Fig 1.
A holistic view of the engineering route in S. cerevisiae to effective 2-PE producer through Ehrlich pathway.
Four categories of methods for manipulating the flux from L-phenylalanine to 2-phenylethanol were grouped and shown respectively. Metabolite abbreviations: L-Phe, L-phenylalanine; PPY, phenylpyruvate; PAA, phenylacetaldehyde; PAC, phenylacetate; 2-PE, 2-phenylethanol. Genes and enzymes: ARO8, ARO9, tyrB, aromatic aminotransferase; PDC1, PDC5, PDC6, pyruvate decarboxylase; ARO10, THI3, kdcA, phenylpyruvate decarboxylase; ALD2, ALD3, acetaldehyde dehydrogenase; ADH1, ADH2, ADH3, ADH4, ADH5, SFA1, alcohol dehydrogenase; lgox, L-glutamate oxidase.
Table 1.
Strains used in this study.
Fig 2.
Overview of the experimental procedures used to obtain 2-PE tolerant yeast strain.
(A) Cell growth of the control strain WT-A in YEPD medium added with different concentration of 2-PE addition. (B) Schematic diagram of the adaptive laboratory evolution process of S. cerevisiae strain AD032. (C) Cell growth and 2-PE titer of ALE stains in YSM light medium. Data are presented as the averages of the results of three independent experiments. Error bars show standard deviations. Error bars show standard deviations. Statistical analysis was performed by using Student’s t-test (one-tailed; two-sample unequal variance; *p < 0.05, **p < 0.01, ***p < 0.001).
Fig 3.
Improvement of cell growth and 2-PE production by adaptive laboratory evolution of S. cerevisiae strain AD032.
(A) Tolerance of the adaptive strain AD032 and control strain WT-A under 2-PE stress in YEPD medium. (B) Cell growth of control strain WT-A and evolutionary adaptive strain AD032 in the flask fermentation with 4 g/L phenylalanine (a) and 6.7 g/L phenylalanine (b) culture medium. (C) Improvement of 2-PE production of control strain WT-A and evolutionary adaptive strain AD032 in the flask fermentation with 4 g/L phenylalanine (a) and 6.7 g/L phenylalanine (b) culture medium. Data are presented as the averages of the results of three independent experiments. Error bars show standard deviations.
Fig 4.
Effective expression of individual key enzyme and fusion protein in Ehrlich pathway.
(A) Influence on cell growth and 2-PE production by overexpressing enzymes of Ehrlich pathway respectively. The gene types were listed under the bar graph. Enzyme abbreviations: PAT, phenylalanine transaminase; PhDC, phenylpyruvate decarboxylase; ADH, alcohol dehydrogenase. The mutant strains were cultured in YSM light medium (4 g/L phenylalanine) for flask fermentation. (B) Gene ARO8, ARO9, ARO10 and ADH2 transcription levels in the mutant strains. (C) Gene tyrB and KdcA transcription levels in the mutant strains. Data are presented as the averages of the results of three independent experiments. Error bars show standard deviations. Statistical analysis was performed by using Student’s t-test (one-tailed; two-sample unequal variance; *p < 0.05, **p < 0.01, ***p < 0.001).
Fig 5.
Influence on 2-PE production by fusion-expressing mutant strains.
(A) Glucose consumption, ethanol and acetate of JM00 and RM17 (WT-A PTPI-ectyrB-llkdcA-scADH2) in YSM medium. (B) Cell growth, 2-PE titers and L-phenylalanine consumption of JM00 and RM17 in YSM medium. (C) Fermentation view of RM17 in YSM medium with the addition of 20 g/L glucose every 24 h. (D) Fermentation view of RM812 (WT-A PTPI-scARO8-scARO10-scADH2) in YSM medium with the addition of 20 g/L glucose every 24 h. Data are presented as the averages of the results of three independent experiments. Error bars show standard deviations.
Fig 6.
The new expression module of lgox for 2-PE synthesis in Ehrlich pathway.
(A) Influence on cell growth and 2-PE production by L-glutamate oxidase expressing mutant strains. (B) Cell growth, ethanol and glucose consumption of RM17 (WT-A PTPI-ectyrB-llkdcA-scADH2) and RM20 (WT-A PTPI-ectyrB-llkdcA-scADH2 PTPI-strlgox) in YSM medium. (C) Improvement of 2-PE synthesis and L-phenylalanine consumption in RM20 compared to RM17 in YSM medium. (D) Fermentation view of RM20 in YSM medium with the addition of 20 g/L glucose every 24 h. Data are presented as the averages of the results of three independent experiments. Error bars show standard deviations.
Fig 7.
Impact on 2-PE synthesis in PDC5 deleting mutant.
(A) Influence on 2-PE synthesis abilities of yeast cell by mutant strains RM21 (WT-A pdc1Δ), RM22 (WT-A pdc5Δ), RM23 (WT-A pdc6Δ), RM24 (WT-A aro10Δ), and RM25 (WT-A thi3Δ). (B) Fermentation view of RM22 (WT-A pdc5△). (C) Diagram of the genetic methods in the engineered strain RM26. (D) Fermentation view of RM26 (WT-A pdc5△ PTPI-ectyrB-llkdcA-scADH2 PTPI-strlgox) in YSM medium with the addition of 20 g/L glucose every 24 h. Data are presented as the averages of the results of three independent experiments. Error bars show standard deviations. Statistical analysis was performed by using Student’s t-test (one-tailed; two-sample unequal variance; *p < 0.05, **p < 0.01, ***p < 0.001).
Fig 8.
Fermentation view of yeast strains RM26 and RM27.
(A) Cell growth, ethanol and glucose consumption of RM26 and RM27 in YSM medium with the addition of 20 g/L glucose every 24 h. (B) Improvement of 2-PE synthesis and L-phenylalanine consumption of RM26 and RM27 in YSM medium with the addition of 20 g/L glucose every 24 h. (C) Fermentation view of RM27 (AD032 pdc5△ PTPI-ectyrB-llkdcA-scADH2 PTPI-strlgox) in YSM medium with the addition of 20 g/L glucose every 24 h.
Fig 9.
Optimization of 2-PE synthesis in metabolic engineered yeast strain RM27.
(A) Fermentation view of JM00 with L-phenylalanine as the sole nitrogen. (B) Fermentation view of RM27 with L-phenylalanine as the sole nitrogen. (C) Fermentation view of RM27 in YSM medium with the addition of glucose every 12 h. (D) Fermentation view of RM27 in YSM medium with the addition of L-glutamate or α-ketoglutarate. (a) One mmol/L α-ketoglutarate was added to the medium at 24 h. (b) One mmol/L α-ketoglutarate was added to the medium every 24 h. (c) One mmol/L L-glutamate was added to the medium at 24 h. (d) One mmol/L L-glutamate was added to the medium every 24 h. Data are presented as the averages of the results of three independent experiments. Error bars show standard deviations. Statistical analysis was performed by using Student’s t-test (one-tailed; two-sample unequal variance; *p < 0.05, **p < 0.01, ***p < 0.001).