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Catabolism and Biotechnological Applications of Cholesterol Degrading Bacteria bs_bs_banner Microbial Biotechnology (2012) 5(6), 679–699 doi:10.1111/j.1751-7915.2012.00331.x Minireview Catabolism and biotechnological applications of cholesterol degrading bacteria J. L. García,* I. Uhía and B. Galán according to its physiological relevance, an imbalance in Environmental Biology Department, Centro de its blood level causes serious diseases in humans. Investigaciones Biológicas, CSIC, C/ Ramiro de Maeztu, Cholesterol is frequently found in the biosphere, not 9, 28040 Madrid, Spain. only because of its natural abundance, but also due to its high resistance to microbial degradation. Cholesterol is a recalcitrant molecule to biodegradation because of its low Summary number of functional groups (one CC double bond and Cholesterol is a steroid commonly found in nature a single hydroxyl group), its low solubility in water with a great relevance in biology, medicine and chem- (3 ¥ 10-8 M) and its complex spatial conformation consti- istry, playing an essential role as a structural compo- tuted by four alicyclic rings and two quaternary carbon nent of animal cell membranes. The ubiquity of atoms. The high hydrophobicity and low volatility of cho- cholesterol in the environment has made it a refer- lesterol leads to a high absorption to solid phases. ence biomarker for environmental pollution analysis Because of their high rate of persistence, cholesterol and and a common carbon source for different microor- some derived compounds, such as coprostanol, have ganisms, some of them being important pathogens been used as reference biomarkers for environmental such as Mycobacterium tuberculosis. This work pollution analysis (Veiga et al., 2005). revises the accumulated biochemical and genetic Steroids, some of them derived from cholesterol, con- knowledge on the bacterial pathways that degrade or stitute a new class of pollutants discharged into the envi- transform this molecule, given that the characteriza- ronment as a result of human activity (Gagné et al., 2006). tion of cholesterol metabolism would contribute not The potent metabolic activities of these compounds affect only to understand its role in tuberculosis but also to a large number of cellular processes and thus, their pres- develop new biotechnological processes that use this ence and accumulation in water wastes and in certain and other related molecules as starting or target ecological niches can affect the endocrine system of materials. animals and humans (Hotchkiss et al., 2008). Moreover, cholesterol is the main component of lanolin, and this and other related sterols are natural contaminants fairly resis- Introduction tant to the anaerobic treatment that is carried out on the Cholesterol is a steroid, i.e. a class of terpenoid lipids, effluents from wool-processing industry (Poole and Cord- with a carbon skeleton virtually flat and relatively rigid Ruwisch, 2004). formed by four fused alicyclic rings (Fig. 1). This polycyclic The ubiquity of cholesterol and related sterols in the sterol has a great importance in biology, medicine and environment has made them a common carbon source for chemistry, being one of the most common molecules in many different microorganisms, some of them being nature, as an essential structural component of animal important pathogens as Mycobacterium tuberculosis.On cell membranes (Staytor and Bloch, 1965), although it has the other hand, the bacterial metabolism of steroids has been also exceptionally found in some prokaryotes attracted also considerable attention as low-cost natural (Hayami et al., 1979). steroids like cholesterol can be used as starting materials The metabolism of cholesterol is critical in eukaryotes for many bioactive synthetic steroids (Fernandes et al., as precursor of vitamins, steroid hormones and bile acids 2003). Therefore, the characterization of the bacterial (Miller, 1988; Björkhem and Eggertsen, 2001) (Fig. 1), but metabolism of cholesterol could be useful not only to understand its influence in pathological processes like tuberculosis, but also to develop new organisms by meta- Received 11 October, 2011; revised 28 December, 2011; accepted 2 January, 2012. *For correspondence. E-mail [email protected]; Tel. bolic engineering with potential use as biotechnological (+34) 918 373 112; Fax (+34) 91 536 04 32. tools. In a recent work we reviewed the accumulated © 2012 The Authors Microbial Biotechnology © 2012 Society for Applied Microbiology and Blackwell Publishing Ltd Cholesterol degradation 680 Fig. 1. Chemical structures of cholesterol and some derived natural molecules. biochemical and genetic information on the bacterial path- century, it was observed that several species of Mycobac- ways that degrade or transform biochemically different terium could use cholesterol as the sole source of carbon steroids (García et al., 2011). This review is mainly and energy (Söhngen, 1913; Tak, 1942). Later on, it was focused in cholesterol catabolism and its biotechnological reported that microorganisms belonging to the genus Pro- applications including the development of cholesterol actinomyces, like Rhodococcus erythropolis (formerly biosensors for clinical diagnosis and food industry, micro- Nocardia erythropolis), have the ability to degrade choles- bial steroid biotransformations for the production of terol and a potential capacity for the synthesis of new steroid drugs and hormones, probiotics with cholesterol- steroid derivatives (Turfitt, 1944; 1948) Moreover, some lowering effects, and enzymes for insecticide and fungi- species of Azotobacter transform cholesterol in cholest- cide purposes. 4-en-3-one (cholestenone) or 7-dehydrocholesterol, and have the ability to hydrolyse the side-chain of cholesterol Catabolism of cholesterol generating methylheptanone (Horvath and Kramli, 1947). Thereafter, other bacteria belonging to the genera Historical issues Nocardia, Arthrobacter, Bacillus, Brevibacterium, Coryne- The search for microorganisms capable of degrading cho- bacterium, Streptomyces, Microbacterium, Serratia, lesterol was started over 70 years ago. In the early 20th Achromobacter, Pseudomonas or Protaminobacter, © 2012 The Authors Microbial Biotechnology © 2012 Society for Applied Microbiology and Blackwell Publishing Ltd, Microbial Biotechnology, 5, 679–699 681 J. L. García, I. