Testosterone is a key crucial hormone synthesized by steroidogenic enzymes that initiate and maintain spermatogenesis and secondary sexual characteristics in adult males. The taste receptor family 1 subunit 3 (T1R3) is reported to be associated with male reproduction. T1R3 can regulate the expressions of steroidogenic enzymes and affect testosterone synthesis . In this study, we addressed the question of whether the expression of steroid synthase was associated with T1R3 and its downstream-tasting molecules during testicular development . The results showed an overall upward trend in testosterone and morphological development in testes from Congjiang Xiang pigs from pre-puberty to sexual maturity. Gene expression levels of testicular steroidogenic acute regulatory protein ( StAR ), 3β-hydroxysteroid dehydrogenase ( 3β-HSD ), cytochrome P450c17 ( CYP17A1 ) and 17β-hydroxysteroid dehydrogenase ( 17β-HSD ) were increased from pre-puberty to sexual maturity. Protein expression changes of CYP17A1 and 3β-HSD were consistent with mRNA. The relative abundance of tasting molecules ( TAS1R3 , phospholipase Cβ2, PLCβ2 ) was increased from pre-puberty to puberty ( P < 0.05), with no further significant changes in expression from puberty to sexual maturity. Steroidogenic enzymes (3β-HSD and CYP17A1) were strongly detected in Leydig cells from pre-puberty to sexual maturity, while tasting molecules were localized in Leydig cells and spermatogenic cells. Correlation analysis showed that the genes mentioned above (except for PLCβ2 ) were positively correlated with testosterone levels and morphological characteristics of the testes at different developmental stages of Congjiang Xiang pigs. These results suggest that steroidogenic enzymes regulate testosterone synthesis and testicular development, and that taste receptor T1R3, but not PLCβ2, may associate with this process.
Substrate competition within a metabolic network constitutes a common challenge in microbial biosynthesis system engineering, especially if indispensable enzymes can produce multiple intermediates from a single substrate. Androstenedione (4AD) is a central intermediate in the production of a series of steroidal pharmaceuticals; however, its yield via the coexpression of 3β-hydroxysteroid dehydrogenase (3β-HSD) and 17α-hydroxylase/17,20-lyase (CYP17A1) in a microbial chassis affords a nonlinear pathway in which these enzymes compete for substrates and produce structurally similar unwanted intermediates, thereby reducing 4AD yields. To avoid substrate competition, we split the competing 3β-HSD and CYP17A1 pathway components into two separate Yarrowia lipolytica strains to linearize the pathway. This spatial segregation increased substrate availability for 3β-HSD in the upstream strain, consequently decreasing the accumulation of the unwanted intermediate 17-hydroxypregnenolone (17OHP5) from 94.8 ± 4.4% in single-chassis monocultures to 24.8 ± 12.6% in cocultures of strains expressing 3β-HSD and CYP17A1 separately. Orthologue screening to increase CYP17A1 catalytic efficiency and the preferential production of desired intermediates increased the biotransformation capacity in the downstream pathway, further decreasing 17OHP5 accumulation to 3.9%. Furthermore, nitrogen limitation induced early 4AD accumulation (final titer, 7.71 mg/L). This study provides a framework for reducing intrapathway competition between essential enzymes during natural product biosynthesis as well as a proof-of-concept platform for linear steroid production.
Runlan Wan,Xi Kong,Youzhe Yang,Siwen Tao,Youyou Chen,Alexander Tobias Teichmann,Frank Heinrich Wieland
The clinical use of the steroidal aromatase inhibitor Formestane (4-hydroxandrostenedione, 4-OHA) in the treatment of advanced ER+ breast cancer has been discontinued, and therefore, interest in this remarkable drug has vanished. As a C-19 sterol, 4-OHA can undergo extensive intracellular metabolism depending on the expression of specific enzymes in the corresponding cells. We used the metabolites 4β-hydroxyandrosterone, 4β-hydroxyepiandrosterone and its 17β-reduced derivative as standards for the proof of catalytic activity present in the cell culture medium and expressed by the isolated enzymes. All of the aldo-keto reductases AKR1C1, AKR1C2, AKR1C3 and AKR1C4 catalysed the reduction of the 3-keto-group and the Δ 4,5 double bond of 4-OHA at the same time. Molecular docking experiments using microscale thermophoresis and the examination of the kinetic behaviour of the isolated enzymes with the substrate 4-OHA proved that AKR1C3 had the highest affinity for the substrate, whereas AKR1C1 was the most efficient enzyme. Both enzymes (AKR1C1and AKR1C3) are highly expressed in adipose tissue and lungs, exhibiting 3β-HSD activity. The possibility that 4-OHA generates biologically active derivatives such as the androgen 4-hydroxytestosterone or some 17β-hydroxy derivatives of the 5α-reduced metabolites may reawaken interest in Formestane, provided that a suitable method of administration can be developed, avoiding oral or intramuscular depot-injection administration.
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