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      Toward Developing a Yeast Cell Factory for the Production of Prenylated Flavonoids

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          Abstract

          Prenylated flavonoids possess a wide variety of biological activities, including estrogenic, antioxidant, antimicrobial, and anticancer activities. Hence, they have potential applications in food products, medicines, or supplements with health-promoting activities. However, the low abundance of prenylated flavonoids in nature is limiting their exploitation. Therefore, we investigated the prospect of producing prenylated flavonoids in the yeast Saccharomyces cerevisiae. As a proof of concept, we focused on the production of the potent phytoestrogen 8-prenylnaringenin. Introduction of the flavonoid prenyltransferase SfFPT from Sophora flavescens in naringenin-producing yeast strains resulted in de novo production of 8-prenylnaringenin. We generated several strains with increased production of the intermediate precursor naringenin, which finally resulted in a production of 0.12 mg L –1 (0.35 μM) 8-prenylnaringenin under shake flask conditions. A number of bottlenecks in prenylated flavonoid production were identified and are discussed.

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          Most cited references52

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          Production of the antimalarial drug precursor artemisinic acid in engineered yeast.

          Malaria is a global health problem that threatens 300-500 million people and kills more than one million people annually. Disease control is hampered by the occurrence of multi-drug-resistant strains of the malaria parasite Plasmodium falciparum. Synthetic antimalarial drugs and malarial vaccines are currently being developed, but their efficacy against malaria awaits rigorous clinical testing. Artemisinin, a sesquiterpene lactone endoperoxide extracted from Artemisia annua L (family Asteraceae; commonly known as sweet wormwood), is highly effective against multi-drug-resistant Plasmodium spp., but is in short supply and unaffordable to most malaria sufferers. Although total synthesis of artemisinin is difficult and costly, the semi-synthesis of artemisinin or any derivative from microbially sourced artemisinic acid, its immediate precursor, could be a cost-effective, environmentally friendly, high-quality and reliable source of artemisinin. Here we report the engineering of Saccharomyces cerevisiae to produce high titres (up to 100 mg l(-1)) of artemisinic acid using an engineered mevalonate pathway, amorphadiene synthase, and a novel cytochrome P450 monooxygenase (CYP71AV1) from A. annua that performs a three-step oxidation of amorpha-4,11-diene to artemisinic acid. The synthesized artemisinic acid is transported out and retained on the outside of the engineered yeast, meaning that a simple and inexpensive purification process can be used to obtain the desired product. Although the engineered yeast is already capable of producing artemisinic acid at a significantly higher specific productivity than A. annua, yield optimization and industrial scale-up will be required to raise artemisinic acid production to a level high enough to reduce artemisinin combination therapies to significantly below their current prices.
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            Phytoalexins in defense against pathogens.

            Plants use an intricate defense system against pests and pathogens, including the production of low molecular mass secondary metabolites with antimicrobial activity, which are synthesized de novo after stress and are collectively known as phytoalexins. In this review, we focus on the biosynthesis and regulation of camalexin, and its role in plant defense. In addition, we detail some of the phytoalexins produced by a range of crop plants from Brassicaceae, Fabaceae, Solanaceae, Vitaceae and Poaceae. This includes the very recently identified kauralexins and zealexins produced by maize, and the biosynthesis and regulation of phytoalexins produced by rice. Molecular approaches are helping to unravel some of the mechanisms and reveal the complexity of these bioactive compounds, including phytoalexin action and metabolism. Copyright © 2011 Elsevier Ltd. All rights reserved.
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              Extraction of genomic DNA from yeasts for PCR-based applications.

              We have developed a quick and low-cost genomic DNA extraction protocol from yeast cells for PCR-based applications. This method does not require any enzymes, hazardous chemicals, or extreme temperatures, and is especially powerful for simultaneous analysis of a large number of samples. DNA can be efficiently extracted from different yeast species (Kluyveromyces lactis, Hansenula polymorpha, Schizosaccharomyces pombe, Candida albicans, Pichia pastoris, and Saccharomyces cerevisiae). The protocol involves lysis of yeast colonies or cells from liquid culture in a lithium acetate (LiOAc)-SDS solution and subsequent precipitation of DNA with ethanol. Approximately 100 nanograms of total genomic DNA can be extracted from 1 × 10(7) cells. DNA extracted by this method is suitable for a variety of PCR-based applications (including colony PCR, real-time qPCR, and DNA sequencing) for amplification of DNA fragments of ≤ 3500 bp.
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                Author and article information

                Journal
                J Agric Food Chem
                J. Agric. Food Chem
                jf
                jafcau
                Journal of Agricultural and Food Chemistry
                American Chemical Society
                0021-8561
                1520-5118
                24 April 2019
                11 December 2019
                : 67
                : 49 , Advances in Bioflavor Research
                : 13478-13486
                Affiliations
                [1] Laboratory of Plant Physiology and Wageningen Plant Research, Wageningen University & Research , Droevendaalsesteeg 1, 6708 PB Wageningen, Netherlands
                []Laboratory of Food Chemistry, Wageningen University & Research , Bornse Weilanden 9, 6708 WG Wageningen, Netherlands
                [§ ]Department of Biotechnology, Delft University of Technology , Van der Maasweg 9, 2629 HZ Delft, Netherlands
                Author notes
                [* ]Telephone: +31-317-480979. E-mail: jules.beekwilder@ 123456wur.nl .
                Article
                10.1021/acs.jafc.9b01367
                6909231
                31016981
                b5f80ad3-7c6f-475a-81d6-f6daefb5854e
                Copyright © 2019 American Chemical Society

                This is an open access article published under a Creative Commons Non-Commercial No Derivative Works (CC-BY-NC-ND) Attribution License, which permits copying and redistribution of the article, and creation of adaptations, all for non-commercial purposes.

                History
                : 28 February 2019
                : 23 April 2019
                : 24 April 2019
                Categories
                Article
                Custom metadata
                jf9b01367
                jf9b01367

                Food science & Technology
                metabolic engineering,saccharomyces cerevisiae,de novo,prenylated flavonoids,naringenin,8-prenylnaringenin

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