Mycobacterium smegmatis is a suitable cell factory for the production of steroidic synthons

13 p.-3 fig.-3 tab.A number of pharmaceutical steroid synthons are currently produced through the microbial side-chain cleavage of natural sterols as an alternative to multistep chemical synthesis. Industrially, these synthons have been usually produced through fermentative processes using environmental isolated microorganisms or their conventional mutants. Mycobacterium smegmatis mc2155 is a model organism for tuberculosis studies which uses cholesterol as the sole carbon and energy source for growth, as other mycobacterial strains. Nevertheless, this property has not been exploited for the industrial production of steroidic synthons. Taking advantage of our knowledge on the cholesterol degradation pathway of M. smegmatis mc2155 we have demonstrated that the MSMEG_6039 (kshB1) and MSMEG_5941 (kstD1) genes encoding a reductase component of the 3-ketosteroid 9a-hydroxylase (KshAB) and a ketosteroid D1-dehydrogenase (KstD), respectively, are indispensable enzymes for the central metabolism of cholesterol. Therefore, wehave constructed a MSMEG_6039 (kshB1) gene deletion mutant of M. smegmatis MS6039 that transforms efficiently natural sterols (e.g. cholesterol and phytosterols) into 1,4-androstadiene-3,17-dione. In addition,we have demonstrated that a double deletion mutant M. smegmatis MS6039-5941 [DMSMEG_6039 (DkshB1)and DMSMEG_5941 (DkstD1)] transforms natural sterols into 4-androstene-3,17-dione with high yields.These findings suggest that the catabolism of cholesterol in M. smegmatis mc2155 is easy to handle and equally efficient for sterol transformation than other industrial strains, paving the way for valuating this strain as a suitable industrial cell factory to develop à la carte metabolic engineering strategies for the industrial production of pharmaceutical steroids.This study 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).Peer reviewe

The Structure of the Transcriptional Repressor KstR in Complex with CoA Thioester Cholesterol Metabolites Sheds Light on the Regulation of Cholesterol Catabolism in Mycobacterium tuberculosis

Cholesterol can be a major carbon source for Mycobacterium tuberculosis during infection, both at an early stage in the macrophage phagosome and later within the necrotic granuloma. KstR is a highly conserved TetR family transcriptional repressor that regulates a large set of genes responsible for cholesterol catabolism. Many genes in this regulon, including kstR, are either induced during infection or are essential for survival of M. tuberculosis in vivo. In this study, we identified two ligands for KstR, both of which are CoA thioester cholesterol metabolites with four intact steroid rings. A metabolite in which one of the rings was cleaved was not a ligand. We confirmed the ligand-protein interactions using intrinsic tryptophan fluorescence and showed that ligand binding strongly inhibited KstR-DNA binding using surface plasmon resonance (IC50 for ligand = 25 nM). Crystal structures of the ligand-free form of KstR show variability in the position of the DNA-binding domain. In contrast, structures of KstR·ligand complexes are highly similar to each other and demonstrate a position of the DNA-binding domain that is unfavorable for DNA binding. Comparison of ligand-bound and ligand-free structures identifies residues involved in ligand specificity and reveals a distinctive mechanism by which the ligand-induced conformational change mediates DNA release

Electron transfer ferredoxins with unusual cluster binding motifs support secondary metabolism in many bacteria

The proteins responsible for controlling electron transfer in bacterial secondary metabolism are not always known or characterised. Here we demonstrate that many bacteria contain a set of unfamiliar ferredoxin encoding genes which are associated with those of cytochrome P450 (CYP) monooxygenases and as such are involved in anabolic and catabolic metabolism. The model organism Mycobacterium marinum M contains eleven of these genes which encode [3Fe–4S] or [4Fe–4S] single cluster containing ferredoxins but which have unusual iron–sulfur cluster binding motif sequences, CXX?XXC(X)nCP, where ‘?’ indicates a variable amino acid residue. Rather than a cysteine residue, which is highly conserved in [4Fe–4S] clusters, or alanine or glycine residues, which are common in [3Fe–4S] ferredoxins, these genes encode at this position histidine, asparagine, tyrosine, serine, threonine or phenylalanine. We have purified, characterised and reconstituted the activity of several of these CYP/electron transfer partner systems and show that all those examined contain a [3Fe–4S] cluster. Furthermore, the ferredoxin used and the identity of the variable motif residue in these proteins affects the functionality of the monooxygenase system and has a significant influence on the redox properties of the ferredoxins. Similar ferredoxin encoding genes were identified across Mycobacterium species, including in the pathogenic M. tuberculosis and M. ulcerans, as well as in a wide range of other bacteria such as Rhodococcus and Streptomyces. In the majority of instances these are associated with CYP genes. These ferredoxin systems are important in controlling electron transfer across bacterial secondary metabolite production processes which include antibiotic and pigment formation among others

