Establishment of duckweeds as a sustainable heterologous system for the production of antidiabetic flavonol glycoside Montbretin A (FlavoWeeds)

• Applicant: Gopal Singh, Ph.D.
• Subject Area: Plant Biochemistry and Biophysics ; Biochemistry ; Biological and Biomimetic Chemistry ; Biological Process Engineering
• Term: since 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 547989757

Plants produce a diverse range of biologically active specialized metabolites with immense pharmaceutical potential. Often these metabolites accumulate in minute quantities in the natural source plants, and their chemical synthesis is challenging and costly. Thus, a major strategy to meet the industrial demand is to produce these specialized metabolites in engineered heterologous hosts. Plants are being explored as photosynthetic hosts as they endogenously possess core metabolic pathways for most specialized metabolites, and do not rely on external supply of carbohydrates. Nicotiana benthamiana is the most used photosynthetic host, however, the plant is toxic and often accumulates undesirable side products due to its complex endogenous metabolism. Another attractive photosynthetic host that is currently being developed are duckweeds, because they are fast-growing, edible, and have a simpler endogenous metabolism. In this project, we aim to foster the giant duckweed (Spirodela polyrhiza) as a new photosynthetic host. As a proof-of-concept, we will engineer the production of mini-Montbretin A (mini-MbA). Mini-MbA is the first bioactive intermediate of the antidiabetic flavonoid glycoside montbretin A (MbA). Development of MbA as a drug is hindered as the native source plant montbretia (Crocosmia x crocosmiiflora) produces this metabolite only in small quantities. As duckweeds produce large amounts of biochemically closely related compounds, particularly flavones and anthocyanins, they are logical production hosts. To produce mini-MbA in duckweeds, we will introduce biosynthetic genes from montbretia into S. polyrhiza, and subsequently optimize the pathway using a push and pull strategy. First, we will introduce myricetin biosynthesis, the core flavonol of MbA. Based on phytochemical analysis of S. polyrhiza, we will introduce three montbretia genes (CcF3H, CcFLS and CcCYP2) to convert naringenin, which is available in S. polyrhiza, to myricetin. Second, we will introduce MbA assembly genes (CcUGT1, CcUGT2 and CcAT1) into S. polyrhiza to convert myricetin into mini-MbA. Third, we will enhance the production of mini-MbA by blocking competing biosynthetic pathways in S. polyrhiza and enhancing the flux through the flavonol pathway. To block competing pathways, we will use CRISPR-Cas to knock-out S. polyrhiza flavone synthase, which is a key enzyme for the biosynthesis of flavones. Furthermore, we will knock-out the MYB-transcription factor that enhances the flux from dihydroflavonols towards anthocyanins. To improve the flux towards mini-MbA, we will overexpress the MYB transcription factor that activates the flavonol pathway to elevate the levels of myricetin, a limiting building block for MbA production. If successful, these strategies will lead to an efficient production of a potential anti-diabetes drug and will foster S. polyrhiza as a promising photosynthetic host for sustainable and efficient production of valuable specialized metabolites.

• DFG Programme: WBP Position