Unravelling a new catabolic pathway of C‐19 steroids in ......intermediate AD occurs via a central catabolic route called 9,10-seco pathway that results in the generation of...

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Unravelling a new catabolic pathway of C-19 steroids in Mycobacterium smegmatis

Fernández-Cabezón, Lorena; Galán, Beatriz; García, José L.

Published in:Environmental Microbiology

Link to article, DOI:10.1111/1462-2920.14114

Publication date:2018

Document VersionPeer reviewed version

Link back to DTU Orbit

Citation (APA):Fernández-Cabezón, L., Galán, B., & García, J. L. (2018). Unravelling a new catabolic pathway of C-19 steroidsin Mycobacterium smegmatis. Environmental Microbiology, 20(5), 1815-1827. https://doi.org/10.1111/1462-2920.14114

https://doi.org/10.1111/1462-2920.14114

https://orbit.dtu.dk/en/publications/4dd93b79-d438-4272-9762-3de82b073cdc

https://doi.org/10.1111/1462-2920.14114

https://doi.org/10.1111/1462-2920.14114

Unravelling a new catabolic pathway of C-19 steroids in

Mycobacterium smegmatis

Lorena Fernández-Cabezón1†

, Beatriz Galán1 and José L. García

1*

1Department of Environmental Biology. Centro de Investigaciones Biológicas. Consejo

Superior de Investigaciones Científicas. Ramiro de Maeztu 9, 28040 Madrid, Spain.

*For correspondence: E-mail [email protected]; Tel. +34 918373112; Fax +34 915360432

†Present address: The Novo Nordisk Foundation Center for Biosustainability, Technical

University of Denmark, Kemitorvet 220, 2800 Lyngby, Denmark

This article is protected by copyright. All rights reserved.

This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an ‘Accepted Article’, doi: 10.1111/1462-2920.14114

2

Summary

In this work, we have characterized the C-19+ gene cluster (MSMEG_2851 to

MSMEG_2901) of Mycobacterium smegmatis. By in silico analysis we have identified the

genes encoding enzymes involved in the modification of the A/B steroid rings during the

catabolism of C-19 steroids in certain M. smegmatis mutants mapped in the PadR-like

regulator (MSMEG_2868), that constitutively express the C-19+ gene cluster. By using

gene complementation assays, resting-cell biotransformations and deletion mutants, we

have characterized the most critical genes of the cluster, i.e, kstD2, kstD3, kshA2, kshB2,

hsaA2, hsaC2 and hsaD2. These results have allowed us to propose a new catabolic route

named C-19+ pathway for the mineralization of C-19 steroids in M. smegmatis. Our data

suggest that the deletion of the C-19+ gene cluster may be useful to engineer more

robust and efficient M. smegmatis strains to produce C-19 steroids from sterols.

Moreover, the new KshA2, KshB2, KstD2 and KstD3 isoenzymes may be useful to

design new microbial cell factories for the 9α-hydroxylation and/or Δ1-dehydrogenation

of 3-ketosteroids.


3

Introduction

Steroids are naturally occurring hydrophobic molecules that present a structure core formed

by four fused alicyclic rings named gonane. Steroid compounds are frequently found in the

biosphere and can be used as carbon and energy sources by different bacteria (e.g.

Mycobacterium, Pseudomonas, Sterolibacterium, Sphingomonas, Novosphingobium,

Comamonas) (Fujii et al., 2002, 2003; Tarlera and Denner, 2003; Horinouchi et al., 2003a,

2004a; Philipp et al., 2006; van der Geize et al., 2007; Roh and Chu, 2010; Leu et al., 2011;

Merino et al., 2013). Most bacteria capable of degrading aerobically sterols (e.g., cholesterol

and phytosterols), which are one of the most abundant steroid compounds in nature, belong to

the phylum Actinobacteria (e. g., Mycobacterium, Rhodococcus, Gordonia) (van der Geize et

al., 2007; Kendall et al., 2007, 2010; Drzyzga et al., 2009; Uhía et al., 2012; Fernández de las

Heras et al., 2013; Li et al., 2014; Bergstrand et al., 2016). The bacterial catabolism of

cholesterol has been investigated in detail because of its relevance in the pathogenicity of

certain bacteria such as Mycobacterium tuberculosis or Rhodococcus equi. These bacteria are

able to metabolize cholesterol located in the membranes of the host cells during the course of

infection, being the degradation of this sterol crucial for bacterial persistence (van der Geize

et al., 2007, 2011; Pandey and Sassetti, 2008). The investigation of cholesterol catabolism has

also attracted increasing interest because low-cost natural sterols (e.g., phytosterols) are

currently biotransformed at an industrial scale into valuable pharmaceutical steroidal

intermediates such as the C-19 steroids 4-androstene-3,17-dione (AD), 1,4-androstadiene-

3,17-dione (ADD) or 9α-hydroxy-4-androstene-3,17-dione (9OH-AD) (García et al., 2012;

Donova, 2017).

The aerobic 9,10-seco degradation for cholesterol pathway has been postulated based

on the compilation of multiple genetic and biochemical investigations carried out on different

Gram-positive bacteria, but this pathway is also partially shared by some Gram-negative


4

bacteria that degrade bile acids and testosterone (Fig. S1) (Barrientos et al., 2015; García et

al., 2012; Galán et al., 2017a). Briefly, in Actinobacteria, the cholesterol is transported by a

specific ATP-dependent transport system named Mce4 (Casali and Riley 2007; Pandey and

Sassetti, 2008; Mohn et al., 2008; Klepp et al., 2012; García-Fernández et al., 2017a) and is

subsequently transformed into cholest-4-en-3-one (cholestenone) by 3β-hydroxysteroid

dehydrogenase/isomerases enzymes (3β-HSD) and/or cholesterol oxidases (ChOx) (Kreit,

2017). Concomitantly with the formation of cholestenone, at least two P450 cytochromes,

named CYP125 and CYP142, initiate the side-chain degradation by performing sequential

oxidations of the methyl group at C-26 to generate a carboxylic acid (Capyk et al., 2009a;

McLean et al., 2009; Rosłoniec et al., 2009; Driscoll et al., 2010; Johnston et al., 2010;

Ouellet et al., 2010; García-Fernández et al., 2013). This modification enables the β-oxidation

of the side-chain that yields two molecules of propionyl-CoA, an acetyl-CoA and the 17-

ketosteroid AD (Fig. S1). Thereafter, the degradation of the A/B steroid rings of the

intermediate AD occurs via a central catabolic route called 9,10-seco pathway that results in

the generation of 2-hydroxyhexa-2,4.dienoic acid (HHD) and 9,17-dioxo-1,2,3,4,10,19-

hexanorandrostan-5-oic acid (HIP) (Fig. S1) (García et al., 2012; Galán et al., 2017a). The

degradation of HDD probably occurs through the action of enzymes similar to the TesE, TesF

and TesG proteins described in C. testosteroni (Horinouchi et al., 2005), while the

degradation of HIP (i.e., the mineralization of C/D steroid rings) takes place in a lower

catabolic pathway, recently studied in detail in M. tuberculosis and other Actinobacteria,

which leads to the formation of metabolites that enter the central metabolic pathways (Crowe

et al., 2017). The expression of the genes responsible for the catabolism of C/D rings is

controlled by the KstR2 repressor, a TetR-like transcriptional regulator (Kendall et al., 2010;

Casabon et al., 2013; Crowe et al., 2015; García-Fernández et al., 2015), while the expression

of the genes encoding the enzymes involved in the side-chain and the A/B rings degradation


5

as well as those encoding the sterol uptake system, is regulated by another TetR-like

transcriptional regulator, the KstR repressor (Kendall et al., 2007; 2010; Uhía et al., 2011).

Conventionally, it has been proposed that the C-19 steroids AD, ADD and 9OH-AD

are key intermediates of the (chole)sterol catabolic pathway due to multiple lines of evidence

(Fig. S1). Firstly, the C-19 compounds have been found in the culture medium of several

naturally sterols-degrading bacteria (Marsheck et al., 1972; Wei et al., 2010). Recently,

various mutant strains producing C-19 steroids from sterols have also been constructed

through metabolic engineering approaches by redirecting the metabolic flux of sterol

catabolism to the accumulation of C-19 compounds by gene deletions (Wilbrink et al., 2011;

Yeh et al., 2014; Yao et al., 2014; Galán et al., 2017b). On the other hand, several examples

of bacteria capable of using both cholesterol and C-19 steroids as the only carbon and energy

source have been described (e.g., Rhodococcus erythropolis strain SQ1, Rhodococcus ruber

strain Chol-4, Rhodococcus rhodochrous DSM 43269, Gordonia neofelifaecis NRRL B-

59395 (van der Geize et al., 2000; Petrusma et al., 2011; Fernández de las Heras et al., 2012;

Li et al., 2014). However, these bacterial strains have other sets of steroid-degrading

enzymes, different from those participating in the cholesterol catabolism, that are specifically

induced by different C-19 steroids. Therefore, in these strains, the different steroid catabolic

pathways could be partially overlapped creating a complex network of metabolic

intermediates that are degraded by a battery of redundant enzymes that have possibly led to

some misinterpretations of the pathways for sterol catabolism.

