because strain YHM-G was not able to grow well (Figure 2a). Supplementation with yeast extract increased 3-Met production compared with no addition, with the yield increasing as its concentration increased. Strain YHM-G was able to produce higher levels of 3-Met when 2-2.8 g/L of yeast extract was present with the highest production of 2.02 g/L when 2.0 g/L of yeast extract was used (Figure 2a). At a concentration of less than 2.0-2.4 g/L, yeast  
Figure 1: The range of growth temperature and pH (**a**) and the tolerance of glucose, sucrose, NaCl and ethanol (**b**) and 3-Met (**c**) of strain YHM-G.  
extract was able to be introduced as a nutrient to promote the growth of strain YHM-G [8]. While as the concentration of yeast extract increased, the bioproduction of 3-Met decreased. Previous reports have shown that almost all 3-Met is biosynthesized from L-Met as the precursor via the Ehrlich pathway, which can be affected by other nitrogen sources during this process [2; 8]. Thus, under an excessive yeast extract concentration, partial yeast extraction was applied as the yeast assimilable nitrogen to cause nitrogen catabolite repression to the Ehrlich pathway, leading to a decrease in production [5; 8; 57; 58]. An excessive addition would lead to rapid cell growth, resulting in excessive substrate consumption for cell reproduction but reducing the accumulation of metabolites. A similar observation has also been reported in previous studies where the effect of yeast extract on 3-Met bioproduction was higher at low concentrations of yeast extract [2; 3; 8]. It should be noted that the optimum concentration of yeast extract for 3-Met production by different yeasts varies, possibly because of their different metabolic capabilities [2; 3; 5]. Figure 2: Effect of yeast extract concentration (0, 0.4, 0.8, 1.2, 1.6, 2.0 and 2.4 g/L) (**a**), L-Met concentration (0, 2, 4, 6, 8, 10, 12 and 14 g/L) (**b**), time point of L-Met addition (0, 12, 24, 36, 48, 60 and 72 h) (**c**), initial pH (3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5 and 7.0) (**d**), temperature (20, 24, 28, 32, 36 and 40 \({}^{\circ}\)C) (**e**), shaking speed and loading volume (0, 45, 90, 135, 180, 225, 270 rpm and 25, 50, 75, 100 and 125 mL/250 mL) (**f**), surfactant types (control, glycerol, Tween-20, Tween-40, Tween-60, Tween-80, Triton X-100 and Triton X-114) (**g**) and time (0, 12, 24, 36, 48, 60, 72, 84 and 96 h) (**h**) on 3-Met concentration.