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Estudio del transporte de esteroides en Mycobacterium smegmatis mc2155 y sus aplicaciones biotecnológicas

AuthorsGarcía Fernández, Julia
AdvisorGarcía, José Luis ; Galán, Beatriz
Microbiología Molecular
Issue Date2015
PublisherCSIC - Centro de Investigaciones Biológicas (CIB)
Abstract1. Introduction Due to the clinical, industrial and environmental cholesterol relevance, the microorganisms able to degrade it have been selected for more than 70 years (Söhngen, 1913; Turfitt, 1944) despite this fact, nowadays most of the genetic and biochemical basis of its degradation pathways are still unknown. The complete knowledge of these metabolic pathways would lead to the development of new biotechnological processes in the pharmaceutical industry for the production of steroids. Moreover and since cholesterol has been linked to the pathogenesis of Mycobacterium tuberculosis (Pandey and Sassetti, 2008; Joshi et al., 2006), the study of these routes has recently acquired a large interest from a clinical point of view. Although one of the key elements in the bacterial metabolism of cholesterol is the uptake process, the knowledge about the selective transport of steroids in bacteria is still limited. Bacteria able to degrade cholesterol possess an operon called mce4 that encodes an ABC system responsible for its uptake into the cell (Pandey and Sassetti, 2008; Mohn et al., 2008). This operon contains 10 genes, yrbE4ABmce4ABCDEFmas4AB, two of which encode the permeases of the system and the rest, of unknown function, are postulated to encode the substrate binding proteins. Additionally, the ATPase activity of this ABC system is provided by the mceG gene (MSMEG_1366) encoding an Mkl enzyme that is located away from the mce4 operon and whose function is shared with other Mce systems that are present in the same cell (Joshi et al., 2006). 2. Objectives and results Given the great interest in the study of the cholesterol uptake, the objectives of this Thesis have been focused on the study of this process using M. smegmatis as a model and genetic and biochemical analysis to apply the acquired knowledge in biotechnological purposes. The main results obtained have allowed us to determine that the steroid uptake system in M. smegmatis depends on the structure and availability of these compounds. Thus, steroids without side chain such as androstenedione or with a short side chain as pregnenolone are able to diffuse through the membrane without the need of a specific transporter. As for steroids with a more complex structure such as cholesterol and phytosterols, the mechanism of uptake is dependent on its availability in the environment so that when these compounds are found in high concentration levels they are able to enter by passive diffusion but when the availability is low it is required the Mce4 system to actively uptake them. The correct function of this active transport requires all of the 10 proteins that are present in the mce4 operon as well as the MceG ATPase to generate a functional transporter. In addition, this ATPase has a C-terminal extension that appears to be involved in the binding to auxiliary proteins. Despite the already large number of existing components in the mce4 operon, the results obtained in this Thesis seem to extend the complexity of this ATPase suggesting the involvement of a larger number of proteins in the cholesterol uptake process.
The study of the Mce4 system has also allowed us to propose that it carries out additional functions in addition to the cholesterol uptake. In this regard, the appearance of altered morphologies in the specific mutants of this system indicates its involvement in transport processes of lipid components present in the cell envelope of M. smegmatis. While similar functions have been postulated for other Mce systems (Dunphy et al., 2010; Marjanovic et al., 2011; Santangelo et al., 2009b), the relation of the mce4 operon with this type of processes is novel to date and suggests that steroidal compounds probably are able to use the Mce4 system to be transported into the cell due to its structural similarity to lipids present in the cell envelope. Despite the growing knowledge on the cholesterol degradation pathway in the genera Mycobacterium and Rhodococcus, the use of these organisms in biotransformations of industrial interest has several disadvantages arising from the presence of an endogenous steroid metabolism. For this reason, the search of heterologous organisms able to perform these kind of biotransformations is one of the most promising alternatives. In this sense, the studies derived from this Thesis about the uptake mechanisms of different steroids of interest in the biotechnological industry as well as the characterization of the MSMEG_5228 dehydrogenase, have allowed us to verify the potential of Corynebacterium as an heterologous host in the industrial production of steroid compounds. 3. Conclusions - The Mce4 active transport system of cholesterol in M. smegmatis requires at least 11 proteins to generate a functional transport system. These proteins include an ATPase (MceG), two permease subunits (YrbE4) and eight additional proteins of unknown function (Mce4ABCDEFMas4AB). None of these proteins can be replaced by the homologous subunits that are present in other mce operons of M. smegmatis mc2155. - The MceG ATPase has a C-terminal extension, not conserved in other similar ABC ATPases, that could bind to additional proteins to those present in the mce4 operon and whose binding is necessary for the proper active transport of cholesterol in M. smegmatis mc2155. - M. smegmatis has a system of passive transport of steroids that is independent of the Mce4. - Mce systems including the Mce4 whose function has been only associated to date with cholesterol uptake, might be also involved in transport processes of lipid components present in the cell envelope of M. smegmatis mc2155 under stress conditions. This fact suggests that probably the steroidal compounds are transported using the Mce4 system because of its structural similarity to some of the cell envelope lipids. - Corynebacterium glutamicum, a microorganism without an endogenous steroid metabolism, has been proven to have potential as a biotechnological chassis for expression of heterologous genes in steroid biotransformations.
Description238 p.-75 fig.-16 tab.
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