In a previous manuscript, we proposed that the C-19 steroids AD, ADD and 9OH-AD

are not the natural intermediates of the (chole)sterol catabolic pathway based on several lines

of evidence (Fernández-Cabezón et al., 2017). Firstly, different authors showed that certain

modifications introduced into the A/B rings during the catabolism of cholesterol (e.g., 9α-

hydroxylation and Δ1-dehydrogenation) might occur simultaneously with the side-chain


6

degradation. The enzymes involved in these catabolic steps appear to use as preferred

substrates the acyl-CoA derivatives resulting from the β-oxidation of the side-chain (i.e., most

probably compounds that still retain 3 or more carbons of the sterol side-chain) (Capyk et al.,

2011; Penfield et al., 2014). Secondly, by inhibiting the B-ring opening during the

degradation of cholesterol, it is possible to detect in the culture medium, in addition to the C-

19 steroids, other steroid intermediates that still retain part of the side-chain like 3-oxo-23,24-

bisnorchol-1,4-dien-22-oic acid (1,4-BNC), 3-oxo-23,24-bisnorchol-4-en-22-oic acid (4-

BNC), 22-hydroxy-23,24-bisnorchol-1,4-dien-3-one (1,4-HBC), or 22-hydroxy-23,24-

bisnorchol-4-en-3-one (4-HBC) (Szentirmai, 1990; Donova et al., 2005;Wilbrink et al., 2011;

Yeh et al., 2014; Galán et al., 2017b). Thirdly, we demonstrated that the C-19 steroids AD,

ADD and 9OH-AD cannot be efficiently mineralized through the cholesterol pathway in M.

smegmatis mc2155, since neither the wild-type strain nor its unregulated mutant ΔkstR are

able to degrade efficiently these compounds (Fernández-Cabezón et al., 2017). All these

results reinforce the hypothesis that certain modifications introduced into the A/B rings of

cholesterol occur simultaneously with the side-chain degradation and therefore, the C-19

steroids AD, ADD or 9OH-AD are actually side products of the cholesterol catabolic route

(Fig. S1).

On the other hand, we also reported the existence of a new silent and tightly-regulated

catabolic pathway in M. smegmatis, that is not induced by sterols (e.g., cholesterol,

phystosterols) nor by C-19 steroids (e.g., AD, ADD, 9OH-AD), but which, after its

desilencing/activation, confers to this bacterium the capacity to mineralize these and other C-

19 steroids (Fernández-Cabezón et al., 2017). The activation of this catabolic pathway is

found in some M. smegmatis spontaneous mutants mapped in the PadR-like regulator

(MSMEG_2868) that acquire the ability to efficiently metabolize C-19 steroids. The PadR

inactivation allows the expression of a set of genes named C-19+ cluster (MSMEG_2851 to


7

MSMEG_2901) that encode putative steroid-degrading enzymes. The C-19+ cluster would

have evolved independently from the upper cholesterol kstR-regulon, since C-19+ cluster has

an independent regulation, but both C-19+ cluster and kstR-regulon would converge on the

lower cholesterol kstR2-regulon responsible for the metabolism of C/D steroid rings (Fig. S1).

However, this is only a working hypothesis that requires further confirmation. Other steroid-

degrading Actinobacteria have homologous C-19+ clusters (e.g., Mycobacterium neoaurum

ATCC 25795, Rhodococcus jostii RHA1, Gordonia neofelifaecis NRRL-B59395,

Nocardioides simplex VKM Ac-2033D), but it is interesting to note that it is absent in M.

tuberculosis and in certain industrial mycobacterial strains used to produce C-19 steroids

from sterols (e.g., Mycobacterium sp. NRRL B-3805) (Fernández-Cabezón et al., 2017).

In this work, we have characterized in more detail the C-19+ gene cluster in M.

smegmatis. Using different methodologies, we have investigated the biological function

and/or the essentiality of those genes of the C-19+ cluster that encode the enzymes involved

in the modification of A/B rings during the catabolism of C-19 steroids in this bacterium.

Results

Identification of the key genes involved in the degradation of C-19 steroids in M. smegmatis

Based on the knowledge of the 9,10-seco pathway for the bacterial catabolism of steroids

(García et al., 2012; Galán et al., 2017a), we have proposed a catabolic route named C-19+

pathway for the catabolism of C-19 steroids in M. smegmatis (Fig. 1A) (Fernández-Cabezón

et al., 2017). By in silico analysis, we identified in the C-19+ gene cluster homologous genes

for almost all the postulated enzymatic steps involved in the A/B steroid ring modification

(Fig. 1B). The members of the kstR regulon involved in the cholesterol catabolism in M.

smegmatis have been used as reference genes (Kendall et al., 2007; Uhía et al., 2012). As it is

shown in the Fig. 1B, despite the existence of homologous genes in the two steroid catabolic

gene clusters found in M. smegmatis they do not share any synteny.


8

The genes MSMEG_2867 (kstD2) and MSMEG_2869 (kstD3) encode putative 3-

ketosteroid-Δ1-dehydrogenases sharing a 36% and 44% protein identity to KstD1

(MSMEG_5941), respectively. The MSMEG_2893 (kshB2) gene encodes a putative reductase

component of a 3-ketosteroid-9α-hydroxylase showing 55% protein identity to KshB1

(MSMEG_6039), whereas the MSMEG_2870 (kshA2) gene would encode the oxygenase

component of the same enzyme since it has a 59% protein identity to KshA1

(MSMEG_5925). The MSMEG_2892 (hsaA2) and MSMEG_2891 (hsaC2) genes may encode

a 3-HSA monooxygenase (oxygenase component) and a 3,4-HSA dioxygenase, that share

between 40-50% protein identity to HsaA1 (MSMEG_6038) and HsaC1 (MSMEG_6036),

respectively. Finally, the MSMEG_2900 (hsaD2) gene encodes a putative 4-9-DSHA

hydrolase with 41% protein identity to HsaD1 (MSMEG_6037). There are not homologous

genes to the hsaB1 (MSMEG_6035), hsaE (MSMEG_5940), hsaF (MSMEG_5939), and hsaG

(MSMEG_5937) genes within the C19+ cluster.

Determination of the biological function of the kstD2, kstD3, kshA2 and kshB2 genes

To investigate the role of the MSMEG_2893 (kshB2) gene, the mutant M. smegmatis

MS6039 (ΔMSMEG_6039, ΔkshB1) was complemented in trans with an original copy of the

kshB2 gene. Galán et al. (2017b) previously demonstrated that the MS6039 mutant is unable

to efficiently grow in sterols as the sole carbon and energy source, unlike the wild-type strain

of M. smegmatis, since the mutant lacks the KshB1 reductase activity necessary to mineralize

cholesterol. Analogously, to study the biological function of the MSMEG_2867 (kstD3) and

MSMEG_2869 (kstD2) genes, the mutant M. smegmatis MS5941 (ΔMSMEG_5941, ΔkstD1)

was constructed and complemented in trans with an original copy of the kstD2 or kstD3 genes

(Table S2). The KstD1 activity required to mineralize cholesterol is eliminated in the MS5941

mutant, and thus, this mutant is unable to efficiently grow in sterols as the sole carbon and

energy (Fig. 2A). However, when the MS5941 mutant is cultured in minimal media


9

containing glycerol as carbon and energy source and cholesterol (or phytosterols) as

feedstock, a large accumulation of 9OH-AD is observed (data not shown). According to our

hypothesis, the expression of a kshB gene (e.g., kshB1 or kshB2) or a kstD gene (e.g., kstD1,

kstD2 or kstD3) in the MS6039 or MS5941 mutants, respectively, will restore its capability to

grow in cholesterol. For this purpose, the MSMEG_2867 (kstD3), MSMEG_2869 (kstD2) and

MSMEG_2893 (kshB2) genes were cloned into the pMV261 plasmid generating the

corresponding recombinant plasmids pMV2867 (harbouring kstD3), pMV2869 (harbouring

kstD2) and pMV2893 (harbouring kshB2). The recombinant plasmids pMV5941 (harbouring

kstD1) and pMV6039 (harbouring kshB1) were also constructed and used as controls for the

complementation experiments. The plasmids harboring the kshB genes were transformed in

the MS6039 mutant (ΔkshB1), whereas the plasmids harbouring the kstD genes were

transformed into the MS5941 mutant (ΔkstD1).

The overexpression of the kstD2 gene in the MS5941 strain restored its capability of

growing in cholesterol as the sole carbon and energy source, exhibiting a similar growth

pattern to the wild-type strain mc2155 and to the MS5941 (pMV5941) that is complemented

with the kstD1 gene (Fig. 2A). Otherwise, the overexpression of the kstD3 gene in the

MS5941 (pMV2867) recombinant did not restore the ability of MS5941 of growing in

cholesterol, suggesting that KstD3 present another enzymatic activity or a different substrate

specificity. To investigate this hypothesis, we monitored the bioconversion of several steroid

substrates by resting-cell biotransformation using the recombinant MS5941 (pMV2867). The

Figure 3A shows that this recombinant was able to transform AD into ADD, whereas the

control strain MS5941 (pMV261) harboring the empty plasmid was not able to, confirming

that KstD3 present 3-ketosteroid-Δ1-dehydrogenase activity. On the other hand, the

overexpression of the kshB2 gene in the MS6039 strain restored its capability of growing in

1.8 mM cholesterol as the sole carbon and energy source (Fig. 2B; Table S2)). The growth of


10

this recombinant strain MS6039 (pMV2893) was similar to the wild-type strain mc2155 and

to the MS6039 (pMV6039) that is complemented with the kshB1 gene. Finally, to investigate

the activity of MSMEG_2870 (kshA2) gene, we constructed the plasmid pMV2870

(harbouring kshA2) and followed the same resting cell approach. As expected, the

recombinant strain MS5941 (pMV2870) that produces constitutively the KshA2 protein, was

able to transform AD into 9OH-AD (Fig. 3B; Table S2).

