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. 2024 Jun 16;14(6):712.
doi: 10.3390/biom14060712.

De Novo Synthesis of Resveratrol from Sucrose by Metabolically Engineered Yarrowia lipolytica

Gehad G Ibrahim  1   2 , Madhavi Perera  1   3 , Saadiah A Abdulmalek  4 , Jinyong Yan  1 , Yunjun Yan  1
Affiliations

De Novo Synthesis of Resveratrol from Sucrose by Metabolically Engineered Yarrowia lipolytica

Gehad G Ibrahim et al. Biomolecules. .

Abstract

Resveratrol, a phenylpropanoid compound, exhibits diverse pharmacological properties, making it a valuable candidate for health and disease management. However, the demand for resveratrol exceeds the capacity of plant extraction methods, necessitating alternative production strategies. Microbial synthesis offers several advantages over plant-based approaches and presents a promising alternative. Yarrowia lipolytica stands out among microbial hosts due to its safe nature, abundant acetyl-CoA and malonyl-CoA availability, and robust pentose phosphate pathway. This study aimed to engineer Y. lipolytica for resveratrol production. The resveratrol biosynthetic pathway was integrated into Y. lipolytica by adding genes encoding tyrosine ammonia lyase from Rhodotorula glutinis, 4-coumarate CoA ligase from Nicotiana tabacum, and stilbene synthase from Vitis vinifera. This resulted in the production of 14.3 mg/L resveratrol. A combination of endogenous and exogenous malonyl-CoA biosynthetic modules was introduced to enhance malonyl-CoA availability. This included genes encoding acetyl-CoA carboxylase 2 from Arabidopsis thaliana, malonyl-CoA synthase, and a malonate transporter protein from Bradyrhizobium diazoefficiens. These strategies increased resveratrol production to 51.8 mg/L. The further optimization of fermentation conditions and the utilization of sucrose as an effective carbon source in YP media enhanced the resveratrol concentration to 141 mg/L in flask fermentation. By combining these strategies, we achieved a titer of 400 mg/L resveratrol in a controlled fed-batch bioreactor. These findings demonstrate the efficacy of Y. lipolytica as a platform for the de novo production of resveratrol and highlight the importance of metabolic engineering, enhancing malonyl-CoA availability, and media optimization for improved resveratrol production.

Keywords: Yarrowia lipolytica; metabolic engineering; microbial cell factory; resveratrol.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
Resveratrol synthesis pathway and genes and enzymes involved in Y. lipolytica engineering in this study. Genes encoding enzymes in blue were inserted into the yeast. Enzymes encoded by red genes were inhibited using the antibiotic cerulenin. 4CL, 4-coumaroyl-coA ligase; ACC, acetyl-CoA carboxylase; Acetyl-CoA, acetyl-coenzyme A; fabB/fabF/fabH, genes that encode the 3-ketoacyl-ACP synthase I/II; MatB, malonyl-CoA synthetase; MatC, malonate carrier protein; STS, stilbene synthase; TAL, tyrosine ammonia-lyase.
Figure 2
Figure 2
p-CA and resveratrol production in different engineered Y. lipolytica strains after 120 h of cultivation in YPD media. (A) p-CA and resveratrol production in the Sx group strains, derived from the Y. lipolytica Po1f strain and recombinant with the pINA1312-ST4C cassette. (B) p-CA and resveratrol production in the S10x group strains, derived from the S10 strain and recombinant with the pINA1312-ST4C and pINA1269-BCA cassettes. (C) p-CA and resveratrol production in the S39x group strains, derived from the S39 strain and recombinant with the pINA1312-ST4C and pINA1269-BCA cassettes. (D) Comparison of p-CA and resveratrol average concentrations obtained from the three groups. ** indicate significant differences at a 0.01 significance level.
Figure 3
Figure 3
The effect of malonate on resveratrol production in different strains. To increase malonyl-CoA levels through the expression of the matB and matC genes, 20 mM malonate was added to the YPD media. Resveratrol concentrations were estimated after 120 h of cultivation. Control fermentations lacked malonate. Letters represent the significance of differences. Distinct letters indicate significant differences at a 0.05 significance level.
Figure 4
Figure 4
The effect of p-CA and cerulenin on resveratrol production in different strains. Either 2 mM p-CA, 1 mg/L cerulenin, or both were added to YNB media. Resveratrol concentrations were estimated after 120 h of cultivation. Control fermentations lacked p-CA and cerulenin. Letters represent the significance of differences. Distinct letters indicate significant differences at a 0.05 significance level.
Figure 5
Figure 5
The effect of carbon source and its concentration on resveratrol and p-CA production by the S10M31 strain. The strain was fermented for 10 days in YP media supplemented with 20, 50, or 80 g/L of either glucose (G) or sucrose (S) as the carbon source. Resveratrol, p-CA, pH, and OD600 were measured every 24 h. Panels (A–C) show the results for glucose concentrations of 20, 50, and 80 g/L, respectively, while panels (D–F) show the results for sucrose concentrations of 20, 50, and 80 g/L, respectively. Dotted lines indicate the depletion of the carbon source.
Figure 6
Figure 6
Fed-batch fermentation of the S10M31 strain in a 3L bioreactor. The strain was grown at 28 °C in YP medium supplemented with sucrose and malonate. Fermentation was carried out for 10 days. Resveratrol, p-CA, and OD600 were measured every 12 h. The dotted line indicates the time when feeding was started.
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