Application of microbial 3-ketosteroid Δ1-dehydrogenases in biotechnology

Rohman, Ali; Dijkstra, Bauke W.

doi:10.1016/j.biotechadv.2021.107751

Cited by 24 publications

(18 citation statements)

References 233 publications

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“…A second double bond is introduced into the A-ring at C1 by Δ 1 -3-ketosteroid dehydrogenases [ 69 ]. Genes encoding these enzymes are found in diverse steroid-metabolizing bacteria.…”

Section: Bacterial Bile Acid Degradationmentioning

confidence: 99%

Degradation of Bile Acids by Soil and Water Bacteria

et al. 2021

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Bile acids are surface-active steroid compounds with a C5 carboxylic side chain at the steroid nucleus. They are produced by vertebrates, mainly functioning as emulsifiers for lipophilic nutrients, as signaling compounds, and as an antimicrobial barrier in the duodenum. Upon excretion into soil and water, bile acids serve as carbon- and energy-rich growth substrates for diverse heterotrophic bacteria. Metabolic pathways for the degradation of bile acids are predominantly studied in individual strains of the genera Pseudomonas, Comamonas, Sphingobium, Azoarcus, and Rhodococcus. Bile acid degradation is initiated by oxidative reactions of the steroid skeleton at ring A and degradation of the carboxylic side chain before the steroid nucleus is broken down into central metabolic intermediates for biomass and energy production. This review summarizes the current biochemical and genetic knowledge on aerobic and anaerobic degradation of bile acids by soil and water bacteria. In addition, ecological and applied aspects are addressed, including resistance mechanisms against the toxic effects of bile acids.

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“…A second double bond is introduced into the A-ring at C1 by Δ 1 -3-ketosteroid dehydrogenases [ 69 ]. Genes encoding these enzymes are found in diverse steroid-metabolizing bacteria.…”

Section: Bacterial Bile Acid Degradationmentioning

confidence: 99%

Degradation of Bile Acids by Soil and Water Bacteria

et al. 2021

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“…The 1,2-dehydrogenation process can be carried out with either a whole-cell system (native and recombinant bacteria are used) or isolated enzymes [ 7 , 8 ]. The poor solubility of steroids in aqueous solutions is usually circumvented by the application of pseudo-crystallofermentation technique (i.e., steroid substrate in the solid-state) or by the addition of organic solvents and various solubilizing agents, e.g., vegetable oils, phospholipids, glycols, dextrans or cyclodextrins [ 8 – 15 ]. The biocompatibility of cyclodextrins makes them valuable solubilizing agents for scale-up microbial steroid biotransformation.…”

Section: Introductionmentioning

confidence: 99%

Universal capability of 3-ketosteroid Δ1-dehydrogenases to catalyze Δ1-dehydrogenation of C17-substituted steroids

et al. 2021

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Background 3-Ketosteroid Δ1-dehydrogenases (KSTDs) are the enzymes involved in microbial cholesterol degradation and modification of steroids. They catalyze dehydrogenation between C1 and C2 atoms in ring A of the polycyclic structure of 3-ketosteroids. KSTDs substrate spectrum is broad, even though most of them prefer steroids with small substituents at the C17 atom. The investigation of the KSTD’s substrate specificity is hindered by the poor solubility of the hydrophobic steroids in aqueous solutions. In this paper, we used 2-hydroxpropyl-β-cyclodextrin (HBC) as a solubilizing agent in a study of the KSTDs steady-state kinetics and demonstrated that substrate bioavailability has a pivotal impact on enzyme specificity. Results Molecular dynamics simulations on KSTD1 from Rhodococcus erythropolis indicated no difference in ΔGbind between the native substrate, androst-4-en-3,17-dione (AD; − 8.02 kcal/mol), and more complex steroids such as cholest-4-en-3-one (− 8.40 kcal/mol) or diosgenone (− 6.17 kcal/mol). No structural obstacle for binding of the extended substrates was also observed. Following this observation, our kinetic studies conducted in the presence of HBC confirmed KSTD1 activity towards both types of steroids. We have compared the substrate specificity of KSTD1 to the other enzyme known for its activity with cholest-4-en-3-one, KSTD from Sterolibacterium denitrificans (AcmB). The addition of solubilizing agent caused AcmB to exhibit a higher affinity to cholest-4-en-3-one (Ping-Pong bi bi KmA = 23.7 μM) than to AD (KmA = 529.2 μM), a supposedly native substrate of the enzyme. Moreover, we have isolated AcmB isoenzyme (AcmB2) and showed that conversion of AD and cholest-4-en-3-one proceeds at a similar rate. We demonstrated also that the apparent specificity constant of AcmB for cholest-4-en-3-one (kcat/KmA = 9.25∙106 M−1 s−1) is almost 20 times higher than measured for KSTD1 (kcat/KmA = 4.71∙105 M−1 s−1). Conclusions We confirmed the existence of AcmB preference for a substrate with an undegraded isooctyl chain. However, we showed that KSTD1 which was reported to be inactive with such substrates can catalyze the reaction if the solubility problem is addressed.