These results confirmed that the kshA2 and kshB genes encode the oxygenase subunit

(KshA) and the ferredoxin reductase subunit (KshB) of a 3-ketosteroid-9α-hydroxylase

enzyme, while the kstD2 and kstD3 genes encode 3-ketoesteroid-Δ1-dehydrogenase proteins.

Determination of the biological function of the hsaA2, hsaC2 and hsaD2 genes

To investigate the biological role of the hsaA2 (MSMEG_2892), hsaC2 (MSMEG_2891) and

hsaD2 (MSMEG_2900) genes in the catabolism of C-19 steroids in M. smegmatis, we

constructed by double homologous recombination three mutant strains in M. smegmatis

ADD+ named MS2891 (ΔhsaC2), MS2892 (ΔhsaA2) and MS2900 (ΔhsaD2) (Table 1). The

three engineered mutants were unable to efficiently grow in C-19 steroids (e.g., 1.8 mM AD)

as the sole carbon and energy source when compared with the parental strain ADD+ (Fig.

4A), although they perfectly grew using glycerol as a carbon source (data not shown).

Interestingly, a color alteration of the C-19 steroid-containing medium was observed during

the culture of the MS2891 and MS2900 mutants (Fig. 4B). A pink coloration of the medium

was developed in the culture of the MS2891 mutant (ΔhsaC2), possibly indicating the

accumulation of a catecholic derivate (e.g., 3,4-HSA) and its subsequent non-enzymatic

oxidation to quinone derivatives. This phenotype was previously observed in ΔhsaC mutants

of the cholesterol catabolism in R. jostii RHA1 and M. tuberculosis H37Rv (van der Geize et

al., 2007; Yam et al., 2009), as well as in the ΔtesB mutant of C. testosteroni TA441 in the


11

presence of ADD or cholic acid (Horinouchi et al., 2004b). On the other hand, a yellowish-

brown coloration in the medium was observed in the culture of the MS2900 mutant (ΔhsaD2)

likely due to the accumulation of 4,9-HSA (Gibson et al., 1966). This characteristic color was

also described in the mutant ΔtesD of C. testosteroni TA441 in the presence of testosterone

(Horinouchi et al., 2003b).

To confirm the absence of polar effects, the three mutant strains were complemented

in trans with an original copy of the corresponding deleted gene (MSMEG_2891,

MSMEG_2892 or MSMEG_2900). For that, each gene was cloned into the pMV261 plasmid

generating the recombinant plasmids pMV2891, pMV2892 and pMV2900 and then, they

were transformed into the mutant strains MS2891, MS2892 and MS2900, respectively (Table

1). Fig. 5B shows that the complemented MS2891 and MS2900 mutants (MS2891c and

MS2900c, respectively) recovered the capacity to grow in C-19 steroids as the sole carbon

and energy source (e.g., 1.8 mM AD) (Table S2). In contrast, the complemented MS2892

strain (MS2892c) was not able to grow in 1.8 mM AD as the sole carbon and energy source,

although it showed a normal growth in other substrates (e.g., 18 mM glycerol) (Fig. 5A).

However, the same pink coloration in the AD-containing medium described above with the

MS2891 mutant was also observed in the culture broths of MS2892c strain (Fig. 5C). This

result supports the hypothesis that the MSMEG_2892 gene encodes a 3-HSA monooxygenase,

which under overexpression conditions, could generate high amounts of 3,4-HSA to inhibit

bacterial growth. The high toxicity of these compounds to the cells has been previously

described (Yam et al., 2009).

Our results confirmed that the hsaA2, hsaC2 and hsaD2 genes play an essential role in

the catabolism of C-19 steroids in M. smegmatis. In addition, considering that the engineered

mutants were unable to efficiently grow in C-19 steroids, we can conclude that the HsaA2,

HsaC2 and HsaD2 activities cannot be replaced in these conditions by other enzymatic


12

activities. Notice that their corresponding homologous proteins involved in cholesterol

catabolism are not expressed in the tested conditions since the cholesterol pathway is not

induced by C-19 compounds.

Discussion

In this work, we have characterized the C-19+ gene cluster recently identified in M.

smegmatis (Fernández-Cabezón et al., 2017). First, we have identified by in silico analysis

several genes encoding enzymes putatively involved in the modification of the A/B steroid

rings for the catabolism of C-19 steroids in this bacterium. Then, we have used different

genetic and biochemical approaches to determine the biological function of these genes that

has allowed us to postulate the C-19+ pathway in M. smegmatis (Fig. 1A).

According to the classical 9,10-seco pathway, the transformation of AD into ADD is

the first enzymatic step necessary for the opening of A/B rings for the aerobic catabolism of

steroids. This reaction is catalyzed by 3-ketosteroid-Δ1-dehydrogenases named KstD in M.

smegmatis (Brzostek et al., 2005), R. erythropolis (van der Geize et al., 2000, 2001, 2002)

and M. tuberculosis (Brzostek et al., 2009), but named TesH in C. testosteroni (Horinouchi et

al., 2003a). The 3-ketosteroid-Δ1-dehydrogenase are FAD-dependent proteins that catalyze

the trans-axial removal of the hydrogen atoms C-1(α) and C-2(β) of 3-ketosteroids (EC

1.33.99.4) (Knol et al., 2008). We have demonstrated that the KstD2 protein is able to replace

the biological role of KstD1 in the catabolism of cholesterol in M. smegmatis MS5941

(ΔMSMEG_5941, ΔkstD1) (at least, under the tested conditions of kstD2 gene

overexpression), even though KstD2 is not involved in the catabolism of sterols in this

bacterium (Fig. 2A). This result agrees with the previous findings of Brzostek et al. (2005). In

contrast, the overexpresssion of the kstD3 gene (MSMEG_2867) in the MS5941 mutant is not

able to restore this capability (Fig. 2A), although the KstD3 protein has 3-ketosteroid-Δ1-

dehydrogenase activity in resting-cell biotransformations (Fig. 3A). This result suggests that


13

KstD3 is not able to efficiently recognize the intermediates with side-chain that are possibly

the true substrates of KstD1 during the cholesterol catabolism (Fernández-Cabezón et al.,

2017).

Then, the 9α-hydroxylation step takes place by the action of the 3-ketosteroid-9α-

hydroxylases (Andor et al., 2006; van der Geize et al., 2007; Capyk et al., 2009b). These

enzymes are two-component monooxygenases consisting of a terminal oxygenase subunit

(KshA) and a ferredoxin reductase subunit (KshB) (E.C. 1.14.13.142) (Petrusma et al., 2014).

The KshA subunit specifically recognizes the steroid substrates, whereas the KshB

component transfers the reducing power required for the progression of the enzymatic

reaction. Petrusma et al. (2011) described the presence of 5 kshA genes involved in the

catabolism of different classes of steroids (e.g., cholesterol, cholate, AD and progesterone) in

R. rhodochrous DSM43269, each displaying unique steroid induction patterns and substrate

ranges. They demonstrated that the overexpression of only certain kshA genes in a null-kshA

mutant restores its growth on any of the different classes of steroids tested. The KshA/KshB

interaction does not seem to be very specific, since it is possible to find more kshA genes than

kshB genes in the genomes of actinobacterial species (e.g. R. jostii RHA1, N. simplex VKM

Ac-2033D or G. neofelifaecis NRRL B-59395) (Petrusma et al., 2011). This observation

suggests that a particular KshB protein can interact with several KshAs or even that, other

non-specific reductases present in the bacterial cell can act as donors of reducing power. In

agreement with this observation, we have demonstrated that the KshB2 of M. smegmatis is

able to replace KshB1 in the catabolism of cholesterol (Fig. 2B). In the same line, Yuan et al.

(2015) were able to reconstitute a functional KSH in Mycobacterium sp. NRRL B-3805 by

overexpressing the kshA gene of M. smegmatis and the kshB gene of G. neofelifaecis NRRL

B-59395. Moreover, we have observed that the co-expression of a kshB gene does not seem to


14

be always essential for the 9α-hydroxylations of 3-ketosteroids (Fig. 3B), as previously

demonstrated by other authors (Andor et al., 2005; Arnell et al., 2007).

