Wang et al. Microb Cell Fact (2020) 19:187 https://doi.org/10.1186/s12934-020-01442-w Microbial Cell Factories RESEARCH Open Access Whole-genome and enzymatic analyses of an androstenedione-producing Mycobacterium strain with residual phytosterol-degrading pathways Hongwei Wang1, Shikui Song1, Fei Peng1, Fei Yang1, Tian Chen1, Xin Li1, Xiyao Cheng1,2, Yijun He3, Yongqi Huang1 and Zhengding Su1,2* Abstract Mycobacterium neoaurum strains can transform phytosterols to 4-androstene-3,17-dione (4-AD), a key intermediate for the synthesis of advanced steroidal medicines. In this work, we presented the complete genome sequence of the M. neoaurum strain HGMS2, which transforms β-sitosterol to 4-AD. Through genome annotation, a phytosterol-degrad- ing pathway in HGMS2 was predicted and further shown to form a 9,10-secosteroid intermediate by fve groups of enzymes. These fve groups of enzymes included three cholesterol oxidases (ChoM; group 1: ChoM1, ChoM2 and Hsd), two monooxygenases (Mon; group 2: Mon164 and Mon197), a set of enzymes for side-chain degradation (group 3), one 3-ketosteroid-1,2-dehydrogenase (KstD; group 4: KstD211) and three 3-ketosteroid-9a-hydroxylases (Ksh; group 5: KshA226, KshA395 and KshB122). A gene cluster encoding Mon164, KstD211, KshA226, KshB122 and fatty acid β-oxidoreductases constituted one integrated metabolic pathway, while genes encoding other key enzymes were sporadically distributed. All key enzymes except those from group 3 were prepared as recombinant proteins and their activities were evaluated, and the proteins exhibited distinct activities compared with enzymes identifed from other bacterial species. Importantly, we found that the KstD211 and KshA395 enzymes in the HGMS2 strain retained weak activities and caused the occurrence of two major impurities, i.e., 1,4-androstene-3,17-dione (ADD) and 9-hydroxyl- 4-androstene-3,17-dione (9OH-AD) during β-sitosterol fermentation. The concurrence of these two 4-AD analogs not only lowered 4-AD production yield but also hampered 4-AD purifcation. HGMS2 has the least number of genes encoding KstD and Ksh enzymes compared with current industrial strains. Therefore, HGMS2 could be a potent strain by which the 4-AD production yield could be enhanced by disabling the KstD211 and KshA395 enzymes. Our work also provides new insight into the engineering of the HGMS2 strain to produce ADD and 9OH-AD for industrial application. *Correspondence: [email protected] 1 Key Laboratory of Industrial Fermentation (Ministry of Education), Hubei Key Laboratory of Industrial Microbiology, National “111” Center for Cellular Regulation and Molecular Pharmaceutics, Hubei University of Technology, Wuhan 430068, China Full list of author information is available at the end of the article © The Author(s) 2020. 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. 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Microb Cell Fact (2020) 19:187 Page 2 of 19 Keywords: 1,4-Androstene-3,17-dione (ADD), 3-Hydroxy-9,10-secoandrost-1,3,5(10)-triene-9,17-dione (HSA), 3-Ketosteroid-1,2-dehydrogenase (KstD), 3-Ketosteroid-9α-hydroxylase (ksh), 4-androstene-3,17-dione (4-AD), 9-Hydroxyl-4-androstene-3,17-dione (9OH-AD), 21-Hydroxy-20-methylpregn-4-en-3-one (BA), Biotransformation, Cholesterol oxidases (cho), Monooxygenase (mon), Mycobacterium sp. strain Introduction Mycobacterium sp. VKM Ac-1817D, a strain producing Steroids, including phytosterols and cholesterol, are 9OH-AD and genetically close to NRRL B-3805, revealed ubiquitous in nature and signifcant growth substrates for that phytosterols stimulated the increased expression of microorganisms. Steroids are biologically active organic 260 genes, including those related to steroid catabolism compounds that function either as important compo- in mycobacteria [19]. Other bacteria contain either a sim- nents of cell membranes or as signaling molecules [1]. ilar steroid degradation pathway, e.g., in Pseudomonas sp. Typically, steroid medicinal compounds share a tetracy- NCIB 10590 [20], or distinct steroid metabolic gene clus- clic hydrocarbon ring or gonane structure with diferent ters, e.g., in Nocardioides simplex VKM Ac-2033D [4, 21]. functional groups attached to C17-site. Tree andros- Based on the Ref-Seq database, Mohn et al. recently iden- tane steroids, namely, 4-androstene-3,17-dione (4-AD), tifed 265 putative steroid-degrading bacteria that shared 1,4-androstadiene-3,17-dione (ADD) and 9α-hydroxy- 9,10-secosteroid degradation as a converged pathway [13, 4-androstenediol (9-OH-AD), are the most important 22]. Tis pathway was expanded to form cholesterol-, starting materials that are used to synthesize advanced cholate- and testosterone-degrading pathways through steroid medicines through chemical and/or enzymatic gene duplication, horizontal gene transfer and plasmid modifcation [2, 3]. Currently, one of the most attractive facilitation. However, our understanding of the set of strategies producing these materials is to employ bacte- genes for specifc androstane steroids that are involved in ria to transform phytosterols to androstane steroids [3, phytosterol biotransformation remains limited. 