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“…Budesonide, an asthma medication of the corticosteroid type with C 1 =C 2 double bond, had a market value of $1.01 billion as Pulmicort and $2.72 billion as Symbicort (co-marketed with Formoterol) in 2020 ( , accessed date, 5 December 2021). Accordingly, Δ 1 -dehydrogenation of steroid compounds is an important reaction in the synthesis of medically useful steroidal molecules [ 3 , 4 , 5 , 6 ]. Such a transformation may be achieved by chemical or biocatalytic methods.…”

Section: Introductionmentioning

confidence: 99%

“…Such a transformation may be achieved by chemical or biocatalytic methods. While the chemical methods require a complicated series of reaction steps due to the complex nature of the steroidal molecules [ 7 , 8 , 9 ], biocatalytic Δ 1 -dehydrogenation of steroids is usually performed under mild conditions with high regioselectivity [ 5 , 6 , 10 ].…”

Section: Introductionmentioning

confidence: 99%

A New 3-Ketosteroid-Δ1–Dehydrogenase with High Activity and Broad Substrate Scope for Efficient Transformation of Hydrocortisone at High Substrate Concentration

et al. 2022

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3-Ketosteroid-Δ1-dehydrogenases (KstDs [EC 1.3.99.4]) catalyze the Δ1-dehydrogenation of steroids and are a class of important enzymes for steroid biotransformations. In this study, nine putative kstD genes from different origins were selected and overexpressed in Escherichia coli BL21(DE3). These recombinant enzymes catalyzed the Δ1-desaturation of a variety of steroidal compounds. Among them, the KstD from Propionibacterium sp. (PrKstD) displayed the highest specific activity and broad substrate spectrum. The detailed catalytic characterization of PrKstD showed that it can convert a wide range of 3-ketosteroid compounds with diverse substituents, ranging from substituents at the C9, C10, C11 and C17 position through substrates without C4-C5 double bond, to previously inactive C6-substituted ones such as 11β,17-dihydroxy-6α-methyl-pregn-4-ene-3,20-dione. Reaction conditions were optimized for the biotransformation of hydrocortisone in terms of pH, temperature, co-solvent and electron acceptor. By using 50 g/L wet resting E. coli cells harboring PrKstD enzyme, the conversion of hydrocortisone was about 92.5% within 6 h at the substrate concentration of 80 g/L, much higher than the previously reported results, demonstrating the application potential of this new KstD.

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Application of microbial 3-ketosteroid Δ1-dehydrogenases in biotechnology

Cited by 24 publications

References 233 publications

Degradation of Bile Acids by Soil and Water Bacteria

Degradation of Bile Acids by Soil and Water Bacteria

Universal capability of 3-ketosteroid Δ1-dehydrogenases to catalyze Δ1-dehydrogenation of C17-substituted steroids

A New 3-Ketosteroid-Δ1–Dehydrogenase with High Activity and Broad Substrate Scope for Efficient Transformation of Hydrocortisone at High Substrate Concentration

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