17α hydroxylase is a key enzyme for the conversion of progesterone to prepare various progestational drug intermediates. To improve the specific hydroxylation capability of this enzyme in steroid biocatalysis, the CYP260A1 derived from cellulose-mucilaginous bacteria Sorangium cellulosum Soce56 and the Fpr and bovine adrenal-derived Adx_4-108 derived from Escherichia coli str. K-12 were used to construct a new electron transfer system for the conversion of progesterone. Selective mutation of CYP260A1 resulted in a mutant S276I with significantly enhanced 17α hydroxylase activity, and the yield of 17α-OH progesterone reached 58% after optimization of the catalytic system in vitro. In addition, the effect of phosphorylation of the ferredoxin Adx_4-108 on 17α hydroxyl activity was evaluated using a targeted mutation technique, and the results showed that the mutation Adx_4-108T69E transferred electrons to S276I more efficiently, which further enhanced the catalytic specificity in the C17 position of progesterone, and the yield of 17α-OH progesterone was eventually increased to 74%. This study provides a new option for the production of 17α-OH progesterone by specific transformation of bacterial-derived 17α hydroxylase, and lays a theoretical foundation for the industrial production of progesterone analogs using biotransformation method.
Animals ; Cattle ; Progesterone/metabolism* ; Hydroxylation ; Biocatalysis ; Electron Transport ; Mixed Function Oxygenases/metabolism*

Recombinant collagen, as an alternative to natural collagen, has the potential to be widely used in biomaterials, biomedicine, etc. Diverse recombinant collagens and their variants can be industrially produced in a variety of expression systems, which lays a foundation for exploring and expanding the clinical application of recombinant collagens. We reviewed different expression systems for recombinant collagens, such as prokaryotic expression systems, yeast expression systems, as well as plant, insect, mammal, and human cell expression systems, and introduced the advantages, potential applications, and limitations of recombinant collagen. In particularly, we focused on the current progress in the recombinant collagen production, including recombinant expression system construction and hydroxylation strategies of recombinant collagen, and summarized the current biomedical applications of recombinant collagen.
Animals ; Biocompatible Materials ; Collagen/biosynthesis* ; Humans ; Hydroxylation ; Recombinant Proteins/biosynthesis*

Nano-LC MS/MS was used to analyze trypsin digested deer-hide gelatin(DHG) samples, hydroxylation and O-glycosylation on lysine sites of DHG were comprehensive identified by using PEAKS Studio software. The sites, sorts and amounts of hydroxylation and O-glycosylation on Type Ⅰ collagen α1 chain(COL1 A1) and α2 chain(COL1 A2) of DHG were revealed. As a result, 5 284 peptides were identified from DHG samples, which were mainly from COL1 A1 and COL1 A2. Among these peptides, there were 449 peptides with hydroxylysine, 442 with galactosyl-hydroxylysine, 449 with glucosyl-galactosyl-hydroxylysine. The major modified sites of hydroxylation and O-glycosylation in DHG were shown as follow: α1-9 N and α2-5 N in N-telopeptides, α1-87, α1-174, α1-930, α2-87, α2-174, α2-933 in triple helix domain, and α1-16 C in C-telopeptides. These hydroxylation and O-glycosylation were correlated with the formation and stability of collagen molecules and collagen fibrils. It is feasible for the collagens and peptides dissolving from deer skin collagen fibrils under high temperature and pressure decocting, high temperature and pressure also might destroy inter-molecular covalent cross-linking and help those glycol-peptides formations. The present study provided ideas and strategies for the in-depth investigation on DHG chemical constituents, and showed good theoretical significance and application value.
Animals ; Deer/metabolism* ; Gelatin ; Glycosylation ; Hydroxylation ; Lysine/metabolism* ; Protein Processing, Post-Translational ; Tandem Mass Spectrometry

The regio- and stereo-selective hydroxylations of two ingenane diterpenoids, 20-deoxyingenol (1) and 13-oxyingenol dodecanoat (2), by the filamentous fungi Mortierella ramanniana and Gibberella fujikuroi were investigated in the present study. Four undescribed metabolites (3-6) of substrate 1 and two undescribed metabolites (7 and 8) of substrate 2 were isolated. All the metabolites were identified as hydroxylated ingenane derivatives by extensive NMR and HR-ESI-MS data analyses. All the biotransformed compounds and the substrates were evaluated for their cytotoxicities against three human cancer cell lines, including human colon cancer Caco-2, breast cancer MCF-7, and adriamycin (ADM)-resistant MCF-7/ADM cell lines. All ingenane alcohols (1, and 3-6) displayed no significant cytotoxic activities. The substrate 13-oxyingenol dodecanoat (2) showed moderate cytotoxicity with IC values being 35.59 ± 5.37 μmol·L (Caco-2), 24.04 ± 4.70 μmol·L (MCF-7), and 22.24 ± 5.19 μmol·L (MCF-7/ADM). However, metabolites 7 and 8 displayed no significant cytotoxicity. These results indicated that the hydroxylation at the C-13 aliphatic acid ester of substrate 2 can significantly reduce the cytotoxic activity.
Antineoplastic Agents ; chemistry ; metabolism ; Biotransformation ; Cell Line, Tumor ; Diterpenes ; chemistry ; metabolism ; Gibberella ; metabolism ; Humans ; Hydroxylation ; Molecular Structure ; Mortierella ; metabolism ; Stereoisomerism

