Draft Genome Sequence of the Steroid Degrader Rhodococcus ruber Strain Chol-4

Heras, Laura Fernández de las; Alonso, Sergio; Léon, Antonio de la Vega de; Xavier, Daniela; Perera, Julián; Lloréns, Juana María Navarro

doi:10.1128/genomea.00215-13

Cited by 8 publications

(5 citation statements)

References 8 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The three KshAs (GeneBank: ACD11366, BAH31462, AAL96829) identified in R. erythropolis SQ1 [27] and the five isoforms (GeneBank: KshA1: ADY18310, KshA2: ADY181316, KshA3: ADY18318, KshA4: ADY18323, KshA5: ADY18328) of R. rhodochrous [19] were blasted against the R. ruber strain Chol-4 genome [35]. Three different kshA ORFs were found in different genomic contexts (Fig.…”

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Functional characterization of 3-ketosteroid 9α-hydroxylases in Rhodococcus ruber strain chol-4

Guevara

Heras

Perera

et al. 2017

The Journal of Steroid Biochemistry and Molecular Biology

Self Cite

View full text Add to dashboard Cite

The 3-Ketosteroid-9α-Hydroxylase, also known as KshAB [androsta-1,4-diene-3,17-dione, NADH:oxygen oxidoreductase (9α-hydroxylating); EC 1.14.13.142)], is a key enzyme in the general scheme of the bacterial steroid catabolism in combination with a 3-ketosteroid-Δ-dehydrogenase activity (KstD), being both responsible of the steroid nucleus (rings A/B) breakage. KshAB initiates the opening of the steroid ring by the 9α-hydroxylation of the C9 carbon of 4-ene-3-oxosteroids (e.g. AD) or 1,4-diene-3-oxosteroids (e.g. ADD), transforming them into 9α-hydroxy-4-androsten-3,17-dione (9OHAD) or 9α-hydroxy-1,4-androstadiene-3,17-dione (9OHADD), respectively. The redundancy of these enzymes in the actinobacterial genomes results in a serious difficulty for metabolic engineering this catabolic pathway to obtain intermediates of industrial interest. In this work, we have identified three homologous kshA genes and one kshB gen in different genomic regions of R. ruber strain Chol-4. We present a set of data that helps to understand their specific roles in this strain, including: i) description of the KshAB enzymes ii) construction and characterization of ΔkshB and single, double and triple ΔkshA mutants in R. ruber iii) growth studies of the above strains on different substrates and iv) genetic complementation and biotransformation assays with those strains. Our results show that KshA2 isoform is needed for the degradation of steroid substrates with short side chain, while KshA3 works on those molecules with longer side chains. KshA1 is a more versatile enzyme related to the cholic acid catabolism, although it also collaborates with KshA2 or KshA3 activities in the catabolism of steroids. Accordingly to what it is described for other Rhodococcus strains, our results also suggest that the side chain degradation is KshAB-independent.

show abstract

Section: Resultsmentioning

confidence: 99%

“…Manipulation of genomic DNA was carried out according to standard protocols [33]. The BioEdit program vs 7.1.3.0 [34] was used to blast a known ORF against the published genome of R. ruber [35].…”

Section: Dna Preparation Sequencing and In Silico Analysismentioning

confidence: 99%

Functional characterization of 3-ketosteroid 9α-hydroxylases in Rhodococcus ruber strain chol-4

Guevara

Heras

Perera

et al. 2017

The Journal of Steroid Biochemistry and Molecular Biology

Self Cite

View full text Add to dashboard Cite

show abstract

“…DNASTAR (Lasergene) programs were used to analyse sequences and to design primers. The R. ruber strain Chol-4 genomic DNA has been previously sequenced [ 33 ]. BioEdit program was used to perform local-blast alignments within the genome data (NCIB::ANGC01000000).…”