The 3-HSA intermediate obtained after the action of the 3-ketosteroid-Δ1-

dehydrogenase and the 3-ketosteroid-9α-hydroxylase enzymes, is hydroxylated by a two-

component oxygenase (HsaA2B1), similar to TesA1A2 from C. testosteroni (Horinouchi et

al., 2004b) or to HsaAB from M. tuberculosis and R. jostii RHA1 (Dresen et al., 2010)

leading to the production of 3,4-DHSA. This catechol derivative is subsequently cleaved by a

meta-extradiol dioxygenase (HsaC2), similar to TesB in C. testosteroni (Horinouchi et al.,

2001) or to HsaC in R. jostii RHA1 (van der Geize et al., 2007) and M. tuberculosis H37Rv

(Yam et al., 2009), that yields 4,9-DSHA. This compound is subsequently hydrolyzed by a

meta-cleavage product (MCP) hydrolase (HsaD2) similar to TesD in C. testosteroni

(Horinouchi et al., 2003b) or to HsaD in M. tuberculosis and R. jostii RHA1 (van der Geize et

al., 2007; Lack et al., 2008, 2010) yielding HIP and HHD. To investigate the biological role

of the HsaA2, HsaC2 and HsaD2 proteins in the catabolism of C-19 steroids, in this work we

have constructed by double homologous recombination the mutants ADD+ MS2891

(ΔMSMEG_2891; ΔhsaC2), ADD+ MS2892 (ΔMSMEG_2892; ΔhsaA2) and ADD+ MS2900

(ΔMSMEG_2900; ΔhsaD2). The three mutants are unable to efficiently grow in C-19 steroids

as the sole carbon and energy source, confirming that the hsaA2, hsaC2 and hsaD2 genes are

essential for the catabolism of C-19 steroids in M. smegmatis (Fig. 4A). Moreover, the color

changes of culture media observed when the mutant strains are cultured in the presence of C-

19 compounds strongly support the C-19+ pathway postulated above.

Finally, the degradation of HDD should occur through the action of the enzymes

HsaE, HsaF and HsaG encoded by the unique hsaEFG operon of M. smegmatis (Fernández-

Cabezón et al., 2017). These genes are not always present in all steroid-degrading gene

clusters in Actinobacteria, because they encode enzymes that are common in many catabolic


15

pathways of aromatic compounds and thus, these enzymes can replace those (Bergstrand et

al., 2016).

Summarizing, the results presented above confirm that the C-19 steroids AD, ADD

and 9OH-AD are mineralized through the C-19+ catabolic pathway that is constitutively

expressed in the padR mutants of M. smegmatis. Although we have identified some of the key

enzymatic steps of the C-19+ pathway, additional studies are required to determine the roles

of other genes found in the C-19+ gene cluster of M. smegmatis (MSMEG_2851 to

MSMEG_2901), as well as to identify the chemical compound that induces this cluster in

vivo. A better understanding of the complexity of steroid catabolism in M. smegmatis and

other actinobacterial species can also be fundamental to optimize the current industrial

bioproduction of steroidal intermediates from sterols. The deletion of the C-19+ gene cluster

or at least of the most relevant genes located therein (e.g., kstD, kshA, kshB) will be useful to

increase the bioconversion yields in producer strains derived from M. smegmatis and other

cholesterol degrading bacteria. Yao et al. (2014) demonstrated that two residual enzymatic

activities KstD (KstD2 and KstD3) present in the mutant Mut(MN-KSTD1) (∆kstD1) of M.

neoaurum ATCC 25795, markedly decreases the conversion yield of 9OH-AD from sterols.

Interestingly, KstD2 and KstD3 are encoded by genes located in a homologous C-19+ cluster

that is not found in the close phylogenetic strains M. neoaurum VKM Ac-1815D and

Mycobacterium sp. NRRL B-3805, both used in industrial bioprocesses. On the other hand,

given the industrial relevance of the 9α-hydroxylation and Δ1-dehydrogenation reactions of 3-

ketosteroids, the new KshA2, KshB2, KstD2 and KstD3 enzymes identified could be used to

design new microbial cell factories for the production of steroids à la carte.


16

Experimental procedures

Chemicals

4-Androstene-3,17-dione (AD) and 1,4-androstadien-3,17-dione (ADD) were purchased from

TCI America. Cholesterol, Tween 80, tyloxapol, gentamicin and kanamycin were from Sigma

(Steinheim, Germany). Glycerol and glucose were purchased from Merck (Darmstardt,

Germany). 9α-Hydroxy-4-androstene-3,17-dione (9OH-AD) was kindly provided by Gadea

Biopharma (León, Spain).

Bacterial strains, plasmids and culture conditions

The plasmids and bacterial strains used in this work are listed in Table 1. M. smegmatis

strains were grown at 37°C in an orbital shaker at 200 r.p.m. Middlebrook 7H9 broth medium

(Difco) supplemented with 0.4% glycerol and 0.05% Tween 80 was used as rich medium.

7H9 broth without any supplement was used as minimal medium. 7H10 agar (Difco) plates

supplemented with 10% albumin-dextrose-catalase (Becton Dickinson) were used for solid

media. Kanamycin (20 µg ml-1

) or gentamicin (5 µg ml-1

), were used for strain selection when

appropriate. All the steroids were dissolved in 10% tyloxapol prior to its addition to the

minimal medium. Because of the low solubility of the steroid compounds, the stock solutions

were warmed at 80 ºC in agitation, sonicated in a bath for 1 h and then autoclaved.

Escherichia coli DH10B strain was used as a host for cloning. It was grown in LB

medium at 37°C in an orbital shaker at 200 r.p.m. LB agar plates were used for solid media.

Kanamycin (50 µg ml-1

) or Gentamicin (10 µg ml-1

), were used for plasmid selection and

maintenance.


17

DNA manipulations and sequencing

DNA manipulations and other molecular biology techniques were essentially as described by

Sambrook and Russell (2001). Oligonucleotides used at this work were purchased from

Sigma-Aldrich and are listed at Table S1. Genomic DNA purification from mycobacterial

strains was performed as previously described (Uhía et al., 2011). DNA amplification was

performed on a Mastercycler Gradient (Eppendorf) using Phusion High-Fidelity DNA

Polymerase from ThermoFisher Scientific. DNA fragments were purified with High Pure

PCR System Product Purification Kit (Roche). Restriction enzymes were obtained from

various suppliers and were used according to their specifications. Plasmid DNA was prepared

with a High Pure Plasmid Isolation Kit (Roche Applied Science). E. coli was transformed by

the rubidium chloride method (Wirth et al., 1989). M. smegmatis cells were transformed by

electroporation (Gene Pulser; Bio-Rad) (Parish and Stoker, 1998). All cloned inserts and

DNA fragments were confirmed by DNA sequencing through an ABI Prism 377 automated

DNA sequencer (Applied Biosystems Inc.) at Secugen S.L. (Madrid, Spain).

Gene deletion

The knock-out strains were constructed by double homologous recombination using the

plasmid pJQ200x, a derivative of the suicide vector pJQ200 that does not replicate in

Mycobacterium (Jackson et al., 2001). The followed approach consisted in amplifying by

PCR two fragments of ~700 bp containing the upstream region (UP fragment) and the

downstream region (DOWN fragment) of the target gene to be deleted (Table S1). The two

PCR fragments were purified, digested with the corresponding enzymes and ligated. Using

the ligation product as template, the UP+DOWN fragment was amplified by PCR using the

most external primers. The resulting amplicon was cloned into the plasmid pJQ200x using E.

coli DH10B competent cells. Then, the resulting pJQ200x-derivate plasmid was


18

electroporated into competent M. smegmatis ADD+ (Table 1). The single cross-overs were

selected on 7H10 agar plates containing gentamicin. The obtained single colonies were

contra-selected in 10% sucrose and in addition, the presence of the xylE gene marker was

confirmed by the appearance of yellow coloration after spreading catechol over each colony

replica. To select for double cross-overs, a single colony was grown in rich medium up to an

optical density of 0.8–0.9 and several dilutions of this culture were plated onto 7H10 agar

plates with 10% sucrose. Potential double cross-overs (i.e., sucrose-resistant colonies) were

subsequently screened for gentamicin sensitivity and the gene deletion was confirmed by

PCR.

Complementation of the knock-out mutants

To isolate the genes (e.g., MSMEG_2891) from M. smegmatis mc2155 for the

complementation experiments, its genomic DNA was extracted and amplified by PCR using

the corresponding primers forward and reverse (e.g., MSMEG_2891F and MSMEG_2891R)

(Table S1). The resulting amplicons were purified and digested with the corresponding

restriction enzymes to be cloned into the expression vector pMV261 (Stover et al., 1991), a

shuttle plasmid that replicates in E. coli and Mycobacterium. The resulting plasmids (e.g.,

pMV2891) were transformed into E. coli DH10B for cloning purposes and once their

sequences were verified, they were used to transform electrocompetent cells of the

corresponding M. smegmatis mutant strain (e.g., MS2891). The mutant strains harboring the

plasmid pMV261were used as controls.

Resting-cell biotransformations

The recombinant strains were grown in rich medium at 37 °C during 24 h. The cells were

harvested by centrifugation at 5000 x g for 20 min at 4 °C and washed once with 0.85% NaCl.


19

The biotransformation was carried out with an optical density (OD600) of 5 in a 100 mL shake

flask containing 40 mL of reaction mixture: 0.1 M phosphate buffer (pH 8.0), 2 mM AD

(substrate) and 0.05% Tween 80. An additional carbon source (1% glucose) was added in the

9alpha-hydroxylation biotransformations. The substrate AD was incorporated into the

medium as a solution with randomly methylated β-cyclodextrin (1:10.3, molar ratio) (Klein et

al., 1995) or alternatively, was dissolved in 10% tyloxapol as mentioned above. The steroid

bioconversion was monitored by thin layer chromatography (TLC) (García-Fernández et al.,

2017b).

Bioinformatic analysis

Sequence alignments were carried out using Clustal W (Thompson et al., 1994) and different

BLAST algorithms from the National Centre of Biotechnology Information Server (NCBI)

were also used.

