Shtratnikova et al. BMC Biotechnology (2021) 21:7 https://doi.org/10.1186/s12896-021-00668-9 RESEARCH ARTICLE Open Access Different genome-wide transcriptome responses of Nocardioides simplex VKM Ac- 2033D to phytosterol and cortisone 21- acetate Victoria Yu Shtratnikova1* , Mikhail I. Sсhelkunov2,3 , Victoria V. Fokina4,5 , Eugeny Y. Bragin4 , Andrey A. Shutov 4,5 and Marina V. Donova4,5 Abstract Background: Bacterial degradation/transformation of steroids is widely investigated to create biotechnologically relevant strains for industrial application. The strain of Nocardioides simplex VKM Ac-2033D is well known mainly for its superior 3-ketosteroid Δ1-dehydrogenase activity towards various 3-oxosteroids and other important reactions of sterol degradation. However, its biocatalytic capacities and the molecular fundamentals of its activity towards natural sterols and synthetic steroids were not fully understood. In this study, a comparative investigation of the genome-wide transcriptome profiling of the N. simplex VKM Ac-2033D grown on phytosterol, or in the presence of cortisone 21-acetate was performed with RNA-seq. Results: Although the gene patterns induced by phytosterol generally resemble the gene sets involved in phytosterol degradation pathways in mycolic acid rich actinobacteria such as Mycolicibacterium, Mycobacterium and Rhodococcus species, the differences in gene organization and previously unreported genes with high expression level were revealed. Transcription of the genes related to KstR- and KstR2-regulons was mainly enhanced in response to phytosterol, and the role in steroid catabolism is predicted for some dozens of the genes in N. simplex. New transcription factors binding motifs and new candidate transcription regulators of steroid catabolism were predicted in N. simplex. Unlike phytosterol, cortisone 21-acetate does not provide induction of the genes with predicted KstR and KstR2 sites. Superior 3-ketosteroid-Δ1-dehydrogenase activity of N. simplex VKM Ac-2033D is due to the kstDs redundancy in the genome, with the highest expression level of the gene KR76_27125 orthologous to kstD2, in response to cortisone 21-acetate. The substrate spectrum of N. simplex 3-ketosteroid-Δ1-dehydrogenase was expanded in this study with progesterone and its 17α-hydroxylated and 11α,17α-dihydroxylated derivatives, that effectively were 1(2)- dehydrogenated in vivo by the whole cells of the N. simplex VKM Ac-2033D. (Continued on next page) * Correspondence: [email protected] 1Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Leninskie gory, h. 1, b. 40, Moscow, Russian Federation 119991 Full list of author information is available at the end of the article © The Author(s). 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Shtratnikova et al. BMC Biotechnology (2021) 21:7 Page 2 of 20 (Continued from previous page) Conclusion: The results contribute to the knowledge of biocatalytic features and diversity of steroid modification capabilities of actinobacteria, defining targets for further bioengineering manipulations with the purpose of expansion of their biotechnological applications. Keywords: Nocardioides simplex, Arthrobacter simplex, Pimelobacter simplex, Biocatalysts, Transcriptome, Phytosterol, Cortisone acetate, Progesterone Key points pathogenicity of Mycobacterium tuberculosis, and estab- lished applications of the non-pathogenic species and Role in phytosterol catabolism is predicted for some their engineered derivatives in biotechnology for produc- dozens of N. simplex genes tion of high-value steroids for the pharmaceutical Four kstD genes are induced that provide superior industry. 