The use of the growth factor is an economical way to improve PMLA production(Cao et al. 2019b). However, less study on the metabolomics changes under the different growth factor has limited the better understanding of the mechanism for PMLA biosynthesis from the metabolic aspect.
In this study, the addition of 3 g/L CSL facilitates cell growth and PMLA production in A. melanogenum. The maximal PMLA production and biomass increased by 32.76% and 41.82%, respectively. Furthermore, the specific growth rate revealed a rapid increase during the early phase of fermentation, and Yp/x in the CSL group was higher than that of the control in the late stage. Moreover, CSL contained various amino acids and vitamins (Table 1) that could function as an effective stimulatory agent of cell growth and PMLA biosynthesis. It was found that the rich nutrients of CSL became a source of nitrogen, essential minerals, and cofactors required for Pichia pastoris cell growth(Zheng et al. 2012). Moreover, the addition of CSL increased the biomass of Trametes versicolor in the initial 12 h of fermentation, and an accelerated growth rate was observed(Wang et al. 2014). The addition of 3 g/L CSL provided various amino acids and vitamins (Table 1), indicating that the CSL could be an effective stimulatory agent of cell growth in A. melanogenum. The high cell growth of A. melanogenum led to a high level of PMLA production. Therefore, we speculated that various nutritional substances provided by CSL benefited cell growth, which in turn benefited PMLA production.
With the help of metabolomics technology, the working mechanism underlying the effect of the addition of 3 g/L CSL on the metabolism of A. melanogenum was analyzed. The metabolomics data showed that the relative concentrations of metabolites involved in amino and organic acid metabolism were changed during fermentation after the addition of 3 g/L CSL (Fig. 5). And the PLS-DA showed a clear separation between 2 groups. In addition, the Tyrosine metabolism, Glycine and serine metabolism, and TCA cycle were up-regulated after the 3 g/L CSL addition. Among them, for the tyrosine metabolism, tyrosine is first converted to 4-hydroxyphenylpyruvate then to homogentisate that is further converted to fumarlacetoacetate, and fumarlacetoacetate is finally transferred to fumarate and enter the TCA cycle(Wang et al. 2019). The metabolomics data showed that the relative concentration of homogentisate increased by 1.44-fold at 144 h compared to the control, and that of fumarate increased by 1.52-fold. Moreover, the concentration of 5-aminolevulinate, which related to the Glycine and serine metabolism, increased by 1.2-fold under the CSL. And a 1.2-fold change on 2-aminomuconate semialde hyde concentration resulted in the Tryptophan up-regulated metabolism that leads tryptophan into the glutaric acid pathway and then the TCA cycle(Fukuwatari et al. 2001). The result also proved that deoxyinosine, involved in purine metabolism, increased by 1.2-fold, and this metabolism is related to the cell’s nitrogen absorption, which can provide molecules that are essential to DNA and RNA biosynthesis, energy metabolism and signal transduction(Jessica and James 2017). Combined with the fermentation data(Fig. 2), the result showed that PMLA production sustained and PMLA yield decreased after 120 h. it was consistent with the metabolomics data that the metabolites related to the PMLA biosynthesis reached the highest at 120 h then it began to decrease.
Metabolomics data can express the metabolic changes under different conditions. However, after the peak alignment and the data normalization, some of the target-metabolites may lose in the data. Therefore, we speculated that the concentrations of tyrosine, glycine, serine, and tryptophan, which related to the up-regulated Tyrosine pathway, Glycine and serine pathway, and Tryptophan pathway, were improved in the CSL group. These pathways may further cause the PMLA production increased. Consequently, we tested the nutritional substances of CSL and found seventeen kinds of amino acids and three types of vitamins (Table 1). Among them, serine is 1.28%, tyrosine is 0.62%, tryptophan is 0.17%, and glycine is 2.05% of the total CSL weight.
In order to figure out which amino acid most benefiting PMLA production in the CSL, different amino acids from the up-regulated pathway (Fig. 5) were added to the fermentation broth based on the ratio of their quality to the total CSL quality to evaluate the effect on PMLA production. The result demonstrated that all these four amino acids increased PMLA production by 29.5%, 21.9%, 9.3%, and 7.6%, respectively. Apart from that, a significant change between tyrosine and the control(P < 0.01) was observed, and this result is consistent with the enrichment analysis, indicating that the CSL significantly influenced the Tyrosine metabolism(P < 0.05). The data obtained from the PMLA-related pathway (Fig. 5) suggested that tyrosine flows to the TCA cycle by converted to fumarate, which is further converted to malic acid and increases the production of PMLA. Meanwhile, it also illustrated that all of the four amino acids, tyrosine, glycine, serine, and tryptophan, eventually flows to the TCA cycle(Fig. 5). However, due to the relative concentration changes of the metabolites (pyruvate and fumarate), we can speculate that the tyrosine probably plays a crucial role in increasing PMLA production under the CSL.
The results, as mentioned above, showed that the TCA cycle was up-regulated after the addition of 3 g/L CSL and led to an increase in malic acid production, which would further increase PMLA production. Therefore, we can conclude that the up-regulated TCA cycle is the key metabolic pathway under the 3 g/L CSL for the increase of PMLA production among three speculated metabolic pathways which are the glyoxylate acid pathway, the reductive TCA pathway, and the TCA pathway.(Zou et al. 2019). Meanwhile, the energy provided by the up-regulated purine metabolism may accelerate this process.
The improvement in amino acid and organic acid metabolism in A. melanogenum from the addition of 3 g/L CSL generated various amino acids and organic acids to improve cell growth. The conversions of the metabolites related to the TCA cycle were enhanced after the addition of 3 g/L CSL. Therefore, we speculated that CSL is an effective stimulatory agent for cell growth and PMLA biosynthesis in A. melanogenum. Meanwhile, CSL could be used as an economic nitrogen source due to its high nutrition and low cost. As a potential replacement of peptone and yeast extract in PMLA production, CSL has satisfactory use prospects in the PMLA industry.