Zhang et al. Microb Cell Fact (2018) 17:77 https://doi.org/10.1186/s12934-018-0916-9 Microbial Cell Factories RESEARCH Open Access Identifcation, function, and application of 3‑ketosteroid Δ1‑dehydrogenase isozymes in Mycobacterium neoaurum DSM 1381 for the production of steroidic synthons Ruijie Zhang1,2,3,4, Xiangcen Liu1, Yushi Wang1, Yuchang Han1, Junsong Sun1,2, Jiping Shi1,2* and Baoguo Zhang1* Abstract Background: 3-Ketosteroid-Δ1-dehydrogenase (KstD) is a key enzyme in the metabolic pathway for chemical modi- fcations of steroid hormones. Only a few KstDs have thus far been characterized biochemically and applied for the production of steroidal pharmaceutical intermediates. Three KstDs, KstD1, KstD2, and KstD3, were identifed in Myco- bacterium neoaurum DSM 1381, and they shared up to 99, 85 and 97% amino acid identity with previously reported KstDs, respectively. In this paper, KstDs from M. neoaurum DSM 1381 were investigated and exemplifed their potential application for industrial steroid transformation. Results: The recombinant KstD2 from Bacillus subtilis exhibited higher enzymatic activity when 4-androstene-3,17-di- one (AD) and 22-hydroxy-23, 24-bisnorchol-4-ene-3-one (4HP) were used as the substrates, and resulted in specifc 1 activities of 22.40 and 19.19 U mg− , respectively. However, the specifc activities of recombinant KstD2 from Escheri- chia coli, recombinant KstD1 from B. subtilis and E. coli, and recombinant KstD3, also fed with AD and 4HP, had sig- nifcantly lower specifc activities. We achieved up to 99% bioconversion rate of 1,4-androstadiene-3,17-dione (ADD) 1 from 8 g L− AD after 15 h of fermentation using E. coli transformant BL21-kstD2. And in vivo transcriptional analysis revealed that the expression of kstD1 in M. neoaurum DSM 1381 increased by 60.5-fold with phytosterols as the sub- strate, while the mRNA levels of kstD2 and kstD3 were bearly afected by the phytosterols. Therefore, we attempted to 1 1 create a 4HP producing strain without kstD1, which could covert 20 g L− phytosterols to 14.18 g L− 4HP. Conclusions: In vitro assay employing the recombinant enzymes revealed that KstD2 was the most promising candidate for biocatalysis in biotransformation of AD. However, in vivo analysis showed that the cellular regulation of kstD1 was much more active than those of the other kstDs in response to the presence of phytosterols. Based on the fndings above, we successfully constructed E. coli transformant BL21-kstD2 for ADD production from AD and M. neoaurum DSM 1381 ΔkstD1 strain for 4HP production using phytosterols as the substrate. Keywords: Mycobacterium, 3-Ketosteroid-∆1-dehydrogenase, 22-Hydroxy-23,24-bisnorchol-4-ene-3-one (4HP), 1,4-Androstadiene-3,17-dione (ADD) steroids Background Many actinobacteria, including Mycobacterium, Strep- tomyces, and Rhodococcus, can utilize natural sterols as carbon and energy sources [1–3], and interruption *Correspondence: [email protected]; [email protected] of those organisms’ unique catabolic pathways often 1 Lab of Biorefnery, Shanghai Advanced Research Institute, Chinese Academy of Sciences, No. 99 Haike Road, Pudong 201210, Shanghai, led to the accumulation of steroid hormone deriva- China tives [4, 5], some of which are important precursors, Full list of author information is available at the end of the article such as C19-steroids (4-androstene-3,17-dione [AD], © The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat​iveco​mmons​.org/licen​ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creat​iveco​mmons​.org/ publi​cdoma​in/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Zhang et al. Microb Cell Fact (2018) 17:77 Page 2 of 16 1,4-androstadiene-3,17-dione [ADD], and 9α-hydroxy- Bacillus subtilis, Pichia pastoris, and Corynebacterium 4-androsten-3,17-dione [9-OHAD]), for the production crenatum to accomplish biotransformation of sterols of steroidal drugs [6–8]. Phytosterols are found in plant [20–24]. For example, an E. coli transformant yielded seeds and can be utilized for the production of AD, ADD, 5.6 g L−1 ADD during fed-batch fermentation [23]. A B. and 9-OHAD using Mycobacterium sp. NRRL 3805B [9], subtilis, expressing codon-optimized kstD gene from M. Mycobacterium sp. NRRL 3683B [1], and Mycobacterium neoaurum JC-12, produced 8.76 g L−1 ADD by whole-cell neoaurum NwIB-yV [10], respectively. However, besides biocatalysis [24]. Prominently, a KstD overexpressing C. the low conversion rate [11], another major drawback of crenatum can transform 83.87% of the AD to ADD [22]. microbial transformation of phytosterols is the concomi- It has been reported that Mycobacterium neoaurum tant accumulation of byproducts due to the excessive DSM 1381 (Mycobacterium parafortuitum complex MCI enzymatic bioprocessing of the industrial strains [12]. 