| 1. |
- Konzock, Oliver, 1991, et al.
(författare)
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Cinnamic acid and p-coumaric acid are metabolized to 4-hydroxybenzoic acid by Yarrowia lipolytica
- 2023
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Ingår i: AMB Express. - 2191-0855. ; 13:1
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Tidskriftsartikel (refereegranskat)abstract
- Yarrowia lipolytica has been explored as a potential production host for flavonoid synthesis due to its high tolerance to aromatic acids and ability to supply malonyl-CoA. However, little is known about its ability to consume the precursors cinnamic and p-coumaric acid. In this study, we demonstrate that Y. lipolytica can consume these precursors through multiple pathways that are partially dependent on the cultivation medium. By monitoring the aromatic acid concentrations over time, we found that cinnamic acid is converted to p-coumaric acid. We identified potential proteins with a trans-cinnamate 4-monooxygenase activity in Y. lipolytica and constructed a collection of 15 knock-out strains to identify the genes responsible for the reaction. We identified YALI1_B28430g as the gene encoding for a protein that converts cinnamic acid to p-coumaric acid (designated as TCM1). By comparing different media compositions we found that complex media components (casamino acids and yeast extract) induce this pathway. Additionally, we discover the conversion of p-coumaric acid to 4-hydroxybenzoic acid. Our findings provide new insight into the metabolic capabilities of Y. lipolytica and hold great potential for the future development of improved strains for flavonoid production.
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| 2. |
- Mao, Jiwei, 1990, et al.
(författare)
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Fine-tuning of p-coumaric acid synthesis to increase (2S)-naringenin production in yeast
- 2023
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Ingår i: Metabolic Engineering. - 1096-7176 .- 1096-7184. ; 79, s. 192-202
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Tidskriftsartikel (refereegranskat)abstract
- (2S)-Naringenin is a key precursor for biosynthesis of various high-value flavonoids and possesses a variety of nutritional and pharmaceutical properties on human health. Systematic optimization approaches have been employed to improve (2S)-naringenin production in different microbial hosts. However, very few studies have focused on the spatiotemporal distribution of (2S)-naringenin and the related pathway intermediate p-coumaric acid, which is an important factor for efficient production. Here, we first optimized the (2S)-naringenin biosynthetic pathway by alleviating the bottleneck downstream of p-coumaric acid and increasing malonyl-CoA supply, which improved (2S)-naringenin production but significant accumulation of p-coumaric acid still existed extracellularly. We thus established a dual dynamic control system through combining a malonyl-CoA biosensor regulator and an RNAi strategy, to autonomously control the synthesis of p-coumaric acid with the supply of malonyl-CoA. Furthermore, screening potential transporters led to identification of Pdr12 for improved (2S)-naringenin production and reduced accumulation of p-coumaric acid. Finally, a titer of 2.05 g/L (2S)-naringenin with negligible accumulation of p-coumaric acid was achieved in a fed batch fermentation. Our work highlights the importance of systematic control of pathway intermediates for efficient microbial production of plant natural products.
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| 3. |
- Tartik, Musa, et al.
(författare)
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Optimizing yeast for high-level production of kaempferol and quercetin
- 2023
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Ingår i: Microbial Cell Factories. - 1475-2859. ; 22:1
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: Two important flavonoids, kaempferol and quercetin possess remarkably potent biological impacts on human health. However, their structural complexity and low abundance in nature make both bulk chemical synthesis and extraction from native plants difficult. Therefore microbial production via heterologous expression of plant enzymes can be a safe and sustainable route for their production. Despite several attempts reported in microbial hosts, the production levels of kaempferol and quercetin still stay far behind compared to many other microbial-produced flavonoids. RESULTS: In this study, Saccharomyces cerevisiae was engineered for high production of kaempferol and quercetin in minimal media from glucose. First, the kaempferol biosynthetic pathway was reconstructed via screening various F3H and FLS enzymes. In addition, we demonstrated that amplification of the rate-limiting enzyme AtFLS could reduce the dihydrokaempferol accumulation and improve kaempferol production. Increasing the availability of precursor malonyl-CoA further improved the production of kaempferol and quercetin. Furthermore, the highest amount of 956 mg L- 1 of kaempferol and 930 mg L- 1 of quercetin in yeast was reached in fed-batch fermentations. CONCLUSIONS: De novo biosynthesis of kaempferol and quercetin in yeast was improved through increasing the upstream naringenin biosynthesis and debugging the flux-limiting enzymes together with fed-batch fermentations, up to gram per liter level. Our work provides a promising platform for sustainable and scalable production of kaempferol, quercetin and compounds derived thereof.
