Metabolic reprogramming and computation-aided protein engineering for high-level de novo biosynthesis for 2-phenylethanol in Pichia pastoris

Abstract

2-Phenylethanol (2-PE), an aromatic compound with a characteristic rose fragrance, is extensively used in the food and cosmetic industries as a flavoring and fragrance agent. Due to limitations in obtaining 2-PE from natural plant sources, microbial cell factories offer a promising alternative for sustainable biosynthesis. In this study, Pichia pastoris was engineered to efficiently synthesize 2-PE. Using computer-assisted predictions of interactions between the key phenylpyruvate decarboxylase KDC2 and its substrates or products, an optimal enzyme variant was rationally designed to boost 2-PE production. Additionally, the shikimic acid pathway was enhanced, and a dynamic regulation promoter was employed to reduce competition from alternative pathways. These strategies significantly increased metabolic flux toward 2-PE production, achieving a titer of 2.81 g/L and 45.8-fold improvement over the non-engineered strain. By integrating controlled carbon feeding and in situ extraction to alleviate acetic acid inhibition and product toxicity, the recombinant strain achieved a final 2-PE titer of 7.10 g/L and a yield of 0.14 g/g glucose, the highest reported microbial production to date. This study highlights the significant potential of P. pastoris as a versatile cell factory for the green biosynthesis of 2-PE and other natural products.

Keywords: 2-Phenylethanol; In situ product removal; Metabolic engineering; Pichia pastoris; Protein engineering.

Graphical abstract

Fig. 1

Schematic diagram of multiple strategies for 2-PE overproduction in P. pastoris. The overexpressed gene is shown in red, the down-regulated gene is shown in green. ALD4 gene was fused with a C-terminal ePTS1 tag. Bbxfpk, codon-optimized Bbxfpk gene; G6P, glucose-6-phosphate; F6P, Fructose-6-phosphate; PEP, phosphoenolpyruvate; E4P, erythrose-4-phosphate; DAHP, 3-deoxy-d-arabino-heptulosonate-7-phosphate; SHIK, shikimate; CHR, chorismate; PPA, prephenate; PPY, phenylpyruvate; TYR, tyrosine; PAC, phenylacetaldehyde; PYR, pyruvate; TKL2, transketolase; ARO4^K219L, feedback-resistant DAHP; ARO1, pentafunctional; ARO2, chorismate synthase; ARO7^G139S, feedback-resistant chorismate mutase; TYR1, prephenate dehydrogenase; PHA2, prephenate dehydratase; KDC2, phenylpyruvate decarboxylase; ALD4, phenylacetaldehyde dehydrogenase.

Fig. 2

The biosynthetic routes of aromatic compound.

Fig. 3

The production of 2-PE was increased by strengthening shikimic acid pathway. (A) Schematic of the 2-PE synthesis pathway in P. pastoris. The genes shown in red are overexpressed. (B) 2-PE production by overexpressing phenylpyruvate decaroxylase genes. (C) 2-PE production by adding precursor shikimic acid. (D–E) Optimizing the shikimic acid pathway increased 2-PE production. Data represent the mean ± S.D. of three biological replicates. Statistical analysis was performed by using one-way ANOVA (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001).

Fig. 4

Regulating the accumulation of E4P and PEP for 2-PE production. (A) The schematic diagram of regulating the supply of E4P and PEP by reforming the pathway. (B) 2-PE production by overexpressing TKL2 gene. (C) Different strength promoters drive Bbxfpk gene to increase E4P concentration to produce 2-PE. Data represent the mean ± S.D. of three biological replicates. Statistical analysis was performed by using one-way ANOVA (∗p < 0.05, ∗∗p < 0.01).

Fig. 5

Directed evolution of phenylpyruvate decarboxylase KDC2. (A–B) Virtual mutant of potential hot spots using Discovery Studio and the average mutation energy of the candidate residues. (C) Variable destabilizing residues are selected. (D) The relative activity of KDC2 variants screened based on random mutagenesis. (E) Evolutionary pathways from WT to the best performing mutant. (F) Reinforcement the 2-PE downstream pathway. Data represent the mean ± S.D. of three biological replicates. Statistical analysis was performed by using one-way ANOVA (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001).

Fig. 6

Modulation of competitive pathway expression. (A) The screening of promoters resulted in a reduction of by-product tyrosol accumulation while enhancing 2-PE production. (B) The schematic of acetic acid metabolic pathway after the introduction of Bbxfpk. (C) Effects of 2-PE and acetic acid on the growth of P. pastoris. (D) Effect of adding calcium carbonate on fermentation of P. pastoris. (E) The synthesis of 2-PE and acetic acid by Pp17–10p strain under two culture strategies. The initial sugar concentrations of batch culture and fed-batch culture were 70 g/L and 20 g/L, respectively. Fed-batch culture was supplemented with 5 g/L glucose every 12 h. Data represent the mean ± S.D. of three biological replicates. Statistical analysis was performed by using one-way ANOVA (∗∗p < 0.01, ∗∗∗p < 0.001).

Fig. 7

The diagram of 2-PE fed-batch two-phase fermentation. (A) The ability of different organic solvents to extract 2-PE. (B) The effect of two-phase extraction technology on the synthesis of 2-PE by strain Pp18.