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

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.

Use of 3-Deoxy-D-arabino-heptulosonic acid 7-phosphate Synthase (DAHP Synthase) to Enhance the Heterologous Biosynthesis of Diosmetin and Chrysoeriol in an Engineered Strain of Streptomyces albidoflavus.

Flavonoids are a large family of polyphenolic compounds with important agro-industrial, nutraceutical, and pharmaceutical applications. Among the structural diversity found in the flavonoid family, methylated flavonoids show interesting characteristics such as greater stability and improved oral bioavailability. This work is focused on the reconstruction of the entire biosynthetic pathway of the methylated flavones diosmetin and chrysoeriol in Streptomyces albidoflavus. A total of eight different genes (TAL, 4CL, CHS, CHI, FNS1, F3'H/CPR, 3'-OMT, 4'-OMT) are necessary for the heterologous biosynthesis of these two flavonoids, and all of them have been integrated along the chromosome of the bacterial host. The biosynthesis of diosmetin and chrysoeriol has been achieved, reaching titers of 2.44 mg/L and 2.34 mg/L, respectively. Furthermore, an additional compound, putatively identified as luteolin 3',4'-dimethyl ether, was produced in both diosmetin and chrysoeriol-producing strains. With the purpose of increasing flavonoid titers, a 3-Deoxy-D-arabino-heptulosonic acid 7-phosphate synthase (DAHP synthase) from an antibiotic biosynthetic gene cluster (BGC) from Amycolatopsis balhimycina was heterologously expressed in S. albidoflavus, enhancing diosmetin and chrysoeriol production titers of 4.03 mg/L and 3.13 mg/L, which is an increase of 65% and 34%, respectively. To the best of our knowledge, this is the first report on the de novo biosynthesis of diosmetin and chrysoeriol in a heterologous host.