Acknowledgements

The technical work of A. Valencia is greatly appreciated. This work was supported by grants

from the Ministry of Science and Innovation (BFU2006-15214-C03-01, BFU2009-11545-

C03-03) and Ministry of Economy and Competitiveness (BIO2012-39695-C02-01). LFC was

supported by an FPU fellowship from the Spanish Ministry of Education, Culture and Sports.

Conflict of interest

None declare


20

References

Andor, A., Jekkel, A., Hopwood, D.A, Jeanplong, F., Ilkoy, E., Kónya, A., Kurucz, I., and

Ambrus, G. (2006) Generation of useful insertionally blocked sterol degradation pathway

mutants of fast-growing mycobacteria and cloning, characterization, and expression of

the terminal oxygenase of the 3-ketosteroid 9alpha-hydroxylase in Mycobacterium

smegmatis mc(2)155. Appl Environ Microbiol 72: 6554-6559.

Arnell, R., Johannisson, R., Lindholm, J., Fornstedt, T., Ersson, B., Ballagi, A., et al. (2007).

Biotechnological approach to the synthesis of 9alpha-hydroxylated steroids. Prep.

Biochem Biotechnol 3: 309-321.

Barrientos, A., Merino, E., Casabon, I., Rodríguez, J., Crowe, A.M., Holert, J., et al. (2015)

Functional analyses of three acyl-CoA synthetases involved in bile acid degradation in

Pseudomonas putida DOC21. Environ Microbiol 17: 47-63.

Bergstrand, L.H., Cardenas, E., Holert, J., Van Hamme, J.D., and Mohn, W.W. (2016)

Delineation of Steroid-Degrading Microorganisms through Comparative Genomic

Analysis. MBio 7: e00166.

Brzostek, A., Sliwiński, T., Rumijowska-Galewicz, A., Korycka-Machała, M., and Dziadek,

J. (2005) Identification and targeted disruption of the gene encoding the main 3-

ketosteroid dehydrogenase in Mycobacterium smegmatis. Microbiology 151: 2393-2402.

Brzostek, A., Pawelczyk, J., Rumijowska-Galewicz, A., Dziadek, B., and Dziadek, J. (2009)

Mycobacterium tuberculosis is able to accumulate and utilize cholesterol. J Bacteriol 191:

6584-6591.

Capyk, J.K., Kalscheuer, R., Stewart, G.R., Liu, J., Kwon, H., Zhao, R., et al. (2009a)

Mycobacterial cytochrome P450 125 (Cyp125) catalyzes the terminal hydroxylation of

C27-steroids. J Biol Chem 284: 35534–35542.


21

Capyk, J.K., D'Angelo, I., Strynadka, N.C., and Eltis, L.D. (2009b) Characterization of 3-

ketosteroid 9α-hydroxylase, a Rieske oxygenase in the cholesterol degradation pathway

of Mycobacterium tuberculosis. J Biol Chem 284: 9937-9946.

Capyk, J.K., Casabon I., Gruninger R., Strynadka N.C., and Eltis L.D. (2011) Activity of 3-

Ketosteroid 9α-hydroxylase (KshAB) indicates cholesterol side chain and ring

degradation occur simultaneously in Mycobacterium tuberculosis. J Biol Chem 286:

40717–40724.

Casabon, I., Zhu, S.H., Otani, H., Liu, J., Mohn, W.W., and Eltis, L.D. (2013) Regulation of

the KstR2 regulon of Mycobacterium tuberculosis by a cholesterol catabolite. Mol

Microbiol 89: 1201-1212.

Casali, N., and Riley, L.W. (2007) A phylogenomic analysis of the Actinomycetales mce

operons. BMC Genomics 8: 60.

Crowe, A.M., Stogios, P.J., Casabon, I., Evdokimova, E., Savchenko, A., and Eltis, L.D.

(2015) Structural and functional characterization of a ketosteroid transcriptional regulator

of Mycobacterium tuberculosis. J Biol Chem 290: 872-882.

Crowe, A.M., Casabon, I., Brown, K.L., Liu, J., Lian, J., Rogalski, J.C., et al. (2017)

Catabolism of the Last Two Steroid Rings in Mycobacterium tuberculosis and Other

Bacteria. MBio 8 pii: e00321-17. doi: 10.1128/mBio.00321-17.

Donova, M.V. (2017). Steroid Bioconversions. Methods Mol Biol. 1645: 1-13.

Donova, M.V., Gulevskaya, S.A., Dovbnya, D.V., and Puntus,I.F. (2005) Mycobacterium sp .

mutant strain producing 9alpha-hydroxyandrostenedione from sitosterol. Appl Microbiol

Biotechnol 67: 671–678.


22

Dresen, C., Lin, L.Y., D'Angelo, I., Tocheva, E.I., Strynadka, N., and Eltis, L.D. (2010) A

flavin dependent monooxygenase from Mycobacterium tuberculosis involved in

cholesterol catabolism. J Biol Chem 285: 22264-22275.

Driscoll, M.D., McLean, K.J., Levy, C., Mast, N., Pikuleva, I.A., Lafite, P., et al. (2010)

Structural and biochemical characterization of Mycobacterium tuberculosis CYP142:

evidence for multiple cholesterol 27-hydroxylase activities in a human pathogen. J Biol

Chem 285: 38270-38282.

Drzyzga, O., Navarro Llorens, J.M., Fernández de Las Heras, L., García Fernández, E., and

Perera, J. (2009) Gordonia cholesterolivorans sp. nov., a cholesterol-degrading

actinomycete isolated from sewage sludge. Int J Syst Evol Microbiol 59: 1011-1015.

Fernández de las Heras, L., van der Geize, R., Drzyzga, O., Perera, J., and Navarro-Llorens,

M.J. (2012) Molecular characterization of three 3-ketosteroid-Δ(1)-dehydrogenase

isoenzymes of Rhodococcus ruber strain Chol-4. J Steroid Biochem Mol Biol 132: 271-

281.

Fernández de Las Heras, L., Alonso, S., de la Vega de León, A., Xavier, D., Perera, J., and

Navarro Llorens, J.M. (2013) Draft Genome Sequence of the Steroid Degrader

Rhodococcus ruber Strain Chol-4. Genome Announc 16. pii: e00215-13.

Fernández-Cabezón, L., García-Fernández, E., Galán, B., and García, J.L. (2017). Molecular

characterization of a new gene cluster for steroid degradation in Mycobacterium

smegmatis. Environ Microbiol 19: 2546-2563.

Fujii, K., Kikuchi, S., Satomi, M., Ushio-Sata, N., and Morita, N. (2002) Degradation of

17beta-estradiol by a gram-negative bacterium isolated from activated sludge in a sewage

treatment plant in Tokyo, Japan. Appl Environ Microbiol 68: 2057-2060.


23

Fujii, K., Satomi, M., Morita, N., Motomura, T., Tanaka, T., and Kikuchi, S. (2003)

Novosphingobium tardaugens sp. nov., an oestradiol-degrading bacterium isolated from

activated sludge of a sewage treatment plant in Tokyo. Int J Syst Evol Microbiol 53: 47-

52.

Galán, B., García-Fernández, J., Felpeto-Santero, C., Fernández-Cabezón, L., and García, J.L.

(2017a). Bacterial metabolism of Steroids. In Handbook of Hydrocarbon and Lipid

Microbiology Series. Aerobic Utilization of Hydrocarbons, Oils and Lipids. Fernando

Rojo (ed). doi: 10.1007/978-3-319-39782-5.

Galán, B., Uhía, I., García-Fernández, E., Martínez, I., Bahíllo, E., de la Fuente, J.L. et al.

(2017b). Mycobacterium smegmatis is a suitable cell factory for the production of

steroidic synthons. Microb Biotechnol 10: 138-150.

García, J.L., Uhía, I., and Galán, B. (2012) Catabolism and biotechnological applications of

cholesterol degrading bacteria. Microb Biotechnol 5: 679-699.

García-Fernández, E., Frank, D.J., Galán, B., Kells, P.M., Podust, L.M., García, J.L., and

Ortiz de Montellano, P.R. (2013) A highly conserved mycobacterial cholesterol catabolic

pathway. Environ Microbiol 15: 2342-2359.

García-Fernández, J., Galán, B., Medrano, F.J., and García, J.L. (2015) Characterization of

the KstR2 regulator responsible of the lower cholesterol degradative pathway in

Mycobacterium smegmatis. Environ Microbiol Rep 7: 155-163.

García-Fernández, J., Papavinasasundaram, K., Galán, B., Sassetti, C.M., and García, JL.

(2017a). Molecular and functional analysis of the mce4 operon in Mycobacterium

smegmatis. Environ Microbiol 19: 3689-3699.


24

García-Fernández, J., Martínez, I., Fernández-Cabezón, L., Felpeto-Santero, C., García, J.L.,

and Galán, B. (2017b). Bioconversion of Phytosterols into Androstadienedione by

Mycobacterium smegmatis CECT 8331. Methods Mol Biol 1645: 211-225.

Gibson, D.T.K.C., Wang, C.J., Sih, C.J., and Whitlock, H. Jr. (1966). Mechanisms of steroid

oxidation by microorganisms. IX. On the mechanism of ring A cleavage in the

degradation of 9,10-seco steroids by microorganisms. J Biol Chem 241: 551–559.