Erythromycin A is a clinically important macrolide antibiotic with broad-spectrum activity. Its biosynthesis involves the formation of the 14-membered skeleton catalyzed by polyketide synthases, and the modification steps such as hydroxylation, glycosylation and methylation. Based on the understanding of the biosynthetic mechanism, it is reliable to genetically manipulate the erythromycin A-producing strain for production improvement and structure modification. In this paper, we reviewed the progress regarding erythromycin A in high-producing strain construction and chemical structure derivation, to provide insights for further development.
Anti-Bacterial Agents ; biosynthesis ; chemistry ; Erythromycin ; biosynthesis ; chemistry ; Glycosylation ; Hydroxylation ; Methylation ; Multigene Family ; Polyketide Synthases ; metabolism

Combined with method of ketoconazole resistance screening, a 7alpha,15alpha-diOH-DHEA high-producing mutant Colletotrichum lini ST-1 was obtained by compound mutation of NTG and low energy N+ ion beam implantation. With the substrate concentration of 10 g/L DHEA, the molar yield of 7alpha,15alpha-diOH-DHEA reached 34.2%, increased by 46.2% than that of the original strain. Then we optimized the medium. First, Plackett-Burman design was used to evaluate the effects of medium components on molar yield of the product. Results show that glucose, yeast extract and MgSO4 x 7H2O were the important parameters for the biotransformation process. Subsequently, the path of steepest ascent was used to approach the optimal levels. To obtain the optimal levels, central composite design and response surface analysis were carried out. The optimal medium was as follows (g/L): glucose 26.34, yeast extract 12.15, corn flour 3.00, FeSO4 x 7H2O 0.015, MgSO4 x 7H2O 0.14, KH2PO4 0.90. Under the optimal conditions, the molar yield of 7alpha,15alpha-diOH-DHEA reached 49.3%, which was 44.2% higher than that of using the medium before optimization.
Biotransformation ; Colletotrichum ; metabolism ; Dehydroepiandrosterone ; chemistry ; Fermentation ; Hydroxylation ; Industrial Microbiology ; Mutation

Prolyl-4-hydroxylase domain (PHDs) family is one of the most important regulatory factors in hypoxic stress. PHD2 plays a critical role in cells and tissues adaptation to the low oxygen environment. Its hydroxylation activity regulates the stability and transcriptional activity of the hypoxia-inducible factor 1 (HIF-1), which is the key factor in response to hypoxic stress. Subsequently, PHD2 acts as an important factor in oxygen homeostasis. Studies have shown that PHD2, through its regulation on HIF-1, plays an important role in the post-ischemic neovascularization. Furthermore, under hypoxic condition, PHD2 also regulates other pathways that positively regulate angiogenesis factors HIF-1 independently. Moreover, recently, several evidences have also shown that PHD2 also affects tumor growth and metastasis in a tumor microenvironment. Based on these facts, PHD2 have been considered as a potential therapeutic target both in treating ischemic diseases and tumors. Here, we review the molecular regulation mechanism of PHD2 and its physiological and pathological functions. We focus on the role of PHD2 in both therapeutic angiogenesis for ischemic disease and tumor angiogenesis, and the current progress in utilizing PHD2 as a therapeutic target.
Animals ; Humans ; Hydroxylation ; Hypoxia-Inducible Factor 1 ; metabolism ; Hypoxia-Inducible Factor-Proline Dioxygenases ; antagonists & inhibitors ; physiology ; Neoplasms ; blood supply ; metabolism ; pathology ; therapy ; Neovascularization, Pathologic ; metabolism ; pathology ; Tumor Microenvironment ; Vascular Diseases ; pathology ; therapy

Animals ; Carps ; genetics ; Cloning, Molecular ; Fish Proteins ; genetics ; Hydroxylation ; Hypoxia-Inducible Factor 1, alpha Subunit ; genetics ; Lakes

In order to discover the steroid biotransformation ability of filamentous fungus Aspergillus niger TCCC41650, we studied the fermentation of 4-androstene-3,17-dione with A. niger TCCC41650. The transformation product was purified, crystallized and determined as 16β-hydroxy-androst-4-ene-3,17-dione by X-ray single crystal diffraction method. The best fermentation condition was found to be pH 6.0, ethanol amount 2% with a substrate concentration of 1 per thousand, the transformation rate is 85.81% after 72 h. Based on the best of our knowledge, 16β-hydroxylation rarely occurs in microbial transformations of steroid. This study laid the foundation for the research of 16β-hydroxylation steroids
Androstenedione ; metabolism ; Aspergillus niger ; metabolism ; Biotransformation ; Fermentation ; Hydroxylation ; Industrial Microbiology

OBJECTIVETo compare the nature of the metabolites formed from the phase I metabolism (hydroxylation and oxidation) and phase II metabolism (glutathionyl conjugation) of PCBs that have different chlorine substitution patterns. To discuss the structure-activity relationships and metabolic mechanisms of PCBs.

METHODS4-Cl-biphenyl (PCB3), 4,4'-Cl-biphenyl (PCB15), 3,4,3',4'-Cl-biphenyl (PCB77) were used for in vitro metabolic study. LC/MS and UV-Vis studies were performed for metabolites identification.

RESULTSThe cytochrome P-450 catalyzed hydroxylation rate decreased as the number of chlorine substitutions increased. In this reaction, PCB3 was fully metabolized, approximately half of the PCB15 was metabolized and PCB77 was not metabolized at all. The oxidation rate of PCB15-HQ was higher than that of PCB3-HQ under various oxidation conditions. The LC/MS and UV-Vis data suggest that in the conjugation reaction of PCB15-Q and GSH, the Michael addition reaction occurs preferentially over the displacement reaction.

CONCLUSIONThe metabolic profiles of polychlorinated biphenyls (PCBs) are dramatically affected by chlorine substitution patterns. It is suggested that the metabolic profiles of PCBs are related to their chlorine substitution patterns, which may have implications for the toxicity of PCB exposure.