Conference Agenda

Cytochrome P450 and Steroidogenesis

Walter L. Miller

 

Department of Pediatrics, University of California, San Francisco

 

Many steroids were isolated and characterized chemically by 1950, but their biosynthetic pathways were unknown. Ryan & Engel reported steroid 21-hydroxylation by adrenal microsomes, inhibited by CO and reversed by light (1956, 57); Klingenberg reported P450 spectra in 1957;  and Cooper, Estabrook et al., showed 21-hydroxylation was P450-mediated (1963-65). Harding et al., reported P450 spectra in adrenal mitochondria in 1964, and in 1966 Omura et al., reported mitochondrial steroid 11-hydroxylation required two proteins now known as ferredoxin (FDX) and ferredoxin reductase (FDXR). These specific steroidogenic steps in different organelles were the first reactions shown to be P450-catalyzed.

 

Steroidogenic pathways, enzymes, and genes vary among species. Most studies of steroidogenesis utilize the adrenal, as steroidogenic cells comprise >80% of its mass, facilitating preparation of specific factors. The adrenal has functional zones that express mitochondrial P450scc (CYP11A1 gene), catalyzing the 3 reactions that convert cholesterol to pregnenolone; a disease where this activity is diminished led to the discovery of the steroidogenic acute regulatory protein, StAR. The Zona Glomerulosa (ZG), converts pregnenolone to the potent mineralocorticoid, aldosterone, because the ZG expresses aldosterone synthase (P450c11AS, CYP11B2) but does not express 17-hydroxylase (P450c17, CYP17A1), thus excluding synthesis of glucocorticoids and sex steroids. The Zona Fasciculata (ZF) expresses P450c11b (CYP11B1) and P450c17, but not P450c11AS, permitting synthesis of cortisol in most vertebrates (the rodent ZF fails to express P450c17, resulting in corticosterone production). P450c17 may also catalyze 17,20-lyase activity, converting 21-carbon (C21) steroids to C19 precursors of sex steroids. Whether 17,20-lyase activity occurs depends on factors favoring electron transfer to P450c17: i) the abundance of P450 oxidoreductase (POR), the redox partner for all Type 2 P450s; ii) the presence of cytochrome b₅ (b5); iii) Ser/Thr phosphorylation of P450c17 (probably by p38a). The mature primate adrenal has a Zona Reticularis (ZR) that produces abundant C19 steroids at the time of  ‘adrenarche’, the onset of adrenal androgen synthesis beginning before puberty. However, the well-studied C19 steroids DHEA, DHEAS, and androstenedione do not activate the androgen receptor: they identify but do not mediate adrenarche.

 

Work from 2013-2018 identified the adrenal androgen as 11keto-testosterone (11KT), made in the ZR by a newly described pathway requiring P450c11b. Also recently described is the adrenal/testicular ‘backdoor pathway’ to dihydrotestosterone that bypasses DHEA/S and androstenedione; this pathway is important in fetal sexual development and in hyperandrogenic disorders. Genetic diseases have been central to identifying enzymes and pathways and continue to reveal new physiology.

The large scale sustainable global production system chosen by nature is based on photosynthesis. We should learn from nature! The chloroplast organelle represents a light-driven power house synthesizing basic metabolites such as amino acids, lipids and diterpene backbones. These may be channeled into the production of high value compounds by targeted transfer of their biosynthetic pathways into the chloroplast with electron transfer to cytochrome P450s proceeding directly from reduced ferredoxin. Pathway discovery for even structurally complex diterpenoids such as forskolin, triptolide, ginkgolides and paclitaxel is facilitated e.g. by the discovered organization of the biosyntetic genes in gene clusters and by access to tissue and cell type specific transcriptomics data. The remarkable multifunctional catalytic properties of many P450s open the doors to functional expression of entire pathways into heterologous hosts and their chloroplasts based on a remarkable low number of genes. An additional benefit of using chloroplasts as a production system is their in nature demonstrated ability to accumulate molar levels of natural products as demonstrated by the accumulation of large amounts of vanillin in chloroplast-derived phenyloplasts in the mature pods of vanilla orchids. The storage of the high value natural products produced in confined dense biocondensates at seemingly impossible molar concentrations may be orchestrated by the use of natural deep eutectic solvents thereby avoiding auto-toxicity issues and disruption of cell homeostasis. Multi-stream product lines based on separate commercialization of the isolated high value compounds and of improved bulk products increase the economic potential of light driven production systems and speed-up commercial scale-up and thereby the transition to a biobased society.. If the production is made in engineered microalgae, these may be grown environmentally contained on ocean-based floating platforms e.g. anchored to the artificial energy islands to be built in the North Sea. All the process steps discussed above need a lot of optimizations over the next few years to make synthetic biology the envisioned basis for environmental benign green future production systems. On the research side, this requires cross-disciplinary discussions between scientists from the natural sciences, humanities, social sciences and law. In this way, we may secure more opmimal uses and thus social acceptance of synthetic biology as one of the technologies that may help us to adress many of the challenges humanities are facing.