Sensing, Uptake and Catabolism of L-Phenylalanine During 2-Phenylethanol Biosynthesis via the Ehrlich Pathway in Saccharomyces cerevisiae

Abstract

2-Phenylethanol (2-PE) is an important flavouring ingredient with a persistent rose-like odour, and it has been widely utilized in food, perfume, beverages, and medicine. Due to the potential existence of toxic byproducts in 2-PE resulting from chemical synthesis, the demand for "natural" 2-PE through biotransformation is increasing. L-Phenylalanine (L-Phe) is used as the precursor for the biosynthesis of 2-PE through the Ehrlich pathway by Saccharomyces cerevisiae. The regulation of L-Phe metabolism in S. cerevisiae is complicated and elaborate. We reviewed current progress on the signal transduction pathways of L-Phe sensing, uptake of extracellular L-Phe and 2-PE synthesis from L-Phe through the Ehrlich pathway. Moreover, the anticipated bottlenecks and future research directions for S. cerevisiae biosynthesis of 2-PE are discussed.

Keywords: 2-phenylethanol; Ehrlich pathway; Saccharomyces cerevisiae; sensing of L-phenylalanine; uptake of L-phenylalanine.

Figure 1

Extracellular L-Phenylalanine (L-Phe) signalling pathway mediated by the Ssy1-Ptr3-Ssy5 (SPS) sensor system. When L-Phe is the sole nitrogen source, Ssy1 may react to it and recruit casein kinase Yck1/2 to phosphorylate the N-terminus of Ssy5. The N-terminus of Ssy5 is ubiquitinated and degraded to free the Cat domain, which cleaves the N-terminal regulatory domains of Stp1 and Stp2. The shorter forms of Stp1 and Stp2 target the nucleus and bind the UAS_AA elements AGP1 and BAP2, activating expression of these genes. Then, Agp1p and Bap2p are secreted into the membrane of the endoplasmic reticulum, where they are processed, modified, and transferred to the Golgi apparatus for further processing and packaging. Finally, Agp1p and Bap2p are localized to the cell membrane and transport extracellular L-Phe into cells.

Figure 2

Sensing and regulation of intracellular L-Phe levels through the target of rapamycin (TOR) pathway. The TOR signalling cascade includes the EGOC complex, TOR complex 1 (TORC1), and downstream effectors (Tap42-PPase and Sch9). Intracellular L-Phe is sensed by the EGOC complex, and TORC1 can be inhibited. However, upon rapamycin treatment or in the presence of a poor nitrogen source, the activity of TORC1 can be restrained, which results in the dephosphorylation of Tap42, freeing it from the vacuole membrane. Gln3 is dephosphorylated by freed Tap42 and is freed from Ure2; then, it targets the nucleus and binds to the GATAA/G motif of nitrogen catabolite repression (NCR)-sensitive genes, activating the transcription of NCR-sensitive genes. In the presence of glutamate, TORC1 is activated and Gln3 cannot be dephosphorylated and resides in the cytoplasm, which represses the expression of NCR-sensitive genes.

Figure 3

Metabolic pathway of 2-Phenylethanol (2-PE) production in Saccharomyces cerevisiae. The Ehrlich pathway (red) and phenylpyruvate pathway (the combination of red and blue) produce 2-PE. PEP, phosphoenolpyruvate; PYR, pyruvate; E4P, erythrose-4-phosphate; DAHP, 3-deoxy-D-arabinoheptulosonate; DHQ, 3-dehydroquinate; DHS, 3-dehydroshikimate; SHK, shikimate; S3P, shikimate-3-phosphate; EPSP, 5-enolpyruvyshikimate-3-phosphate; CHR, chorismite; L-Phe, L-phenylalanine; PPA, phenylpyruvate; PAAL, phenylacetaldehyde; and 2-PE, 2-phenylethanol. 1, phospho-2-dehydro-3-deoxyheptonate aldolase ARO3/4; 2, pentafunctional AROM polypeptide ARO1; 3, chorismate synthase ARO2; 4, chorismate mutase ARO7; 5, prephenate dehydratase PHA2; 6, aminotransferase ARO8/9; 7, decarboxylase ARO10; 8, dehydrogenase ADH; 9, citrate T-cell target antigen CTT1/2; 10, aconitase ACO1; 11, isocitrate dehydrogenase IDH1/2; 12, NAD-dependent glutamate dehydrogenase 2 GDH2; 13, NADP-dependent glutamate dehydrogenase 3 GDH3; 14, NADH-dependent glutamate synthase 1 GLT1; and 15, glutamate-ammonia ligase GLN1.

Figure 4

Promoter sequences of Aro80 target genes. CCG triplets, the binding sites of Aro80, are underlined, and potential GATA factor binding sites (GATAA/G) are indicated in bold.