Horinouchi, M., Yamamoto, T., Taguchi, K., Arai, H., and Kudo, T. (2001) Metacleavage

enzyme gene tesB is necessary for testosterone degradation in Comamonas testosteroni

TA441. Microbiology 147: 3367-3375.

Horinouchi, M., Hayashi, T., Yamamoto, T., and T. Kudo. (2003a). A new bacterial steroid

degradation gene cluster in Comamonas testosteroni TA441 which consists of aromatic-

compound degradation genes for seco-steroids and 3-ketosteroid dehydrogenase genes.

Appl Environ Microbiol 69: 4421–4430.

Horinouchi, M., Hayashi, T., Koshino, H., Yamamoto, T., and Kudo, T. (2003b) Gene

encoding the hydrolase for the product of the meta-cleavage reaction in testosterone

degradation by Comamonas testosteroni. Appl Environ Microbiol 69: 2139-2152.

Horinouchi, M., Kurita, T., Yamamoto, T., Hatori, E., Hayashi, T., and Kudo, T. (2004a)

Steroid degradation gene cluster of Comamonas testosteroni consisting of 18 putative

genes from meta-cleavage enzyme gene tesB to regulator gene tesR. Biochem Biophys

Res Commun 324: 597-604.

Horinouchi, M., Hayashi, T., and Kudo, T. (2004b) The genes encoding the hydroxylase of 3-

hydroxy-9,10-secoandrosta-1,3,5(10)-triene-9,17-dione in steroid degradation in

Comamonas testosteroni TA441. J Steroid Biochem Mol Biol 92: 143-154.


25

Horinouchi, M., Hayashi, T., Koshino, H., Kurita, T., and Kudo, T. (2005) Identification of

9,17-dioxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid, 4-hydroxy-2-oxohexanoic acid,

and 2-hydroxyhexa-2,4-dienoic acid and related enzymes involved in testosterone

degradation in Comamonas testosteroni TA441. Appl Environ Microbiol 71: 5275-5281.

Jackson, M., Reinaldo Camacho, L., Gicquel, B., and Guilhot, C. (2001) Gene replacement

and transposon delivery using the negative selection marker sacB. Methods Mol Med 54:

59-75.

Johnston, J.B., Ouellet, H., and Ortiz de Montellano, P.R. (2010) Functional redundancy of

steroid C26-monooxygenase activity in Mycobacterium tuberculosis revealed by

biochemical and genetic analyses. J Biol Chem 285: 36352-36360.

Kendall, S.L., Withers, M., Soffair, C.N., Moreland, N.J., Gurcha, S., Sidders, B., et al.

(2007) A highly conserved transcriptional repressor controls a large regulon involved in

lipid degradation in Mycobacterium smegmatis and Mycobacterium tuberculosis. Mol

Microbiol 65: 684–699.

Kendall, S.L., Burgess, P., Balhana, R., Withers, M., Ten Bokum, A., Lott, J.S., et al. (2010)

Cholesterol utilization in mycobacteria is controlled by two TetR-type transcriptional

regulators: kstR and kstR2. Microbiology 156: 1362–1371.

Klein, U., Gimpl, G., and Fahrenholz, F. (1995) Alteration of the myometrial plasma

membrane cholesterol content with b-cyclodextrin modulates the binding affinity of the

oxytocin receptor. Biochemistry 34: 13784–13793.

Klepp, L.I., Forrellad, M.A., Osella, A.V., Blanco, F.C., Stella, E.J., Bianco, M.V., et al.

(2012) Impact of the deletion of the six mce operons in Mycobacterium smegmatis.

Microbes Infect 14: 590-599.


26

Knol, J., Bodewits, K., Hessels, G.I., Dijkhuizen, L., and van der Geize, R. (2008) 3-Keto-

5alpha-steroid Delta(1)-dehydrogenase from Rhodococcus erythropolis SQ1 and its

orthologue in Mycobacterium tuberculosis H37Rv are highly specific enzymes that

function in cholesterol catabolism. Biochem J 410: 339-346.

Kreit, J. (2017) Microbial catabolism of sterols: focus on the enzymes that transform the

sterol 3β-hydroxy-5-en into 3-keto-4-en. FEMS Microbiol Lett 364. doi:

10.1093/femsle/fnx007.

Lack, N., Lowe, E.D., Liu, J., Eltis, L.D., Noble, M.E., Sim, E., and Westwood, I.M. (2008)

Structure of HsaD, a steroid-degrading hydrolase, from Mycobacterium tuberculosis.

Acta Crystallogr Sect F Struct Biol Cryst Commun 64: 2-7.

Lack, N.A., Yam, K.C., Lowe, E.D., Horsman, G.P., Owen, R.L., Sim, E., and Eltis, L.D.

(2010) Characterization of a carbon-carbon hydrolase from Mycobacterium tuberculosis

involved in cholesterol metabolism. J Biol Chem 285: 434-443.

Leu, Y.L., Wang, P.H., Shiao, M.S., Ismail, W., and Chiang, Y.R. (2011) A novel

testosterone catabolic pathway in bacteria. J Bacteriol 193: 4447-4455.

Li, W., Ge, F., Zhang, Q., Ren, Y., Yuan, J., He, J., et al. (2014). Identification of gene

expression profiles in the actinomycete Gordonia neofelifaecis grown with different

steroids. Genome 57: 345-353.

Marsheck, W.J., Kraychy, S., and Muir, R.D. (1972) Microbial degradation of sterols. Appl

Microbiol 23: 72-77.

McLean, K.J., Lafite, P., Levy, C., Cheesman, M.R., Mast, N., Pikuleva IA, et al. (2009) The

Structure of Mycobacterium tuberculosis CYP125: molecular basis for cholesterol

binding in a P450 needed for host infection. J Biol Chem 284: 35524-35533.


27

Merino, E., Barrientos, A., Rodríguez, J., Naharro, G., Luengo, J.M., and Olivera, E.R. (2013)

Isolation of cholesterol- and deoxycholate-degrading bacteria from soil samples:

evidence of a common pathway. Appl Microbiol Biotechnol 97: 891-904.

Mohn, W.W., van der Geize, R., Stewart, G.R., Okamoto, S., Liu, J., Dijkhuizen, L., and

Eltis, L.D. (2008) The actinobacterial mce4 locus encodes a steroid transporter. J Biol

Chem 283: 35368–35374.

Ouellet, H., Guan, S., Johnston, J.B., Chow, E.D., Kells, P.M., Burlingame, A.L., et al. (2010)

Mycobacterium tuberculosis CYP125A1, a steroid C27 monooxygenase that detoxifies

intracellularly generated cholest-4-en-3-one. Mol Microbiol 77: 730–742.

Pandey, A.K., and Sassetti, C.M. (2008) Mycobacterial persistence requires the utilization of

host cholesterol. Proc Natl Acad Sci USA 105: 4376–4380.

Parish, T., and Stoker, N.G. (1998) Electroporation of mycobacteria. Methods Mol Biol 101:

129-144.

Penfield, J.S., Worrall, L.J., Strynadka, N.C., and Eltis, L.D. (2014) Substrate specificities

and conformational flexibility of 3-ketosteroid 9α-hydroxylases. J Biol Chem 289:

25523-25536.

Petrusma, M., Hessels, G., Dijkhuizen, L., and van der Geize, R. (2011) Multiplicity of 3-

Ketosteroid-9α-Hydroxylase enzymes in Rhodococcus rhodochrous DSM 43269 for

specific degradation of different classes of steroids. J Bacteriol 193: 3931-3940.

Petrusma, M., van der Geize, R., and Dijkhuizen, L. (2014) 3-Ketosteroid 9α-hydroxylase

enzymes: Rieske non-heme monooxygenases essential for bacterial steroid degradation.

Antonie Van Leeuwenhoek 106: 157-172.

Philipp, B., Erdbrink, H., Suter, M.J., and Schink, B. (2006) Degradation of and sensitivity to

cholate in Pseudomonas sp. strain Chol1. Arch Microbiol 185: 192-201.


28

Roh, H., and Chu, K. H. (2010) A 17beta-estradiol-utilizing bacterium, Sphingomonas strain

KC8: part I- characterization and abundance in wastewater treatment plants. Environ Sci

Technol 44: 4943-4950.

Rosłoniec, K.Z., Wilbrink, M.H., Capyk, J.K., Mohn, W.W., Ostendorf, M., van der Geize,

R., et al. (2009) Cytochrome P450 125 (CYP125) catalyzes C26-hydroxylation to initiate

sterol side chain degradation in Rhodococcus jostii RHA1. Mol Microbiol 74: 1031–

1043.

Sambrook, J., and Russell, D.W. (2001) Molecular Cloning: A Laboratory Manual, 3rd edn.

New York, NY, USA: Cold Spring Harbor Laboratory Press.

Snapper, S.B., Melton, R.E., Mustafa, S., Klesser, T., and Jacobs, W.R. Jr. (1990) Isolation

and characterizaton of efficient plasmid transformation mutants of Mycobacterium

smegmatis. Mol Microbiol 4: 1911-1919.

Stover, C.K., de la Cruz, V.F., Fuerst, T.R., Burlein, J.E., Benson, L.A., Bennett, L.T., et al.

(1991) New use of BCG for recombinant vaccines. Nature 351: 456-460.

Szentirmai, A. (1990) Microbial physiology of sidechain degradation of sterols. J Ind

Microbiol 6: 101-115.

