steroids and deoxycholic
acid (DCA)?
Steroidal drugs were born in the 40s of the 20th century and occupy an important position in the chemical drug system. At present, more than 400 steroids have been produced worldwide, making it the second largest class of chemical drugs after antibiotics. The Chinese steroidal drug market accounts for only one-third of the global market. China's research on steroid drugs, compared with the world's advanced countries, there is still a big gap. The development of new steroid drug resources is one of the key development directions of the pharmaceutical industry.
In addition to immunosuppressive, anti-inflammatory, antirheumatic, pregnancy-inducing, diuretic, sedative, anabolic, and contraceptive effects, steroid drugs can also treat cancer, osteoporosis, HIV infection, and other diseases[1]. Steroidal compounds are characterized by their small molecular weight and high-fat solubility, making them easy to enter cells and produce a series of physiological changes.
Deoxycholic acid (DCA) is a bile acid. It is also a novel injectable medication approved by the FDA for the treatment of submental fat. DCA has gained popularity in recent years because of its proven efficacy, fewer side effects, and limited downtime[2]. When injected into submental fat, deoxycholic acid helps destroy (adipocytes) fat cells, which are metabolized by the body for several months.
hydroxylation?
Hydroxylation is one of the important reactions for the functionalization of steroidal compounds,
which refers to the introduction of hydroxyl groups on various groups of organic compounds.
On one hand, hydroxylation can provide intermediates for chemical synthesis; On the other hand, the
hydroxylation reaction using microorganisms can reach parts that cannot be reached by general
chemical reactions, and steroidal drugs with different efficacy can be formed by hydroxylation in
different positions or different spaces. In addition, hydroxylation can increase the polarity
of sterols, affect their cell secretion, toxicity, and efflux across cell membranes, and enhance
the biological activity of compounds. Hydroxylated steroids generally exhibit higher biological
activity than non-hydroxylated steroids with low polarity.
6β-OH-DCA?
Numerous studies have shown that bile acids are enhanced in function and value when they are hydroxylated.
Given the significant effects of hydroxylation, we hope to obtain 3a, 6b, 12a-trihydroxy-5b-cholan-24-oic-acid (6β-OH DCA) with more application potential through hydroxylation modification of deoxycholic acid.
choose Olep?
Cytochrome P450 is abbreviated as CYP450. It represents a large family of self-oxidizing ferrous heme proteins, belonging to the class of monooxygenases and named for its specific absorption peak at 450 nm.
The P450 enzyme is one of the most versatile biocatalysts in nature. It can not only catalyze a wide range of reaction types but also have a wide substrate profile. Its diverse functions have great potential for synthetic biology applications. P450 has two main biological functions: one is the metabolism of heterologous substances, and fat-soluble drugs must be bioconverted in the kidneys to allow excretion. P450 can reduce the hydrophobicity of compounds, forming intermediate metabolites for easy excretion. The second is the biosynthesis of bioactive molecules, including the metabolism of steroids, vitamins, and fatty acids[3].
As a terminal oxygenase, cytochrome P450 is involved in processes such as sterol hormone synthesis in organisms. In recent years, the structure and function of cytochrome P450, especially its role in drug metabolism, have made great progress. Studies have shown that cytochrome P450 is a key enzyme in drug metabolism and has important effects on cytokines and thermoregulation.
In reading the literature about the P450 enzyme, we found the P450 monooxygenase CYP107D1(Olep), which is a promising enzyme for the production of bile acid derivatives. In the course of extensive reading of the literature on the hydroxylation of sterols, we found that UDCA has been synthesized by expressing the P450 monooxygenase CYP107D1 (Olep) hydroxylated in Escherichia coli (E. coli) to modify lithophanic acid LCA[4], significantly enhancing the efficiency of the biocatalytic method for the preparation of UDCA. In a further review of the literature on Olep, we learned that Olep can also hydroxylate testosterone at the positions 6β, 7β, 12β, and 15β[5], bile acids like LCA and deoxycholic acid (DCA) are hydroxylated exclusively at the 6β-position, forming MDCA and 3α-, 6β-, 12α-trihydroxy-5β-cholan-24-oic acid, respectively[4]. We were therefore inspired to use Olep to hydroxylate deoxycholic acid DCA to produce 6β-OH DCA.
the soluble
expression of Olep?
When a foreign gene is expressed in prokaryotic cells (especially highly expressed in E. coli), the concentration of newly generated peptides is high, and there is not enough time to fold them, thus forming amorphous protein aggregates[6]. When studying the properties of P450, we found that there were many inclusions so that its soluble expression was poor, which critically affected the catalytic efficiency of P450 enzyme. So we hope to improve the soluble expression of P450 enzyme to improve its catalytic efficiency.
heme supply in E. coli ?
