Further Studies on the 3-Ketosteroid 9α-Hydroxylase of Rhodococcus ruber Chol-4, a Rieske Oxygenase of the Steroid Degradation Pathway

Abstract

The biochemistry and genetics of the bacterial steroid catabolism have been extensively studied during the last years and their findings have been essential to the development of biotechnological applications. For instance, metabolic engineering of the steroid-eater strains has allowed to obtain intermediaries of industrial value. However, there are still some drawbacks that must be overcome, such as the redundancy of the steroid catabolism genes in the genome and a better knowledge of its genetic regulation. KshABs and KstDs are key enzymes involved in the aerobic breakage of the steroid nucleus. Rhodococcus ruber Chol-4 contains three kshAs genes, a single kshB gene and three kstDs genes within its genome. In the present work, the growth of R. ruber ΔkshA strains was evaluated on different steroids substrates; the promoter regions of these genes were analyzed; and their expression was followed by qRT-PCR in both wild type and ksh mutants. Additionally, the transcription level of the kstDs genes was studied in the ksh mutants. The results show that KshA2B and KshA1B are involved in AD metabolism, while KshA3B and KshA1B contribute to the cholesterol metabolism in R. ruber. In the kshA single mutants, expression of the remaining kshA and kstD genes is re-organized to survive on the steroid substrate. These data give insight into the fine regulation of steroid genes when several isoforms are present.

Characterization and Degradation Pathways of Microbacterium resistens MZT7, A Novel 17β-Estradiol-Degrading Bacterium

“…kshB is a monooxygenase widely present in steroid-degrading bacteria. It is essential to open the steroid B-ring, thereby initiating degradation of the steroid ring system [ 31 , 32 ]. In a recent study, Hsiao et al demonstrated that fadD3 gene expression was upregulated during the degradation of E1 by Rhodococcus sp.…”

Recent developments in the enzymatic modifications of steroid scaffolds

“…3-Ketosteroid 9α-hydroxylase (KSH), an essential enzyme in bacterial steroid degradation, consists of two components: a Rieske oxygenase (KshA) and a ferredoxin reductase (KshB). [75][76][77][78] It catalyzes the 9α-hydroxylation of ASD ( 8) to produce 9α-OH-ASD (43). Qin and coworkers reconstituted the NADH regeneration system, utilizing the fusion expression of toluene 2,3-oxidoreductase TDO-R from Pseudomonas putida F17 instead of KshB, along with a Rieske [2Fe-2S] iron-oxidoreductant protein, TDO-F. 79 The reconstitution significantly enhanced the electron transfer efficiency by 176.3%.…”

Rhodococcus strains as a good biotool for neutralizing pharmaceutical pollutants and obtaining therapeutically valuable products: Through the past into the future

“…Biocatalytic transformations begin with the oxidation of steroids to stenones by a cholesterol oxidase (ChOx; Baldanta et al, 2021 ; Figure 6 ). ChOx is an extracellular flavin-dependent bifunctional oxidoreductase catalyzing the oxidation of a Δ 5 -ene-3β-hydroxysterol to Δ 5 -ene-3β-ketosteroid and the isomerization of the latter to yield Δ 4 -ene-3β-ketosteroid ( Fernández de las Heras et al, 2011 , 2014 ).…”

“…The steroid nucleus degradation involves oxygen-dependent rupture and subsequent hydrolytic cleavage of rings A and B following the 9,10-seco pathway due to 3-ketosteroid-dehydrogenase (KstD) and 3-ketosteroid 9α-hydroxylase (KsH). AD – 4-androstene-3,17-dione; ADD – androsta-1,4-diene-3,17-dione; 9OH-AD –9α-hydroxy-4-androstene-3,17-dione; 9OH-ADD – 9 α -hydroxyandrosta-1,4-diene-3,17-dione; 3-HSA – 3-hydroxy-9,10-secoandrosta-1,3,5(10)-triene-9,17-dione; 3,4-DHSA – 3,4-dihydroxy-9,10-secoandrosta-1,3,5(10)-triene-9,17-dione; 4,9-DSHA – 4,5,9,10-diseco-3-hydroxy-5-9-17-trioxoandrosta-1(10),2-diene-4-oic acid; HIP – 3aα-H-4α (3′-propanoate)-7a-β-methylhexahydro-1,5-indanedione; OPCA – oxypentadienecarboxylic acid ( Haußmann et al, 2013 ; Kreit, 2017 ; Baldanta et al, 2021 ).…”

“…( Carpova et al, 2011 ; Nogovitsina et al, 2011 ; Petrusma et al, 2014 ; Guevara et al, 2017 ; Rohman and Dijkstra, 2019 ; Zappaterra et al, 2021 ). As model strains for studying the steroid metabolism, R. erythropolis SQ1, R. ruber Chol-4, R. jostii RHA-1, R. rhodochrous DSM43269 were described ( van der Geize et al, 2002 , 2008 ; Fernández de las Heras et al, 2009 , 2012 , 2014 ; Petrusma et al, 2009 , 2014 ; Yam et al, 2010 ; Mohn et al, 2012 ; van Oosterwijk et al, 2012 ; Costa et al, 2013 ;; Haußmann et al, 2013 ; Guevara et al, 2017 , 2019a ; Chen et al, 2020 ; Baldanta et al, 2021 ; Zappaterra et al, 2021 ). In most studies, cholesterol, less commonly β-sitosterol, campesterol, cortisone, and cholate, are used as an initial substrate ( Kreit, 2017 ).…”