Tarlera, S., and Denner, E.B. (2003) Sterolibacterium denitrificans gen. nov., sp. nov., a

novel cholesterol oxidizing, denitrifying member of the beta-Proteobacteria. Int J Syst

Evol Microbiol 53: 1085-1091.

Thompson, J.D., Higgins, D.G., and Gibson, T.J. (1994) CLUSTAL W: improving the

sensitivity of progressive multiple sequence alignment through sequence weighting,

position-specific gap penalties and weight matrix choice. Nucl Acids Res 22: 4673–4680.

Uhía, I., Galán, B., Morales, V., and García, J.L. (2011) Initial step in the catabolism of

cholesterol by Mycobacterium smegmatis mc2155. Environ Microbiol 13: 943–59.


29

Uhía, I., Galán, B., Kendall, S.L., Stoker, N.G., and García, J.L. (2012) Cholesterol

metabolism in Mycobacterium smegmatis. Environ Microbiol Rep 4: 168-182.

van der Geize, R., Hessels, G.I., van Gerwen, R., Vrijbloed, J.W., van Der Meijden, P., and

Dijkhuizen, L. (2000) Targeted disruption of the kstD gene encoding a 3-kestosteroid

delta(1)-dehydrogenase isoenzyme of Rhodococcus erythropolis strain SQ1. Appl

Environ Microbiol 66: 2029-2036.

van der Geize, R., Hessels, G.I., van Gerwen, R., van der Meijden, P., and Dijkhuizen, L.

(2001) Unmarked gene deletion mutagenesis of kstD, encoding 3-ketosteroid Delta1-

dehydrogenase, in Rhodococcus erythropolis SQ1 using sacB as counter-selectable

marker. FEMS Microbiol Lett 205: 197-202.

van der Geize, R., Hessels, G.I., and Dijkhuizen L. (2002) Molecular and functional

characterization of the kstD2 gene of Rhodococcus erythropolis SQ1 encoding a second

3-ketosteroid Δ1- dehydrogenase isoenzyme. Microbiology 148: 3285-3592.

van der Geize, R., Yam, K., Heuser, T., Wilbrink, M.H., Hara, H., Anderton, M.C., et al.

(2007) A gene cluster encoding cholesterol catabolism in a soil actinomycete provides

insight into Mycobacterium tuberculosis survival in macrophages. Proc Natl Acad Sci

USA 104: 1947-1952.

van der Geize, R., Grommen, A.W., Hessels, G.I., Jacobs, A.A., and Dijkhuizen, L. (2011)

The steroid catabolic pathway of the intracellular pathogen Rhodococcus equi is

important for pathogenesis and a target for vaccine development. PLoS Pathog 7:

e1002181.

Wei, W., Fan, S., Wang, F., and Wei, D. (2010) A new steroid-transforming strain of

Mycobacterium neoaurum and cloning of 3-ketosteroid 9alpha-hydroxylase in NwIB-01.

Appl Biochem Biotechnol 162: 1446-1456.


30

Wilbrink, M.H., Petrusma, M., Dijkhuizen, L., and van derGeize, R. (2011) FadD19 of

Rhodococcus rhodochrous DSM43269, a steroid-coenzyme A ligase essential for deg-

radation of C-24 branched sterol side chains. Appl EnvironMicrobiol 77: 4455–4464.

Wirth, R., Friesenegger, A., and Fieldler, S. (1989) Transformation of various species of

gram-negative bacteria belonging to 11 different genera by electroporation. Mol Gen

Genet 216: 175–177.

Yam, K.C, D'Angelo, I., Kalscheuer, R., Zhu, H., Wang, J.X., Snieckus, V., et al. (2009)

Studies of a ring-cleaving dioxygenase illuminate the role of cholesterol metabolism in the

pathogenesis of Mycobacterium tuberculosis. PLoS Pathog. 5: e1000344.

Yao, K., Xu, L.Q., Wang, F.Q., and Wei, D.Z. (2014) Characterization and engineering of 3-

ketosteroid-△1-dehydrogenase and 3-ketosteroid-9α-hydroxylase in Mycobacterium

neoaurum ATCC 25795 to produce 9α-hydroxy-4-androstene-3,17-dione through the

catabolism of sterols. Metab Eng 24: 181-191.

Yeh, C.H., Kuo, Y.S., Chang, C.M., Liu, W.H., Sheu, M.L., and Meng, M. (2014) Deletion of

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

Rhodococcus equi USA-18 disrupts sterol catabolism, leading to the accumulation of 3-

oxo-23,24-bisnorchola-1,4-dien-22-oic acid and 1,4-androstadiene-3,17-dione. Microb

Cell Fact 13: 130.

Yuan, J., Chen, G., Cheng, S., Ge, F., Qiong, W., Li, W., and Li, J. (2015) Accumulation of

9α-hydroxy-4-androstene-3,17-dione by co-expressing kshA and kshB encoding

component of 3-ketosteroid-9α-hydroxylase in Mycobacterium sp. NRRL B-3805. Sheng

Wu Gong Cheng Xue Bao 31: 523-533 (article in Chinese).


31

TABLES

Table 1. Bacterial strains and plasmids used in this study.

Strain Genotype and/or description Source or reference

Mycobacterium

smegmatis

mc2155 ept-1, mc26 mutant efficient for

electroporation Snapper et al. (1990)

MS6039 mc2155 ΔMSMEG_6039 (ΔkshB1); high-ADD

producer Galán et al. (2017)

ADD+

mc2155 mutant mapped in the PadR-like

regulator (MSMEG_2868) that can efficiently

metabolize all C-19 steroids (i.e., AD, ADD,

9OH-AD)

Fernández-Cabezón et

al. (2017)

MS5941 mc2155 ΔMSMEG_5941 (ΔkstD1); high-9OH-

AD producer This study

MS2891

ADD+ ΔMSMEG_2891 (ΔhsaC2) This study

MS2892 ADD+ ΔMSMEG_2892 (ΔhsaA2) This study

MS2900 ADD+ ΔMSMEG_2900 (ΔhsaD2) This study

mc2155 (pMV261) mc2155 strain harbouring plasmid pMV261 This study

MS2891 (pMV261) MS2891 strain harbouring plasmid pMV261 This study





32








MS6039 (pMV261) MS6039 strain harbouring plasmid pMV261 Galán et al. (2017)


MS6039 (pMV6039) MS6039 strain harbouring plasmid pMV6039 Galán et al. (2017)

Escherichia coli

DH10B

F-, mcrA, Δ (mrrhsdRMS-mcrBC),

Φ80dlacZΔM15, ΔlacX74, deoR, recA1,

araD139, Δ(ara-leu)7697, galU, galK, λ-,

rpsL, endA1, nupG

Invitrogen

Plasmids

pMV261

Mycobacterium/E. coli shuttle vector with the

kanamycin resistance aph gene from

transposon Tn903 and the promoter from the

hsp60 gene from M. tuberculosis

Stover et al. (1991)

pMV2867 pMV261 harbouring the MSMEG_2867 gene

encoding KstD3 This study




33


encoding KshA2 This study


encoding HsaC2 This study


encoding HsaA2 This study


encoding KshB2 This study


encoding HsaD2 This study




encoding KshB1 Galán et al. (2017)

pJQ200x

Suicide vector used to perform allelic

exchange mutagenesis in mycobacteria, P15A

ori, sacB, GmR, xylE

Jackson et al. (2001)

pJQ2891

pJQ200x containing one fragment upstream

and another fragment downstream of

MSMEG_2891 gene

This study

pJQ2892



MSMEG_2892 gene

This study

pJQ2900



MSMEG_2900 gene

This study


34

pJQ5941



MSMEG_5941 gene

Galán et al. (2017)


35

FIGURE LEGENDS

Fig. 1. Catabolic pathway of C-19 steroids in M. smegmatis mc2155. A) Scheme of the C-19+

route proposed for the degradation of C-19 steroids (i.e., AD, ADD and 9OH-AD) in this

bacterium. The upper catabolic pathway consisting of the degradation of A/B rings of C-19

steroids is carried out by enzymes encoded by the C-19+ gene cluster. The resulting metabolic

intermediates (HHD and HIP) converge with the cholesterol catabolic pathway where the

degradation of C/D steroid rings occurs. The upper catabolic pathway is transcriptionally

regulated by the PadR repressor (padR regulon), while the lower catabolic pathway is

controlled by the KstR2 regulator (kstR2 regulon). B) Comparative genomic analysis of genes

encoding A/B steroid ring modification enzymes in the catabolism of cholesterol or C-19

steroids in M. smegmatis. Black numbers indicate the relative position of these gene clusters

in the genome of M. smegmatis (NC_008596.1). Homologous genes are shown with the same

color. Gene annotation: MSMEG_(number). Protein identities are indicated in parentheses.

