## 3 Results and Discussion
### Optimization of Culture Conditions by Single Factor Design
Same letters in the column indicates that the data do not differ significantly at 5% probability by [PERSON]’s test. As L-Met is the precursor for 3-Met synthesis in the Ehrlich pathway, its concentration significantly influences 3-Met synthesis by yeasts [5,59]. Like previous reports, the present study found that no 3-Met was produced when L-Met was not present, thus confirming the importance of L-Met for the biosynthesis of 3-Met and the unlikeness of successful 3-Met biosynthesis from de novo sugars by strain YHM-G (Figure 2b) [2]. More 3-Met was synthesized as L-Met increased in the fermentation medium as it is an essential precursor for 3-Met production. It has been reported that increasing the L-Met concentration can enhance the formation of 3-Met, but in the present study, the yield reached a peak of 4 g/L, and when L-Met concentrations were >4 g/L, the production of 3-Met decreased slightly (Figure 2b) [3,5,8]. The reason was that an excessive L-Met concentration would strengthen demethylation, thereby reducing the effective L-Met concentration and intermediate metabolites in the Ehrlich pathway [3,5]. Some studies have also shown that the nitrogen content is positively correlated with the fused acid content, so more 3-methylhiopropionic acid was produced via the Ehrlich pathway at high L-Met concentrations, which was more toxic to the cells [3,5,60,61]. Regarding the economics of production, the optimum L-Met concentration in most studies was between 1 and 5 g/L. The results of the present study were also in this range, and the differing optimal L-Met concentrations for 3-Met production with different yeasts may have been caused mainly by differences in the characteristics of the strains and also by the slight differences between the initial fermentation mediums [3,5,6,8]. Although our results have confirmed the importance of L-Met for 3-Met synthesis by strain YHM-G, the question arises as to when adding L-Met could promote 3-Met synthesis. At present, no information detailing the optimal time for L-Met addition has been reported. The optimal time at which L-Met should be added for 3-Met production by strain YHM-G was determined by adding it at different culture times. 3-Met production was significantly higher when L-Met was added after 24 h of culture compared with adding at the start of culture, with no significant increase in production at 12 or 36 h but lower production at other times (Figure 2c). The results also showed that 3-Met production decreased gradually as the time of addition was greater than 24 h (Figure 2c). This was due to the overall effect of several factors: (1) L-Met is not only required for 3-Met production but is also an amino acid required for optimum growth of yeast; (2) adding L-Met later would shorten the time available for the transformation and utilization of L-Met to 3-Met; (3) the conversion of L-Met to 3-Met via the Ehrlich pathway relies on either ATP or the electrochemical gradient so that adding L-Met at the beginning of fermentation would inhibit cell growth and adding too late would lead to insufficient ATP because of the high consumption of glucose and dissolved oxygen [5]; and (4) yeast extract contained in the fermentation medium can take over the role of L-Met as an amino acid for growth when added later. For strain YHM-G, when L-Met was added at 24 h of culture, L-Met can be fully utilized as a precursor and amino acid, so that the state of the cells and energy storage in the system was optimal. Studies have shown that fluctuations in the environmental pH can stress microorganisms, thus changing the permeability of cell membranes and the activity of enzymes. The pH can change the state of nutrients in the system and affect the utilization of nutrients by microorganisms, resulting in different growth states and products for the microorganisms [8,25,62,63]. The optimum pH for 3-Met production was measured over a range from pH 3 to 7.