Section: Methodsmentioning

confidence: 99%

Functional differentiation of 3-ketosteroid Δ1-dehydrogenase isozymes in Rhodococcus ruber strain Chol-4

et al. 2017

Self Cite

View full text Add to dashboard Cite

BackgroundThe Rhodococcus ruber strain Chol-4 genome contains at least three putative 3-ketosteroid Δ1-dehydrogenase ORFs (kstD1, kstD2 and kstD3) that code for flavoenzymes involved in the steroid ring degradation. The aim of this work is the functional characterization of these enzymes prior to the developing of different biotechnological applications.ResultsThe three R. ruber KstD enzymes have different substrate profiles. KstD1 shows preference for 9OHAD and testosterone, followed by progesterone, deoxy corticosterone AD and, finally, 4-BNC, corticosterone and 19OHAD. KstD2 shows maximum preference for progesterone followed by 5α-Tes, DOC, AD testosterone, 4-BNC and lastly 19OHAD, corticosterone and 9OHAD. KstD3 preference is for saturated steroid substrates (5α-Tes) followed by progesterone and DOC. A preliminary attempt to model the catalytic pocket of the KstD proteins revealed some structural differences probably related to their catalytic differences. The expression of kstD genes has been studied by RT-PCR and RT-qPCR. All the kstD genes are transcribed under all the conditions assayed, although an additional induction in cholesterol and AD could be observed for kstD1 and in cholesterol for kstD3. Co-transcription of some correlative genes could be stated. The transcription initiation signals have been searched, both in silico and in vivo. Putative promoters in the intergenic regions upstream the kstD1, kstD2 and kstD3 genes were identified and probed in an apramycin-promoter-test vector, leading to the functional evidence of those R. ruber kstD promoters.ConclusionsAt least three putative 3-ketosteroid Δ1-dehydrogenase ORFs (kstD1, kstD2 and kstD3) have been identified and functionally confirmed in R. ruber strain Chol-4. KstD1 and KstD2 display a wide range of substrate preferences regarding to well-known intermediaries of the cholesterol degradation pathway (9OHAD and AD) and other steroid compounds. KstD3 shows a narrower substrate range with a preference for saturated substrates. KstDs differences in their catalytic properties was somehow related to structural differences revealed by a preliminary structural modelling. Transcription of R. ruber kstD genes is driven from specific promoters. The three genes are constitutively transcribed, although an additional induction is observed in kstD1 and kstD3. These enzymes have a wide versatility and allow a fine tuning-up of the KstD cellular activity.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-017-0657-1) contains supplementary material, which is available to authorized users.

show abstract

“…Among them, R. ruber DSM 43338, R. aetherivorans DSM 44752 and R. rhodochrous DSM 43269 can only grow on a single type of steroid hormone (testosterone), whereas R. ruber strain Chol‐4 (= DSM 45280) displayed a broad range of catabolic capacities for cholesterol, testosterone and progesterone (Fernández de las Heras et al , ). This strain and R. rhodochrous DSM 43269 have been used to identify degradation genes responsible for different steroid substrates in actinobacteria (Petrusma et al , , , ; Fernández de las Heras et al , ; Fernández de Las Heras et al , ; Guevara et al , ). On the other hand, information on progesterone degraders is relatively scant.…”

Section: Microorganisms Involved In the Degradation Of Steroid Sex Homentioning

confidence: 99%

Microbial degradation of steroid sex hormones: implications for environmental and ecological studies

Chiang

Wei

Wang

et al. 2019

Microbial Biotechnology

View full text Add to dashboard Cite

Steroid hormones modulate development, reproduction and communication in eukaryotes. The widespread occurrence and persistence of steroid hormones have attracted public attention due to their endocrine-disrupting effects on both wildlife and human beings. Bacteria are responsible for mineralizing steroids from the biosphere. Aerobic degradation of steroid hormones relies on O 2 as a co-substrate of oxygenases to activate and to cleave the recalcitrant steroidal core ring. To date, two oxygen-dependent degradation pathwaysthe 9,10-seco pathway for androgens and the 4,5-seco pathways for oestrogens have been characterized. Under anaerobic conditions, denitrifying bacteria adopt the 2,3-seco pathway to degrade different steroid structures. Recent meta-omics revealed that microorganisms able to degrade steroids are highly diverse and ubiquitous in different ecosystems. This review also summarizes culture-independent approaches using the characteristic metabolites and catabolic genes to monitor steroid biodegradation in various ecosystems.

show abstract

Draft Genome Sequence of the Steroid Degrader Rhodococcus ruber Strain Chol-4

Cited by 8 publications

References 8 publications

Functional characterization of 3-ketosteroid 9α-hydroxylases in Rhodococcus ruber strain chol-4

Functional characterization of 3-ketosteroid 9α-hydroxylases in Rhodococcus ruber strain chol-4

Functional differentiation of 3-ketosteroid Δ1-dehydrogenase isozymes in Rhodococcus ruber strain Chol-4

Microbial degradation of steroid sex hormones: implications for environmental and ecological studies

Contact Info

Product

Resources

About