De novo biosynthesis of myricetin, kaempferol and quercetin in Streptomyces albus and Streptomyces coelicolor

Abstract

Flavonols are a flavonoid subfamily widely distributed in plants, including several ones of great importance in human and animal diet (apple, tomato, broccoli, onion, beans, tea). These polyphenolic nutraceuticals exert potent antimicrobial (membrane potential disruptors), antioxidant (free-radical scavengers), pharmacokinetic (CYP450 modulators), anti-inflammatory (lipoxygenase inhibitors), antiangiogenic (VEGF inhibitors) and antitumor (cyclin inhibitors) activities. Biotechnological production of these nutraceuticals, for example via heterologous biosynthesis in industrial actinomycetes, is favored since in plants these polyphenols appear as inactive glycosylated derivatives, in low concentrations or as part of complex mixtures with other polyphenolic compounds. In this work, we describe the de novo biosynthesis of three important flavonols, myricetin, kaempferol and quercetin, in the industrially relevant actinomycetes Streptomyces coelicolor and S. albus. De novo biosynthesis of kaempferol, myricetin and quercetin in actinomycetes has not been described before.

Fig 1. Engineered flavonoid biosynthetic pathway in Streptomyces sp., including the different feeding experiments with naringenin (dashed lines).

Enzyme abbreviations: TAL, Tyrosine ammonia-lyase; 4CL, 4-coumaroyl CoA ligase; CHS, chalcone synthase; CHI, chalcone isomerase; N3DOX, naringenin 3-dioxygenase; FLS1, flavonol synthase 1; F3’H, flavonoid 3’-hydroxylase; F3’5’H, flavonoid 3’,5’-hydroxylase.

Fig 2

A: HPLC-MS chromatogram obtained after MRM analysis of the flavonols extracted from S. coelicolor-pKF. It shows the m/z peaks corresponding to dihydrokaempferol (DHK: <0.1 μM) and quercetin (QR: <0.1 μM). B: HPLC-MS chromatogram obtained after MRM analysis of the flavonols extracted from S. albus-pKF. It shows the m/z peaks corresponding to dihydrokaempferol (DHK: 0.039 μM), quercetin (QR: <0.2 μM) and kaempferol (KF: 0.212 μM).

Fig 3

A: HPLC-MS chromatogram obtained after MRM analysis of the flavonols extracted from S. coelicolor-pQR. The m/z peaks correspond to dihydrokaempferol (DHK: <0.1 μM) and to quercetin (QR: <0.1 μM). B: HPLC-MS chromatogram obtained after MRM analysis of the flavonols extracted from S. albus-pQR. The peaks correspond to dihydrokaempferol (DHK: 0.047 μM), quercetin (QR: 0.340 μM) and kaempferol (KF: 0.155 μM).

Fig 4

A: HPLC-MS chromatogram obtained after MRM analysis of the flavonols extracted from S. coelicolor-pMYR. The peaks correspond to myricetin (MYR: <0.1 μM), dihydrokaempferol (DHK: <0.1 μM) and quercetin (QR: <0.1 μM). B: chromatogram obtained after MRM analysis of the flavonols extracted from S. albus-pMYR. The peaks correspond to myricetin (MYR: 0.146 μM), dihydrokaempferol (DHK: 0.024 μM), quercetin (QR: 1.984 μM) and kaempferol (KF: 0.034 μM). C: chromatogram obtained from extracts of S. albus-pMYR-pREC4. The peaks correspond to apigenin (0.3 μM) and luteolin (below 0.1 μM).