Metabolomics-Guided Analysis of the Biocatalytic Conversion of Sclareol to Ambradiol by Hyphozyma roseoniger

The biocatalytic conversion of sclareol to ambradiol, a valuable component in the fragrance industry, using whole-cell biotransformation by the dimorphic yeast Hyphozyma roseoniger, was investigated using metabolomics tools. An integrated approach was used to identify and quantify the participating intermediates in this bioconversion using both nuclear magnetic resonance (NMR) spectroscopy and liquid chromatography coupled to mass spectrometry (LC–MS). This study entailed growth stage-dependent analysis of H. roseoniger suspensions grown in batch culture over a 14-day period, beginning with a three-day induction period using 20 mg/200 mL sclareol, followed by a further 1 g/200 mL sclareol dose to enable ambradiol production. The progress of the bioconversion and the resulting dynamic changes to the metabolome were monitored using NMR analysis and semi-targeted LC–MS metabolomics. This outlined the molecular conversions occurring within the matrix and no novel intermediates participating in the sclareol to ambradiol conversion could be identified. This study presents new findings about the transformative capabilities of H. roseoniger as a whole cell biocatalyst, highlighting its potential utility in similar applications.

Engineering of CYP153A33 With Enhanced Ratio of Hydroxylation to Overoxidation Activity in Whole-Cell Biotransformation of Medium-Chain 1-Alkanols

α,ω-Dodecanediol is a versatile material that has been widely used not only as an adhesive and crosslinking reagent, but also as a building block in the pharmaceutical and polymer industries. The biosynthesis of α,ω-dodecanediol from fatty derivatives, such as dodecane and dodecanol, requires an ω-specific hydroxylation step using monooxygenase enzymes. An issue with the whole-cell biotransformation of 1-dodecanol using cytochrome P450 monooxygenase (CYP) with ω-specific hydroxylation activity was the low conversion and production of the over-oxidized product of dodecanoic acid. In this study, CYP153A33 from Marinobacter aquaeolei was engineered to obtain higher ω-specific hydroxylation activity through site-directed mutagenesis. The target residue was mutated to increase flux toward α,ω-dodecanediol synthesis, while reducing the generation of the overoxidation product of dodecanoic acid and α,ω-dodecanedioic acid. Among the evaluated variants, CYP153A33 P136A showed a significant increase in 1-dodecanol conversion, i.e., 71.2% (7.12 mM from 10 mM 1-dodecanol), with an increased hydroxylation to over-oxidation activity ratio, i.e., 32.4. Finally, the applicability of this engineered enzyme for ω-specific hydroxylation against several 1-alkanols, i.e., from C6 to C16, was investigated and discussed based on the structure-activity relationship.

Biotransformation of Daidzein, Genistein, and Naringenin by Streptomyces Species Isolated from High-Altitude Soil of Nepal

Flavonoids have achieved widespread importance in pharmaceutical, food, and cosmetics industries. Furthermore, modification of these naturally occurring flavonoids to structurally diverse compounds through whole cell biotransformation with enhanced biological activities has numerous biotechnological applications. The present study investigated the biotransformation potential of Streptomyces species isolated from a high-altitude-soil sample towards selected flavonoid molecules. The biotransformed metabolites were confirmed by comparing the HPLC chromatogram with authentic compounds and LC-MS/MS analysis. Of these isolates, Streptomyces species G-18 (Accession number: MW663767.1) catalyzed isoflavone molecules daidzein and genistein to produce hydroxylated products at 24 h of reaction condition in a whole cell system. The hydroxylation of daidzein (4′,7-dihydroxyisoflavone) was confirmed at 3′-position of the B ring to produce 3′,4′,7-trihydroxyisoflavone. In addition, Streptomyces species G-14 (Accession number: MW663770.1) and Streptomyces species S4L (Accession number: MW663769.1) also revealed the transformation of daidzein (4′,7-dihydroxyisoflavone) to hydroxy daidzein at a distinct position than that of G-18 isolates, whereas thee Streptomyces species S4L reaction mixture with naringenin as a substrate also revealed the hydroxylated product. Our results demonstrated that microorganisms isolated from different ecological niches have broad application.

Corticosteroid Biosynthesis Revisited: No Direct Hydroxylation of Pregnenolone by Steroid 21-Hydroxylase

Cytochrome P450s (CYPs) are an essential family of enzymes in the human body. They play a crucial role in metabolism, especially in human steroid biosynthesis. Reactions catalyzed by these enzymes are highly stereo- and regio-specific. Lack or severe malfunctions of CYPs can cause severe diseases and even shorten life. Hence, investigations on metabolic reactions and structural requirements of substrates are crucial to gain further knowledge on the relevance of different enzymes in the human body functions and the origin of diseases. One key enzyme in the biosynthesis of gluco- and mineralocorticoids is CYP21A2, also known as steroid 21-hydroxylase. To investigate the steric and regional requirements of substrates for this enzyme, we performed whole-cell biotransformation assays using a strain of fission yeast Schizosaccharomyces pombe recombinantly expressing CYP21A2. The progestogens progesterone, pregnenolone, and their 17α-hydroxy-derivatives were used as substrates. After incubation, samples were analyzed using gas chromatography coupled to mass spectrometry. For progesterone and 17α-hydroxyprogesterone, their corresponding 21-hydroxylated metabolites 11-deoxycorticosterone and 11-deoxycortisol were detected, while after incubation of pregnenolone and 17α-hydroxypregnenolone, no hydroxylated product was observed. Findings were confirmed with authentic reference material. Molecular docking experiments agree with these results and suggest that interaction between the 3-oxo group and arginine-234 of the enzyme is a strict requirement. The presented results demonstrate once more that the presence of an oxo-group in position 3 of the steroid is indispensable, while a 3-hydroxy group prevents hydroxylation in position C-21 by CYP21A2. This knowledge may be transferred to other CYP21A2 substrates and hence help to gain essential insights into steroid metabolism.