Biosynthesis of Hesperetin, Homoeriodictyol, and Homohesperetin in a Transcriptomics-Driven Engineered Strain of Streptomyces albidoflavus
Journal article, 2024

Flavonoids exhibit various bioactivities including anti-oxidant, anti-tumor, anti-inflammatory, and anti-viral properties. Methylated flavonoids are particularly significant due to their enhanced oral bioavailability, improved intestinal absorption, and greater stability. The heterologous production of plant flavonoids in bacterial factories involves the need for enough biosynthetic precursors to allow for high production levels. These biosynthetic precursors are malonyl-CoA and l-tyrosine. In this work, to enhance flavonoid biosynthesis in Streptomyces albidoflavus, we conducted a transcriptomics study for the identification of candidate genes involved in l-tyrosine catabolism. The hypothesis was that the bacterial metabolic machinery would detect an excess of this amino acid if supplemented with the conventional culture medium and would activate the genes involved in its catabolism towards energy production. Then, by inactivating those overexpressed genes (under an excess of l-tyrosine), it would be possible to increase the intracellular pools of this precursor amino acid and eventually the final flavonoid titers in this bacterial factory. The RNAseq data analysis in the S. albidoflavus wild-type strain highlighted the hppD gene encoding 4-hydroxyphenylpyruvate dioxygenase as a promising target for knock-out, exhibiting a 23.2-fold change (FC) in expression upon l-tyrosine supplementation in comparison to control cultivation conditions. The subsequent knock-out of the hppD gene in S. albidoflavus resulted in a 1.66-fold increase in the naringenin titer, indicating enhanced flavonoid biosynthesis. Leveraging the improved strain of S. albidoflavus, we successfully synthesized the methylated flavanones hesperetin, homoeriodictyol, and homohesperetin, achieving titers of 2.52 mg/L, 1.34 mg/L, and 0.43 mg/L, respectively. In addition, the dimethoxy flavanone homohesperetin was produced as a byproduct of the endogenous metabolism of S. albidoflavus. To our knowledge, this is the first time that hppD deletion was utilized as a strategy to augment the biosynthesis of flavonoids. Furthermore, this is the first report where hesperetin and homoeriodictyol have been synthesized from l-tyrosine as a precursor. Therefore, transcriptomics is, in this case, a successful approach for the identification of catabolism reactions affecting key precursors during flavonoid biosynthesis, allowing the generation of enhanced production strains.

flavonoid

substrate flexibility

methyltransferase

l-tyrosine feeding

Author

Álvaro Pérez-Valero

Instituto de Investigación Sanitaria del Principado de Asturias

Instituto Universitario de Oncologia del Principado de Asturias

University of Oviedo

Juan Serna-Diestro

Instituto Universitario de Oncologia del Principado de Asturias

University of Oviedo

Instituto de Investigación Sanitaria del Principado de Asturias

Albert Tafur Rangel

Chalmers, Life Sciences, Systems and Synthetic Biology

Novo Nordisk Foundation

Simona Barbuto Ferraiuolo

University of Campania Luigi Vanvitelli

C. Schiraldi

University of Campania Luigi Vanvitelli

Eduard Kerkhoven

Chalmers, Life Sciences, Systems and Synthetic Biology

Novo Nordisk Foundation

Claudio J. Villar

Instituto Universitario de Oncologia del Principado de Asturias

Instituto de Investigación Sanitaria del Principado de Asturias

University of Oviedo

Felipe Lombó

Instituto Universitario de Oncologia del Principado de Asturias

Instituto de Investigación Sanitaria del Principado de Asturias

University of Oviedo

International Journal of Molecular Sciences

16616596 (ISSN) 14220067 (eISSN)

Vol. 25 7 4053

Synthetic microbial consortia-based platform for flavonoids production using synthetic biology (Synbio4Flav)

European Commission (EC) (EC/H2020/814650), 2019-01-01 -- 2023-02-28.

Subject Categories

Biochemistry and Molecular Biology

Microbiology

DOI

10.3390/ijms25074053

More information

Latest update

8/7/2024 9