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| 4. |
- Tous Mohedano, Marta, 1995
(författare)
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Metabolic engineering of S. cerevisiae for the production of flavonoids
- 2023
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The interest in the production of natural products such as flavonoids has been increasing during the last decade. Flavonoids have several attractive bioactivities including antitumoral, antioxidant or antimicrobial properties. To produce these high-value products, we usually recur to chemical synthesis or plant extraction. However, these two options are costly and not environmentally friendly, and microbial production is therefore preferred. S. cerevisiae is a thoroughly characterized model organism with a wide range of available tools for engineering, making it an ideal organism for this challenge. The aim of this thesis was to apply different strategies to engineer S. cerevisiae to establish and optimize the production of the flavonoids pinocembrin and naringenin, and their derivatives. Different approaches were used: different heterologous genes were screened, their copy number was increased to achieve the highest production, the competing pathways were eliminated, and the precursors availability was increased. Furthermore, the bottlenecks of the pathways were identified. For pinocembrin production, I established that the accumulation of the toxic intermediate cinnamic acid limits production. Therefore, the transcriptional changes that S. cerevisiae undergoes under aromatic acid stress were investigated. My findings indicate that by employing transcription factor engineering it is possible to develop strains that are tolerant to aromatic compounds that can be utilized for the production of valuable natural products. When analysing the naringenin biosynthetic pathway it was found that the distribution of the pathway intermediates in the cell is a major issue. The spatiotemporal distribution of p-coumaric acid (a key pathway intermediate) and naringenin was assessed and it was determined that p-coumaric acid accumulates extracellularly and cannot be fully utilized. Therefore, a dual dynamic control system that combines a malonyl-CoA biosensor regulator and an RNAi strategy was established, to autonomously control the synthesis of p-coumaric acid and downregulate the fatty acid pathways that compete directly for the precursor malonyl-CoA. Finally, the production of naringenin and pinocembrin derivatives was established including kaempferol, quercetin and baicalein which present valuable bioactivities. Overall, this thesis employs diverse strategies for constructing and optimizing yeast factories for flavonoid production.
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| 5. |
- Tous Mohedano, Marta, 1995, et al.
(författare)
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Optimization of Pinocembrin Biosynthesis in Saccharomyces cerevisiae
- 2023
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Ingår i: ACS Synthetic Biology. - : American Chemical Society (ACS). - 2161-5063. ; 12:1, s. 144-152
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Tidskriftsartikel (refereegranskat)abstract
- The flavonoid pinocembrin and its derivatives have gained increasing interest for their benefits on human health. While pinocembrin and its derivatives can be produced in engineered Saccharomyces cerevisiae, yields remain low. Here, we describe novel strategies for improved de novo biosynthesis of pinocembrin from glucose based on overcoming existing limitations in S. cerevisiae. First, we identified cinnamic acid as an inhibitor of pinocembrin synthesis. Second, by screening for more efficient enzymes and optimizing the expression of downstream genes, we reduced cinnamic acid accumulation. Third, we addressed other limiting factors by boosting the availability of the precursor malonyl-CoA, while eliminating the undesired byproduct 2′,4′,6′-trihydroxy dihydrochalcone. After optimizing cultivation conditions, 80 mg/L pinocembrin was obtained in a shake flask, the highest yield reported for S. cerevisiae. Finally, we demonstrated that pinocembrin-producing strains could be further engineered to generate 25 mg/L chrysin, another interesting flavone. The strains generated in this study will facilitate the production of flavonoids through the pinocembrin biosynthetic pathway.
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| 6. |
- Tous Mohedano, Marta, 1995, et al.
(författare)
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Strategies to increase tolerance and robustness of industrial microorganisms
- 2022
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Ingår i: Synthetic and Systems Biotechnology. - : Elsevier BV. - 2405-805X. ; 7:1, s. 533-540
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Tidskriftsartikel (refereegranskat)abstract
- The development of a cost-competitive bioprocess requires that the cell factory converts the feedstock into the product of interest at high rates and yields. However, microbial cell factories are exposed to a variety of different stresses during the fermentation process. These stresses can be derived from feedstocks, metabolism, or industrial production processes, limiting production capacity and diminishing competitiveness. Improving stress tolerance and robustness allows for more efficient production and ultimately makes a process more economically viable. This review summarises general trends and updates the most recent developments in technologies to improve the stress tolerance of microorganisms. We first look at evolutionary, systems biology and computational methods as examples of non-rational approaches. Then we review the (semi-)rational approaches of membrane and transcription factor engineering for improving tolerance phenotypes. We further discuss challenges and perspectives associated with these different approaches.
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