Effects of multiple metal ions for biomass and CHA production
The effects of metal ions were usually beneficial to the growth of microbe and the biosynthesis of active metabolites. In the present work, the different content (0.5 and 1 mM) of five categories metal ions (Al3+, Ca2+, Cu2+, Mn2+ and Zn2+) were individually researched for selection the dominant metal ion on CHA production and biomass in the Fig. 1. The effect of 0.5 mM Al3+ could effectively promote the CHA production, which reached 52.3 mg/L as 1.5 times that of the control. The higher concentration of Al3+ played little role in the biosynthesis of CHA production. The production of CHA was achieved respectively 55.31 and 40.18 mg/L by addition of 0.5 and 1 mM Ca2+. Ca2+ and Al3+ could enhance CHA production, but had no effect on biomass of A. fumigatus. The effect of Cu2+ on CHA production was decreased by the increased content, meanwhile, the effect of Cu2+ on biomass was increased. The similar phenomenon was showed by the effect of Mn2+, in which the inhibitory effect of Mn2+ was stronger than Cu2+. The Zn2+ completely inhibited CHA biosynthesis and reduced biomass of A. fumigatus. Comparation to the effects of five metal ions, the low concentration of Ca2+ and Al3+ could significantly improve the CHA production, which indicated Ca2+ and Al3+ were dominant metal ions.
Optimization of addition time and concentration by dominant metal ions
For enhancement of CHA production, the addition concentration and time of Ca2+ and Al3+ were investigated, which was based on the researched content of selection of metal ions. The different concentration (0, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7 mM) of Ca2+ was researched for CHA production and biomass of A. fumigatus in the Fig. 2A. The changing tendency of CHA production was gradually rising and then slowly falling by the increasing concentration of Ca2+. When the content of Ca2+ was 0.5 mM, the CHA production reached the maximum value, which was 58.8 mg/L as the optimized concentration. However, the effect of Ca2+ was not obvious in the biomass of A. fumigatus, in which the biomass was relatively lower with 0.5 and 0.55 mM Ca2+ than others. After the addition concentration of Ca2+ was determined, the addition time (0, 72, 120, 168, 216, 264, 312 h) of Ca2+ was subsequently studied, which results were showed in the Fig. 2B. The CHA production was fast increased and reached the peak (62.74 mg/L, at 120 h) following the time from 0-120 h, then tended towards balance. The biomass of A. fumigatus had an analogous tendency as optimization of concentration, which was that the tendency of biomass was not correspond to that of CHA production. Now, the optimized addition concentration and time of Ca2+ were respectively 0.5 mM and 120 h.
The analogous experimental methods were utilized to research the effect of Al3+ on CHA production and biomass. Similarly, the test concentration of Al3+ was designed as that of Ca2+, which results were displayed in Fig. 3A. The tendency of different concentration of Al3+ was increasing slowly from 0 mM to 0.45 mM, then maintaining stability from 0.45 mM to 0.6 mM and decreasing tardily from 0.6 mM to 0.7 mM. When the content of Al3+ was 0.5 and 0.55 mM, the CHA production was getting stabilized, which was respectively 49.45 and 49.15 mg/L. the effect of addition concentration of Al3+ on biomass was minor fluctuations, the trend of which was generally stable. The appropriate addition concentration of Al3+ was defined as 0.5 mM, after that the corresponding addition time of Al3+ was experimented. The addition time of Al3+ was set as 0, 72, 120, 168, 216, 264, 312 h, which results were showed in the Fig. 3B. the effect of addition time of Al3+ was increasing quickly and then decreasing slowly, in which the maximum value was 50.43 mg/L at 120 h same to that of Ca2+. Therefore, the optimization conditions of Ca2+ and Al3+ addition were determined, which was supplied with 0.5 mM Ca2+ and Al3+ at 120 h.
Intermediate metabolites response to metal ions addition
After the addition condition of Ca2+ and Al3+ were optimized (0.5 mM, 120 h), the change trend of intermediate metabolites was researched for exploring the mechanism of mental ions addition. The synthesis trend of pyruvate was rapidly formed in the early stage of fermentation and slowly consumed in the late stage of fermentation, in which the synthesis of pyruvate after addition of Ca2+ and Al3+ was lower than control in Fig. 4A. Before 120 h of fermentation, the change of DAHP were similar both in experimental group and control group. The biosynthesis production of DAHP was higher with addition of metal ions than control form 168 h to 312 h (Fig. 4B). The tendency of chorismate biosynthesis resembled to that of DAHP, in which the biosynthetic capacity of chorismate was also higher than control in Fig. 4C. The production of tryptophan with addition Ca2+ and Al3+ was fast increased from 0 h to 216 h and then gradually decreased from 216 h to 360 h, in which the synthesis amount of tryptophan was far more than control. Visibly, the addition of Ca2+ and Al3+ was advanced in biosynthesis of intermediate metabolites in CHA biosynthetic pathway.