4]. Aerobic side chain degradation of phytosterols is the Genetic manipulation of the phytosterol-transformat- basis for the production of androstane steroids. Bacteria ing strains has been attempted to improve transforma- belonging to the genera Gordonia, Nocardia and Rhodo- tion yield [2]. Knockout of the kasB gene for the cell wall coccus are able to degrade the side chains of phytosterols synthesis of M. neoaurum ATCC 25795 strain enhanced [5–7]. To date, only strains in Mycobacterium genus have the bioconversion of phytosterols to 9OH-AD by 1.4- been found to have the potential to accumulate andros- fold [23]. Null mutation of 3-ketosteroid-9α-hydroxylase tane steroids for industrial applications [2, 3, 8–10]. (Ksh) genes in the M. neoaurum strains led to the sta- Many key enzymes in the steroid degradation pathways ble accumulation of androst-4-ene-3,17-dione (AD) and of pathogenic bacteria have been studied biochemically androst-1,4-diene-3,17-dione (ADD) [24–26]. Muta- [11, 12], although the pathways are not fully understood. tion in 4-AD-producing strain M. phlei M51 blocked Te importance of genomic analyses on steroid degrada- 9α-hydroxylation, strain transformed β-sitosterol to tion pathways in microorganism has recently been drawn 23,24-dinorcholane derivatives, which are useful starting much attention [13]. Many genome sequences of patho- materials for corticosteroid synthesis [27]. genic Mycobacterium tuberculosis strains are available Among the aforementioned three important andros- [14], providing information guidance for understanding tane steroids, 4-AD is one of the most important starting various steroid degradation pathways. However, genomic materials [28] in the synthesis of steroid medicines such data on nonpathogenic mycobacteria, especially andros- as abiraterone [29], testosterone [30], estradiol [31] and tane steroid-producing strains, are still limited so far. M. progesterone [32]. Microbial bioconversion of phytoster- smegmatis MC2 155 is a nonpathogenic model to study ols to 4-AD is still an industrial challenge. Although the drug resistance and shares signifcant genomic similar- complete steroid metabolic pathways of Mycobacterium ity with M. tuberculosis [15]. Mycobacterium neoau- strains are not fully understood, Mycobacterium neoau- rum NRRL B-3805 was the frst industrial strain used rum strains have been successfully utilized for the indus- for the biotransformation of β-sitosterol to 4-AD [16]. trial production of 4-AD and 9OH-AD using β-sitosterol Before its whole genome sequence became available [3, as substrate [2, 33, 34]. However, the transformation 17], biochemical analysis revealed that NRRL B-3805 yields of currently used industrial strains remain unsta- transformed β-sitosterol to 4-AD via eleven catabolic ble, as bioconversion fermentation is often accompa- enzymes acting in fourteen consecutive enzymatic steps nied by either complete degradation of the substrate or to degrade the branched hydrocarbon side-chain of phy- the generation of various metabolic impurities [3, 5]; tosterols [6, 18]. Te global transcriptome analysis of Wang et al. Microb Cell Fact (2020) 19:187 Page 3 of 19 therefore, further exploration of new 4-AD-producing stirred for 30 min. Te mixture was fltrated with auto- strains is required. claved Whatman Grade 4 flter paper (Particle reten- In this work, we attempted to isolate and character- tion > 25 μm, GE, USA). Te fltrate was mixed with 1% ize phytosterol-degrading strains from soils contami- β-sitosterol and 10% skim milk powder and lyophilized nated with vegetable oils. One of the isolates, namely, overnight to reduce the sample volume. Te lyophilized M. neoaurum HGMS2, enabled the accumulation of powder was resuspended in 20 mL of autoclaved Milli- 4-AD in vegetable oil-based media. Terefore, we were Q water. Te suspension was distributed in 0.2 mL ali- initially focused on elucidating the phytosterol deg- quots, and each aliquot was spread on a MOPS agar radation pathway of the strain by genome sequencing plate containing 1% β-sitosterol as the sole carbon.