Cytochrome P450 (CYP450) is a large enzyme superfamily with heme as a cofactor, consisting of many isoenzymes and isoforms[7]. It takes heme as the active center and needs to be combined with heme to be active. During the catalytic process, it was found that the supply of the heme prosthetic group was insufficient due to the efficient expression of the P450 enzyme in E. coli, which severely limited the catalytic efficiency of the P450 enzyme in whole cells[8]. Since E. coli has less intracellular heme content, we hope to increase the activity of the P450 enzyme by increasing the content of heme. However, commercial E. coli generally lacks a heme transport system and requires the expression of heme transporters to utilize exogenously added heme[9]. Therefore, we design to enhance the heme synthesis pathway in host cells.
modify the optimal
redox partners?
In bacteria, most P450 enzymes belong to type I cytochrome P450 enzymes[10]. They require redox partners consisting of ferredoxin reductase (FdR) and ferredoxin (Fdx). The partner (RP) system is responsible for shuttling two electrons from the cofactor NAD(P)H to the heme iron catalytic center of the P450 enzyme along the "NAD(P)H→FdR→Fdx" electron transport chain, activates O2 to produce highly reactive intermediates, and realizes substrate oxidation[11]. However, the native RP of P450 enzymes is often in an unknown state. There are many genes encoding RP proteins in the bacterial genome, while the genes encoding P450 enzymes and their potential endogenous RP genes are often far away. The understanding of the interaction between P450 enzymes and RP has not been systematically studied, which greatly limits the scientific research and practical application of P450 enzymes[12,13]. Therefore, the strategy of constructing heterozygous P450 catalytic systems using alternative forms of RP is often used for functional studies of P450 enzymes. Based on the above analysis, redox partners play an important role in electron transport in P450 enzymes, so we hope to find redox partners that are more suitable for Olep.
optimal whole-cell
catalytic system?
The use of purified P450 enzyme for biotransformation reaction is not convenient. On the one hand, the purification process of pure enzyme-catalyzed reaction requires complex requirements, on the other hand, it also requires expensive NAD(P)H cofactors.
At present, by providing key precursors, cofactors and a suitable environment suitable for multi-step reactions, heterologous expression of the P450 gene in E. coli to construct a whole-cell catalytic system to synthesize target compounds has good application prospects.
Reference:
- Sultana, N. Microbial biotransformation of bioactive and clinically useful steroids and some salient features of steroids and biotransformation. Steroids 2018, 136, 76-92. DOI: 10.1016/j.steroids.2018.01.007
- Watchmaker, J.; Callaghan, D. J., III; Dover, J. S. Deoxycholic Acid in Aesthetic Medicine. Advances in Cosmetic Surgery 2020, 3, 77-87. DOI: 10.1016/j.yacs.2020.01.009
- Albertolle, M. E.; Peter Guengerich, F. The relationships between cytochromes P450 and H(2)O(2): Production, reaction, and inhibition. J Inorg Biochem 2018, 186, 228-234. DOI: 10.1016/j.jinorgbio.2018.05.014
- Grobe, S.; Badenhorst, C. P. S.; Bayer, T.; Hamnevik, E.; Wu, S.; Grathwol, C. W.; Link, A.; Koban, S.; Brundiek, H.; Großjohann, B.; et al. Engineering Regioselectivity of a P450 Monooxygenase Enables the Synthesis of Ursodeoxycholic Acid via 7β-Hydroxylation of Lithocholic Acid. Angew Chem Int Ed Engl 2021, 60, 753-757. DOI: 10.1002/anie.202012675
- Agematu, H.; Matsumoto, N.; Fujii, Y.; Kabumoto, H.; Doi, S.; Machida, K.; Ishikawa, J.; Arisawa, A. Hydroxylation of Testosterone by Bacterial Cytochromes P450 Using the Escherichia coli Expression System. Bioscience, Biotechnology, and Biochemistry 2006, 70, 307-311. DOI: 10.1271/bbb.70.307
- Baneyx, F.; Mujacic, M. Recombinant protein folding and misfolding in E. coli. Nat Biotechnol 2004, 22, 1399-1408. DOI: 10.1038/nbt1029
- Manikandan, P.; Nagini, S. Cytochrome P450 Structure, Function and Clinical Significance: A Review. Current drug targets 2018, 191, 38-54. DOI: 10.2174/1389450118666170125144557
- Hu, B.; Yu, H.; Zhou, J.; Li, J.; Chen, J.; Du, G.; Lee, S. Y.; Zhao, X. Whole-Cell P450 Biocatalysis Using Engineered E. coli with Fine-Tuned Heme Biosynthesis. Adv Sci 2023, 10, e2205580. DOI: 10.1002/advs.202205580
- Villarreal, D. M.; Phillips, C. L.; Kelley, A. M.; Villarreal, S.; Villaloboz, A.; Hernandez, P.; Olson, J. S.; Henderson, D. P. Enhancement of recombinant hemoglobin production in E. coli BL21(DE3) containing the Plesiomonas shigelloides heme transport system. Appl Environ Microbiol 2008, 74, 5854-5856. DOI: 10.1128/aem.01291-08
- Rudolf, J. D.; Chang, C. Y.; Ma, M.; Shen, B. Cytochromes P450 for natural product biosynthesis in Streptomyces: sequence, structure, and function. Nat Prod Rep 2017, 34, 1141-1172. DOI: 10.1039/c7np00034k
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