Enzyme expression response to metal ions addition
After the intermediate metabolites were investigated with addition of metal ions, the activities of enzymes (DAHPs, AroA and TrpC) in CHA biosynthetic pathway were studied. The key enzymes, which played an important role in CHA production with addition of mental ions, were determined as the action sites of metal ions. DAHPs was a key enzyme in shikimate pathway, which was also an important enzyme in CHA biosynthetic pathway. After the metal ions added, the activity of DAHPs was increased first and then decreased, which were all higher than control at every time. The maximum of DAHPs activity was 3.58 folds than control at 216 h (Fig. 5A). AroAs was inducing the synthesis of 3-dehydroquinic acid, which was a key node of CHA biosynthetic pathway. The trend of AroAs activity was analogous to DAHPs in Fig. 5B, in which the AroAs activity was improved by 2.01, 3.60 and 2.10 folds than control at 168, 216 and 264 h. TrpCs induced indoleglycerol phosphate to synthesize tryptophan, which was a rate-limiting enzyme in CHA biosynthetic pathway. The addition effect of metal ions was not stronger than the first two enzymes, but in which the TrpCs activity was increased by 3.34 and 1.81 folds than control at 168 and 216 h in Fig. 5C. the effect of Ca2+ and Al3+ addition was useful in enhancing the activities of key enzymes in CHA biosynthetic pathway.
Gene expression response to metal ions addition
In the previous work, the author explored the biosynthetic way of CHA and found the predicted genes significantly affecting the biosynthesis of CHA (Liu et al. 2018, 2019). To explore further the possible mechanism of addition of Ca2+ and Al3+ on CHA production, transcription levels of four genes laeA (a global regulator), dahp, cs and trpC of CHA biosynthetic pathway were discovered by qRT-PCR. The expression levels of these genes in collaborative supplement of Ca2+ and Al3+ with samples taken at 120, 168, 216, 264, 312 and 360 h were investigated in Fig. 6.
As shown in Fig. 6A, the transcription level of laeA was obviously elevated at 168, 216 and 264 h, which increased multiple were respectively 2.89, 3.22 and 2.46 folds than the group without addition of metal ions. The expression level of dahp was increased about 12.65 folds at 168 h and 5.51 folds at 216 h (Fig. 6B), which were affected stronger than others with supplement of Ca2+ and Al3+. The transcription level of cs gene was up-regulated at 216, 264 and 312 h, and the expressed folds of this gene increased 5.58, 3.32 and 2.67 with feeding Ca2+ and Al3+ in Fig. 6C. The change trend of trpC gene was similar to dahp, but the enhanced effect of trpC gene was worse than dahp. The expression level of trpC was increased about 6.99 folds at 168 h and 2.12 folds at 216 h (Fig. 6D). These results indicated the key genes of CHA biosynthetic pathway were up-regulated with co-addition of Ca2+ and Al3+.
Scale-up CHA production to lab-scale bioreactor
For the supplement effect of Ca2+ and Al3+ was really worked, the scale-up experiment was performed in 5-L stirred bioreactor. As shown in Fig. 7A and B, the time profiles of 5-L bioreactor and shake flask with addition of Ca2+ and Al3+ were investigated. The trend of DCW was similar both in first 5-L bioreactor and shake flask, which was increased slowly finally declined slightly. However, DCW in 5-L bioreactor was litter lower than that in shake flask at the end of fermentation. The rate of sugar consumption was also analogy in 5-L bioreactor and shake flask, and the descent rate of sugar consumption was faster in 5-L bioreactor after feeding metal ions than that in shake flask. The change trend of pH was consistent, which was increased fast then declined and finally around 4 in 5-L bioreactor and shake flask. When Ca2+ and Al3+ were co-added in 5-L bioreactor, the value of pH was rising and then falling quickly at 120 h. The DO continued to decline during 0-120 h in Fig. 6B, then maintained stability at 20% from 120 h to 336 h. The trend of CHA production in 5-L bioreactor and shake flask were both increased then decreased. The maximum of CHA production in 5-L bioreactor was 75.6 mg/L at 336 h, which was higher than that (37.6 mg/L, at 360 h) in shake flask. Therefore, the scale-up experiment in laboratory was successful, in which the sugar consumption, pH and CHA production in 5-L bioreactor were all better than that in shake flask.