0617) was able to transform phytosterols to 4HP and Although actinobacteria strains were reported to be able HPD, and the molar ratio of HPD/4HP reached 16.61:1, to perform chemical modifcations on C22-steroids, such suggesting that M. neoaurum DSM 1381 owned KstDs as 22-hydroxy-23, 24-bisnorchola-4-en-3-one (4HP), with high catalytic activities [11]. In this study, transcrip- 22-hydroxy-23, 24-bisnorchola-1,4-dien-3-one (HPD) tional analysis and heterologous overexpression of KstDs and 9,22-dihydroxy-23,24-bisnorchol-4-ene-3-one were performed to determine the isoenzymes’ biochemi- (9-OHHP) [1, 12, 13] which are all valuable precursors cal roles in the biotransformation of phytosterols to HPD. for the synthesis of progestational and adrenocortical KstD2 can be used to construct recombinant strains hormones, more informations in detailed mechanisms that could efciently transform AD to ADD, and ΔkstD1 are urgently needed for the full industrial application. mutant of M. neoaurum DSM 1381 was found to synthe- Earlier research has, to some extent, clarifed the sterol size 4HP, all of which could bring a substantive impact on metabolic pathways in the actinobacteria based on the the current pharmaceutical industry. identifcation of intermediates [2]. Generally, 3-oxidation and the partial or full removal of aliphatic chains at C17 Methods of the sterols, a process similar to fatty acid β-oxidation, Bacterial strains, plasmids, and reagents are initial steps for the degradation of sterols, lead- Mycobacterium neoaurum DSM 1381 was purchased ing to the synthesis of 3-keto compounds such as 4HP from Deutsche Sammlung von Mikroorganismen und and AD (Fig. 1) [2, 14]. Enzymes, including cholesterol Zellkulturen (DSMZ), and the MP01 medium used to oxidase (CHO), 17β-hydroxysteroid dehydrogenase/β- maintain M. neoaurum DSM 1381 at 30 °C was (g L−1): hydroxyacyl-CoA dehydrogenase (Hsd4A), thiolase corn steep powder 10.0, glucose 6.0, K2HPO4 · 3H2O FadA5 (FadA5), and cytochrome P450 125 (CYP125), 2.0, MgSO4 · 7H2O 1.0, NaNO 3 2.0 and Tween 80 1.0 mL have been reported to be involved in that degradation of (v/v) (adjusted to pH 7.5). 5 g L−1 phytosterols were sterols [12, 15]. After the degradation, as shown in Fig. 1, added to MP01 medium to measure the steroid bio- 4HP and AD can then be converted to HPD and ADD, conversion performances of the M. neoaurum DSM respectively, by 3-ketosteroid-Δ1-dehydrogenase (KstD) 1381 and related transformants. For high steroid con- [10, 12]. And HPD or ADD enter downstream oxidative centration fermentation, phytosterols were prepared in process in cells after the 9α-hydroxylation, which is cata- (2-Hydroxypropyl)-β-cyclodextrin (HP-β-CD) (1:1.5). E. lysed by 3-ketosteroid-9α-hydroxylases (KSH) [16, 17]. coli DH5α, E. coli BL21 (DE3) and B. subtilis 6051a were KstD removes hydrogen atoms of the C-1 and C-2 in cultivated with Luria–Bertani medium (LB medium) at the A-ring of the polycyclic ring structure of 3-ketoster- 37 °C and 200 rpm for molecular cloning and heterolo- oids in substrates including AD, hydrocortisone acetate, gous expression of kstD genes. All the other strains and and 9-OHAD (Fig. 1) [12, 18, 19]. Although recent stud- plasmids were listed in Table 1. Oligonucleotides are ies also focused on the genetic removal of KstD from listed in Additional fle 1: Table S1. All the plasmids were native cells [10], the genetic manipulation in some strains constructed with ClonExpress® II One Step Cloning Kit might be challenging since host cells could contain mul- (Vazyme Biotech Co.Ltd. Nanjing, China). Restriction tiple kstDs with each gene playing a diferent role in engi- enzymes and other molecular biology reagents were pur- neering the bacteria and altering its metabolic pathway chased from Termo Fisher Scientifc. [10, 13]. For example, in M. neoaurum ATCC 25795, Phytosterols were purchased Jiangsu Yuehong KstD3 and KstD1 contributed to the conversion of AD Group Company (Jiangsu, China). AD and ADD were and 9-OHAD, respectively, whereas KstD2 was probably obtained from Sigma. 4HP was from Steraloids (New- involved in Δ1- dehydrogenation of C22 intermediates port, RI, USA). Phenazine methosulphate (PMS) [10, 12]. Besides, enormous attempts have been made to and 2,6-dichlorophenolindophenol (DCPIP) were express these KstDs heterologously in Escherichia coli, obtained from Sigma-Aldrich (Shanghai, China). Zhang et al. Microb Cell Fact (2018) 17:77 Page 3 of 16 Fig. 1 An overview of proposed pathway for phytosterols degradation in mycobacteria. Phytosterols can be transformed to vary valuable intermediates, including C19-steroids (4-androstene-3,17-dione [AD], 1,4-androstadiene-3,17-dione [ADD], and 9α-hydroxy-4-androsten-3,17-dione [9-OHAD]) and C22-steroids (22-hydroxy-23, 24-bisnorchola-4-en-3-one [4HP], 22-hydroxy-23, 24-bisnorchola-1,4-dien-3-one [HPD] and 9,22-dihyd roxy-23,24-bisnorchol-4-ene-3-one [9-OHHP]).