Deletion of the gene encoding the reductase component of 3-ketosteroid 9¿-hydroxylase in

Yeh, Chin-Hsing; Kuo, Yung-Shun; Chang, Che-Ming; Liu, Wen-Hsiung; Sheu, Meei-Ling; Meng, Menghsiao

doi:10.1186/preaccept-1874674672127933

Cited by 2 publications

(3 citation statements)

References 19 publications

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“…Recently it has been proposed that sterols can be catabolised in R. equi USA-18 by two partially different pathways, namely via AD or via Δ1,4-BNC, that converge in the intermediary 9OHADD [74]. Given its substrate preference, the fact that the growth in AD is independent of the KstD3 activity while the growth in cholesterol needs the presence of either KstD2 or KstD3 in R. ruber [24], lead us to suggest that KstD3 may be involved in an alternative AD-independent cholesterol catabolic pathway.…”

Section: Resultsmentioning

confidence: 99%

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

et al. 2017

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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.

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Section: Resultsmentioning

confidence: 99%

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

et al. 2017

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“…Chol-4 to avoid cholesterol mineralization (Fernández de las Heras et al, 2012 ). So far, only Yeh et al ( 2014 ) were able to design an ADD-producing strain of Rhodococcus equi by deleting only one kshB gene. Moreover, the equivalent mutants of the genus Mycobacterium and Rhodococcus accumulate different types and/or amounts of steroidal intermediates from sterols.…”

Section: Current Achievements Of Steroid Biotechnologymentioning

confidence: 99%

“…Moreover, the equivalent mutants of the genus Mycobacterium and Rhodococcus accumulate different types and/or amounts of steroidal intermediates from sterols. For instance, AD/ADD-producing mycobacterial strains accumulate small amounts of 4-HBC/1,4-HBC alcohols, whereas the respective mutants of Rhodococcus accumulate notable amounts of their acid derivatives 3-oxo-23,24-bisnorchol-4-en-22-oic acid (4-BNC) and 3-oxo-23,24-bisnorchol-1,4-dien-22-oic acid (1,4-BNC) (Marsheck et al, 1972 ; Wilbrink et al, 2011 ; Yeh et al, 2014 ; Xu et al, 2016 ; Galán et al, 2017b ). For example, Wilbrink et al ( 2011 ) constructed a mutant strain of R. rhodochrous DSM 43269 (strain RG32) devoid of 9α-hydroxylase activity, which accumulated mostly 1,4-BNC and only small amounts of ADD from sterols.…”

Section: Current Achievements Of Steroid Biotechnologymentioning

confidence: 99%

New Insights on Steroid Biotechnology

2018

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Nowadays steroid manufacturing occupies a prominent place in the pharmaceutical industry with an annual global market over $10 billion. The synthesis of steroidal active pharmaceutical ingredients (APIs) such as sex hormones (estrogens, androgens, and progestogens) and corticosteroids is currently performed by a combination of microbiological and chemical processes. Several mycobacterial strains capable of naturally metabolizing sterols (e.g., cholesterol, phytosterols) are used as biocatalysts to transform phytosterols into steroidal intermediates (synthons), which are subsequently used as key precursors to produce steroidal APIs in chemical processes. These synthons can also be modified by other microbial strains capable of introducing regio- and/or stereospecific modifications (functionalization) into steroidal molecules. Most of the industrial microbial strains currently available have been improved through traditional technologies based on physicochemical mutagenesis and selection processes. Surprisingly, Synthetic Biology and Systems Biology approaches have hardly been applied for this purpose. This review attempts to highlight the most relevant research on Steroid Biotechnology carried out in last decades, focusing specially on those works based on recombinant DNA technologies, as well as outlining trends and future perspectives. In addition, the need to construct new microbial cell factories (MCF) to design more robust and bio-sustainable bioprocesses with the ultimate aim of producing steroids à la carte is discussed.

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Deletion of the gene encoding the reductase component of 3-ketosteroid 9¿-hydroxylase in

Cited by 2 publications

References 19 publications

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

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

New Insights on Steroid Biotechnology

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