Abbreviations of enzymes: 3-ketosteroid-Δ1-dehydrogenase (KstD); 3-ketosteroid-9α-

hydroxylase (KshAB); 3-HSA monooxygenase (HsaAB); 3,4-HSA dioxygenase (HsaC); (4,9-

DSHA) hydroxylase (HsaD); 2-hydroxy-2,4-hexadienoic acid hydratase (HsaE); 4-hydroxy-2-

hydroxy-2-ketovalerate aldolase (HsaF); acetaldehyde dehydrogenase (HsaG). Abbreviations

of metabolites: 4-androstene-3,17-dione (AD); 1,4-androstadiene-3,17-dione (ADD); 9α-

hydroxy-4-androstene-3,17-dione (9OH-AD); 9α-hydroxy-1,4-androstadiene-3,17-dione

(9OH-ADD); 3-hydroxy-9,10-secoandrosta-1,3,5(10)-trien-9,17-dione (3-HSA); 3,4-

dihydroxy-9,10-secoandrosta-1,3,5(10)-trien-9,17-dione (3,4-HSA); 4,5,9,10-diseco-3-

hydroxy-5,9,7-trioxoandrosta-1(10),2-diene-4-oic acid (4,9-DSHA); 2-hydroxy-2,4-

hexadienoic acid (HHD); 3aα-H-4α(3´-propionic acid)-7aβ-methylhexahydro-1,5-indanedione

(HIP).


36

Fig. 2. Gene complementation assays of MS5941 and MS6039 mutant strains in shake flasks

with minimal medium containing 1.8 mM cholesterol as the only carbon and energy source.

(A) Gene complementation of MS5941 (ΔMSMEG_5941, ΔkstD1) mutant: mc2155

(pMV261) [wt, black]; MS5941 (pMV261) [MS5941, red]; MS5941 (pMV5941) [KstD1,

blue]; MS5941 (pMV2869) [KstD2, green]; and MS5941 (pMV2867) [KstD3, grey]. (B)

Gene complementation of the MS6039 (ΔMSMEG_6039, ΔkshB1) mutant: mc2155 (pMV261)

[wt, black]; MS6039 (pMV261) [MS6039, red]; MS6039 (pMV6039) [KshB1, blue]; and

MS6039 (pMV2893) [KshB2, green].

Fig. 3. Determination of the biological function of enzymes KstD and KshA by resting-cell

biotransformations. The bioconversion of 1.8 mM AD into ADD (A) or 9-OHAD (B) by

recombinant strains of M. smegmatis was monitored by thin layer chromatography (TLC).

Nomenclature of M. smegmatis strains used: MS5941 (pMV261) [Control]; MS5941

(pMV2867) [KstD3]; and MS5941 (pMV2870) [KshA2].

Fig. 4. Growth curves of mutant strains of M. smegmatis ADD+ on C-19 steroids. (A) The

strains ADD+ [black], ADD+ MS2891 (ΔMSMEG_2891, ΔhsaC2) [red], ADD+ MS2892

(ΔMSMEG_2892, ΔhsaA2) [blue] and ADD+ MS2900 (ΔMSMEG_2900, ΔhsaD2) [green]

were cultivated in shake flasks with minimal medium containing 1.8 mM AD as the sole

carbon and energy source. (B) Photograph taken of a final sample of the bacterial cultures in

the presence of AD.

Fig. 5. Gene complementation of MS2891, MS2892 and MS2900 mutant strains. The knock-

out mutants were complemented in trans with an original copy of the corresponding deleted


37

gene [MS2891c, MS2892c and MS2900c strains; dotted lines] or transformed with the empty

plasmid as control [MS2891v, MS2892v and MS2900v strains; continuous lines]. The

recombinant strains were cultured in shake flasks with minimal medium containing 18 mM

glycerol (A) or 1.8 mM AD (B) as the sole carbon and energy sources. (C) Photograph taken

of a final sample of the bacterial cultures in the presence of AD. (D) Scheme of several key

enzymatic steps postulated in the catabolism of C-19 steroids in M. smegmatis.


38

Supplementary information

Table S1. Oligonucleotides used in this study.

Fig. S1. Scheme of the cholesterol catabolic pathway conventionally proposed in

Actinobateria. The cholesterol pathway has been traditionally considered to take place

through three consecutive stages. Firstly, the side-chain degradation and 3β-dehydrogenation

of the cholesterol molecules occurs in the upper catabolic pathway apparently yielding two

molecules of propionyl-CoA, an acetyl-CoA and the 17-ketosteroid AD. Then, the

modification of A/B rings of AD in the central catabolic pathway leads to the generation of

the HHD and HIP acids. This last compound is mineralized through the lower catabolic

pathway. The expression of the genes encoding the enzymes involved in the side-chain and

the A/B rings degradation as well as those encoding the sterol uptake system, is regulated by

the KstR repressor (kstR regulon), while the expression of the genes responsible for the

catabolism of C/D rings is controlled by the KstR2 repressor (kstR2 regulon). Abbreviations

of enzymes: 3-ketosteroid-Δ1-dehydrogenase (KstD); 3-ketosteroid-9α-hydroxylase

(KshAB); 3-HSA monooxygenase (HsaAB); 3,4-HSA dioxygenase (HsaC); (4,9-DSHA)

hydroxylase (HsaD). Abbreviations of metabolites: 4-androstene-3,17-dione (AD); 1,4-

androstadiene-3,17-dione (ADD); 9α-hydroxy-4-androstene-3,17-dione (9OH-AD); 9α-

hydroxy-1,4-androstadiene-3,17-dione (9OH-ADD); 3-hydroxy-9,10-secoandrosta-1,3,5(10)-

trien-9,17-dione (3-HSA); 3,4-dihydroxy-9,10-secoandrosta-1,3,5(10)-trien-9,17-dione (3,4-

HSA); 4,5,9,10-diseco-3-hydroxy-5,9,7-trioxoandrosta-1(10),2-diene-4-oic acid (4,9-DSHA);

2-hydroxyhexa-2,4.dienoic acid (HHD); 3aα-H-4α(3´-propionic acid)-7aβ-methylhexahydro-

1,5-indanedione (HIP).

Fig. S2. Chemical structures of relevant steroidal intermediates detected in the culture

medium of several naturally sterols-degrading bacteria. Abbreviations: AD (4-androstene-


39

3,17-dione); ADD (1,4-androstadiene-3,17-dione); 9OH-AD (9α-hydroxy-4-androstene-3,17-

dione); 4-HBC (22-hydroxy-23,24-bisnorchol-4-en-3-one); 1,4-HBC (22-hydroxy-23,24-

bisnorchol-1,4-dien-3-one); 4-BNC (3-oxo-23,24-bisnorchol-4-en-3-oic acid); 1,4-BNC (3-

oxo-23,24-bisnorchol-1,4-dien-3-oic acid).


KstD2/ KstD3

HsaA2B1

ADD AD

9OH-ADD 9OH-AD 3-HSA

KshA2B2

HsaC2

3,4-HSA HHD

KshA2B2

KstD2/ KstD3 HsaD2

HIP

LOWER

CATABOLIC

PATHWAY

Propionyl-CoA

Pyruvate

HsaE, HsaF,

HsaG

4,9-DSHA

Non-

enzymatic

Fig. 1 Catabolic pathway of C-19 steroids in M. smegmatis mc2155.

(A)

(B) Catabolism of cholesterol

6110566 6102080

ksh

B1

hsa

D1

hsa

B1

60

40

kstR

hsa

C1

hsa

A1

60

41

5997153 6001720

kstD

1

hsa

E

hsa

H

hsa

G

ksh

A1

5985003..5986154

(41)

ksh

A2

kstD

2

cv

c2

pa

dR

kstD

3

iclR

hsa

C2

hsa

A2

ksh

B2

hsa

D2

28

88

2928126

Catabolism of C-19 steroids

2963487..2964347 2957439 2952197 2933769 (55) (40) (50) (44) (59) (36)


(A) (B)

0.0

0.5

1.0

1.5

2.0

2.5

0 20 40 60 80 100

OD

(600 n

m)

Time of culture (h)

wt MS5941 KstD1

KstD2 KstD3

0.0

0.5

1.0

1.5

2.0

0 10 20 30 40 50 60 70

OD

(600 n

m)

Time of culture (h)

wt MS6039 KshB1 KshB2

Fig. 2. Gene complementation assays of MS5941 and MS6039 mutants.


AD

ADD

Control KstD3

MS5941 Control KshA2

MS5941

9OH-AD

AD

5 h 24 h

(A) (B) AD ADD AD 9OH-AD

Fig. 3. Determination of the biological function of enzymes KstD and KshA by

resting-cell biotransformations.


(A) (B)

0.0

0.5

1.0

1.5

2.0

2.5

0 10 20 30 40 50

OD

(600 n

m)

Time of culture (h)

ADD+ MS2891 MS2900 MS2892

ADD+ MS2891 MS2892 MS2900

Fig. 4 Growth curves of mutant strains of M. smegmatis ADD+ on C-19 steroids.


(A) (B)

0.0

1.0

2.0

3.0

4.0

0 10 20 30 40

OD

(600 n

m)

Time of culture (h)

MS2891v MS2892v MS2900v

MS2891c MS2892c MS2900c

0.0

0.5

1.0

1.5

2.0

0 10 20 30 40 50 60 70

OD

(600 n

m)

Time of culture (h)

MS2891v MS2892v MS2900v

MS2891c MS2892c MS2900c

Fig. 5. Gene complementation of MS2891, MS2892 and MS2900 mutant strains.

(C)

MS2891v MS2892v MS2900v MS2891c MS2892c MS2900c

HsaD2 3-HSA 3,4-HSA 4,9-HSA

HsaA2B1 HsaC2

MS2892 MS2891

HHD

+ HIP

MS2900

(D)


Unravelling a new catabolic pathway of C‐19 steroids in ......intermediate AD occurs via a central catabolic route called 9,10-seco pathway that results in the generation of...

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