3.5 Characterization of related genes and biosynthetic pathway of 2-PE by Meyerozyma sp. strain YLG18 through transcriptome analysis
At present, only limited literatures have reported 2-PE production by using M. guilliermondii. To further elaborate the 2-PE production mechanism by strain YLG18, genes involved in substrate consumption, L-phe metabolism and 2-PE production were identified and characterized, which could help speculate the 2-PE biosynthesis pathway and provided clues on the divergence of gene expression level between Shikimate pathway and Ehrlich pathway (Fig. 5). After integrating the annotations of strain genome sequences in GO, COG and KEGG, total 10 main unigenes (aroF, aroM, CM, aroC, pheA, AAT, ARO8, HisC, PDC, ADH ) with functions assigned to 2-PE biosynthesis were identified. According to KEGG annotation, the first five genes are key ones in the Shikimate pathway, which were mainly responsible for conversion of glucose into phenylpyruvate; while the last five genes are key ones in the Ehrlich pathway, which were mainly responsible for conversion of L-phe into 2-PE. As for the Shikimate pathway in strain YLG18, a penta-functional enzyme of AroM was identified, which is different from some prokaryotes, whose Shikimate pathway reactions are mainly catalyzed by enzymes encoded by individual genes (aroB , aroD , aroE ,aroK , aroA ). To further confirm the function of this penta-functional enzyme AroM, the protein sequence was blasted in NCBI website of non-redundant protein sequences (nr) database. Only 59.33% similarity was shown between AroM from strain YLG18 and the penta-functional protein ARO1p from S. cerevisiae S288C (NP_010412.1). Previous studies have reported that the penta-functional protein ARO1 in S. cerevisiae is essential for aromatic amino acid production (Gold et al., 2015; Hassing et al., 2019). Although AroM in M. guilliermondii complicates tuning gene expression at individual activity level, it can convert 3-deoxy-D-arabinoheptulosonate-7-phosphate (DAHP) to 5-enolpyruvyl-shikimate-3-phosphate (EPSP), which is important in Shikimate pathway. This newly identified penta-functional enzyme AroM from strain YLG18 may broaden our knowledge of 2-PE production through Shikimate de novo pathway.
To confirm the reproducibility and accuracy of differential gene expression identified through the Illumina analysis, genes related to phenylalanine biosynthesis and phenylalanine metabolism were selected for qRT-PCR analysis to determine the FPKM value. As can be seen from Fig. 5, the expression levels of genes in Ehrlich pathway were higher than those in Shikimate pathway except for HisC. The overall change in the expression pattern for Ehrlich pathway vs. Shikimate pathway ranged from 0.95-fold to 62.68- fold. This result can effectively explain that the 2-PE production synthesized through Ehrlich pathway was generally higher than that synthesized through Shikimate pathway. It is important to point out that the expression of aspartate aminotransferase (AAT) shows nearly 62 times difference in these two pathways. Although substrates were different, the expression of ARO8, which is also an amino acid aminotransferase was far lower than that of AAT. This means that AAT plays an important role in the Ehrlich pathway of strain YLG18. Different from the existing results on 2-PE production by other yeasts such as S. cerevisiae , various amino acid transaminases (Aro8, Aro9, Bat1, and Bat2) have shown to catalyze the first step of Ehrlich pathway in the catabolism of aromatic amino acids(Kim, Cho, & Hahn, 2014; Yin et al., 2015). However, AAT has rarely been reported for 2-PE production. Shrawder et al. (Shrawder & Martinez-Carrion, 1972) have proved that AAT from porcine heart had phenylalanine transaminase activity, and Cárdenas-Fernández et al. (Cárdenas-Fernández, López, álvaro, & López-Santín, 2012) have successfully synthesized L-phenylalanine with immobilized AAT. Therefore, AAT may be a potential enzyme for 2-PE synthesis, and Meyerozyma sp. strain YLG18 may also serve as a potential candidate for industrial 2-PE production from L-phe. Future studies are needed to elaborate AroM and AAT function, and the tolerance mechanism of strain YLG18.
4. Conclusion
This study presents how a newly identified M. guilliermondiistrain YLG18 can be used as a potential candidate for high 2-PE production from L-phe. After determination of influencing factors, process optimization using RSM and reducing toxicity of 2-PE with ISPR techniques, 3.20 g/L of 2-PE can be produced from 63.54 g/L of glucose and 10.70 g/L of L-phe. The high 2-PE production by using strain YLG18 indicates that it may show great potential for 2-PE production. Candidate genes related to 2-PE biosynthesis and metabolism can be used as target genes for marker-assisted selection through genetic engineering to further improve final 2-PE titer in future studies.