1(2)-dehydrogenase activity of the strain Sterol catabolism is a complicated, multi-step process Gene kstD2 KR76_27125 is highly over-expressed in that included degradation of side chain, rings A/B and response to AcC rings C/D of steroid core oxidation (Fig. 1). This path- Metabolism of cortisone acetate is not regulated by way was reported to be controlled by two TetR-type KstR-repressors transcriptional repressors, KstR and KstR2 [1–4]. New candidate transcription regulators of steroid Cholesterol catabolism has been intensively studied in catabolism were predicted the so-called mycolic acid rich actinobacteria such as pathogenic species: Mycobacterium tuberculosis [5], Rho- Background dococcus strains [6, 7], as well as in the non-pathogenic Steroids represent a huge class of specific organic mole- species such as Mycolicibacterium smegmatis (basonym cules with many of them playing essential roles in all liv- Mycobacterium smegmatis)mc2155 [4], Gordonia choles- ing systems. Diverse bacteria from different ecological terolivorans [8]. The roles of certain enzymes, mecha- niches have been evolved to degrade steroids as sources nisms of their functioning and interactions with of carbon and energy, or detoxify exogenic steroids by substrates were investigated. Cholesterol oxidases their structural modifications. Among bacteria, actino- (ChOs) and/or 3β-hydroxysteroid dehydrogenases (3- bacteria excite a particular interest due to their compre- HsDs) were shown to be responsible for modification of hensive metabolic possibilities and high activity of the 3β-hydroxy-5-ene to 3-keto-4-ene- moiety that is con- enzymatic systems towards various steroids. sidered as a first reaction of sterol degradation [4, 9]. Currently, degradation of sterols (such as cholesterol, Cytochrome P450 monooxygenases encoded by cyp125, or phytosterols) by actinobacteria is in the focus of in- cyp124 and cyp142 account for sterol hydroxylation at tensive researches due to its exclusive role in C-26(27) [10, 11], and aldol lyases encoded by ltp3 and ChoD, KsdI HO O Cyp125, FadD17, FadD19,Ltp2, Ltp3, KstR Ltp4, ChsH1, ChsH2, ChsE2, ChsE3, ChsE4, ChsE5, EchA19, Hsd4A, Hsd4B O O O KstR2 KsdD HsaA, HsaB, O HsaC, HsaD, FadD3, IpdA, IpdB, O O HsaE, HsaF, IpdC, IpdF, FadA6, HsaG FadE30, FadE31, KshA, KshB KshA, KshB COOH O O FadE32, EchA20 KsdD SCoA SCoA OH O + OC OC O HO COOH Fig. 1 Putative scheme of phytosterol catabolism by N. simplex VKM Ac-2033D. “KstR” and “KstR2” in ovals mean regulons Shtratnikova et al. BMC Biotechnology (2021) 21:7 Page 3 of 20 ltp4 play role in the degradation of C24-branched chain capable of hydrolyzing steroid esters such as 21- sterols [12]. Acyl-CoA synthetase FadD19 catalyzes the acetylated steroids, reduction of C17 and C20 carbonyl formation of the cholestanoate CoA-thioester [13] that groups of androstanes and pregnanes, respectively [31– is further oxidized to C-17-ketosteroids by three succes- 33] and able to grow on cholesterol [27]. sive cycles of β-oxidation [4, 14]. 9α-Hydroxylase High KstD activity of N. simplex VKM Ac-2033D had (KshAB, consisting of KshA and KshB subunits) and 3- been earlier demonstrated towards cortisol [37] and its ketosteroid-Δ1-dehydrogenase (KstD) [15, 16] are well- 6-methylated derivative [38], androst-4-ene-3,17-dione known as key enzymes of steroid core degradation (AD) and its 6-aminomethylated derivatives [34], testos- whose cooperative action provides the ring B opening terone [34], androsta-4,9-diene-3,17-dione [39], 21- (Fig. 1). 3-Hydroxy-9,10-seconandrost-1,3,5(10)-triene-9, acetoxy-pregna-4(5),9(11),16(17)-triene-21-ol-3,20-dione 17-dione (3-HSA) formed is further degraded by the en- and pregna-4,9(11)-diene-17α,21-diol-3,20-dione ace- zymes encoded by hsaA, hsaB, hsaC, hsaD, hsaE, hsaF, tates [32, 33], 7α- and 7β-hydroxytestololactone [40], hsaG [17–19]. The degradation of the last two rings etc. (more information is given in the Supplementary (rings C/D) of the steroid core is catalyzed by the prod- Table S1). In this study, the substrate spectrum of ste- ucts of the genes of the so-called KstR2-regulon [20]. roids for bioconversion with N. simplex was expanded Unlike mycobacteria, rhodococci and other above- with progesterone (Pr) and its 17α-hydroxylated and mentioned species related to Corynebacteriales order, 11α,17α-dihydroxylated derivatives (17α-OH-Pr and much less is known on steroid catabolism in actinobac- 11α,17α-di-OH-Pr). teria belonging to Propionibacteriales order such as rep- The strain had originally been isolated from the Pamir resentatives of Nocardioides genus and related genera. highland soils [41] and identified firstly as Mycobacter- The capability of Arthrobacter simplex (syn. Nocar- ium sp. 193 [42], then re-classified as Mycobacterium dioides simplex) to degrade cholesterol has been re- globiforme 193 [43], and later as