A Flavin-dependent Monooxygenase from Mycobacterium tuberculosis Involved in Cholesterol Catabolism

Mycobacterium tuberculosis (Mtb) and Rhodococcus jostii RHA1 have similar cholesterol catabolic pathways. This pathway contributes to the pathogenicity of Mtb. The hsaAB cholesterol catabolic genes have been predicted to encode the oxygenase and reductase, respectively, of a flavin-dependent mono-oxygenase that hydroxylates 3-hydroxy-9,10-seconandrost-1,3,5(10)-triene-9,17-dione (3-HSA) to a catechol. An hsaA deletion mutant of RHA1 did not grow on cholesterol but transformed the latter to 3-HSA and related metabolites in which each of the two keto groups was reduced: 3,9-dihydroxy-9,10-seconandrost-1,3,5(10)-triene-17-one (3,9-DHSA) and 3,17-dihydroxy-9,10-seconandrost-1,3,5(10)-triene-9-one (3,17-DHSA). Purified 3-hydroxy-9,10-seconandrost-1,3,5(10)-triene-9,17-dione 4-hydroxylase (HsaAB) from Mtb had higher specificity for 3-HSA than for 3,17-DHSA (apparent k_(cat)/K_m = 1000 ± 100 M^(−1) s^(−1) versus 700 ± 100 M^(−1) s^(−1)). However, 3,9-DHSA was a poorer substrate than 3-hydroxybiphenyl (apparent k_(cat)/K_m = 80 ± 40 M^(−1) s^(−1)). In the presence of 3-HSA the K_(mapp) for O_2 was 100 ± 10 μM. The crystal structure of HsaA to 2.5-Å resolution revealed that the enzyme has the same fold, flavin-binding site, and catalytic residues as p-hydroxyphenyl acetate hydroxylase. However, HsaA has a much larger phenol-binding site, consistent with the enzyme's substrate specificity. In addition, a second crystal form of HsaA revealed that a C-terminal flap (Val^(367)–Val^(394)) could adopt two conformations differing by a rigid body rotation of 25° around Arg^(366). This rotation appears to gate the likely flavin entrance to the active site. In docking studies with 3-HSA and flavin, the closed conformation provided a rationale for the enzyme's substrate specificity. Overall, the structural and functional data establish the physiological role of HsaAB and provide a basis to further investigate an important class of monooxygenases as well as the bacterial catabolism of steroids

High-Resolution Phenotypic Profiling Defines Genes Essential for Mycobacterial Growth and Cholesterol Catabolism

The pathways that comprise cellular metabolism are highly interconnected, and alterations in individual enzymes can have far-reaching effects. As a result, global profiling methods that measure gene expression are of limited value in predicting how the loss of an individual function will affect the cell. In this work, we employed a new method of global phenotypic profiling to directly define the genes required for the growth of Mycobacterium tuberculosis. A combination of high-density mutagenesis and deep-sequencing was used to characterize the composition of complex mutant libraries exposed to different conditions. This allowed the unambiguous identification of the genes that are essential for Mtb to grow in vitro, and proved to be a significant improvement over previous approaches. To further explore functions that are required for persistence in the host, we defined the pathways necessary for the utilization of cholesterol, a critical carbon source during infection. Few of the genes we identified had previously been implicated in this adaptation by transcriptional profiling, and only a fraction were encoded in the chromosomal region known to encode sterol catabolic functions. These genes comprise an unexpectedly large percentage of those previously shown to be required for bacterial growth in mouse tissue. Thus, this single nutritional change accounts for a significant fraction of the adaption to the host. This work provides the most comprehensive genetic characterization of a sterol catabolic pathway to date, suggests putative roles for uncharacterized virulence genes, and precisely maps genes encoding potential drug targets

Iron-containing monooxygenases in Mycobacterium tuberculosis cholesterol degradation : biochemical and phylogenetic perspectives

Mycobacterium tuberculosis (Mtb) is the human pathogen that causes tuberculosis. A gene cluster encoding a cholesterol degradation pathway plays a role in Mtb virulence. Two iron-containing monooxygenases in this pathway were characterized with respect to their roles in bacterial cholesterol catabolism: the Rieske oxygenase (RO) KshAB, and the cytochrome P450 (P450) Cyp125. These enzymes are predicted to catalyze the first ring-opening step and the first transformation of the steroid side chain, respectively. Cyp125A1 (Mtb) and Cyp125A14P (Rhodococcus jostii RHA1) were expressed in R. jostii RHA1 and characterized in vitro using the Mtb reductase KshB. Both enzymes were purified with the heme iron in a predominantly high spin state and exhibiting thiolate ligation of the heme iron. Both P450s bound cholesterol and 4-cholesten-3-one with apparent submicromolar affinity. Cyp125A1 was demonstrated to catalyze C26-monohydroxylation of both steroids. KshA (a terminal oxygenase) and KshB (an oxygenase reductase) of Mtb were produced in Escherichia coli and characterized in vitro. KshAB had over twenty times the apparent substrate specificity for steroid substrates with isopropionyl-CoA side chains than for the corresponding 17-keto steroids. The apparent KMO₂ with a CoA thioester-bearing steroid was 90 ± 10 μM whereas that for the corresponding 17-keto steroid was in excess of 1.2 mM. These results suggest that the physiological substrate(s) for KshAB is likely a CoA thioester intermediate of cholesterol side chain degradation. A comprehensive phylogenetic analysis was undertaken to consolidate the available RO literature. Six hundred fifty enzymes that are fully representative of the RO terminal oxygenase (RO-O) sequences in the NCBI database were collected and aligned to a structure-based sequence template. The structure-based alignment was also used to objectively define the structurally conserved positions that were included in phylogenetic reconstruction. The resulting analysis revealed a level of RO-O diversity that has been unrecognized in previous literature and that necessitates a different approach to RO-O classification. A classification scheme based on the system currently in use for P450s was proposed. This work provides significant insight into the cholesterol degradation pathway of Mtb and the RO-O protein family and contributes to potential commercial applications in bioremediation, biocatalysis, and Mtb therapeutics.Medicine, Faculty ofBiochemistry and Molecular Biology, Department ofGraduat

The measurements of earthing systems and the role of electrical soil resistivity

The theoretical part of the thesis is focused on explaining the concept of soil resistivity, applicable methods of its measurement, comparison of two methods used to measure the resistance of the earth electrode and a description of the measuring device Metro PU 193 used for the practical part of the thesis. In the practical part, the reader is acquainted with the results of measuring soil resistivity and earth resistance in the field, with their evaluation with respect to various influencing factors and with designs of earth conductors for high voltage poles for which soil resistivity was measured. The thesis contains plotted graphs of various dependencies according to the results of measured values and compares them with theoretical assumptions

Calculation of earth current distribution in OpenDSS

The thesis deals with the ground current distribution in electrification systems. The first part introduces the reader to the electrical parameters of distribution system elements and options of their calculation. Various factors and environmental influences that can affect the parameters of individual network elements are described using graphics dependencies. The second part of the thesis describes the creation of a network model in the OpenDSS program, which is an integral part of the study in question used to make complex calculations for the given issue. Furthermore, this part contains created models of distribution networks at voltage levels of 110 and 0.4 kV on which individual simulations are performed, including a single-phase fault. These network models are used to observe the effects on the magnitude of current distribution through the grounding system, grounding wires and the PEN conductor, in connection with changes in the arrangement of conductors on masts, with changes in wiring, etc. Finally, the obtained simulation values are compared with theoretical assumptions and evaluated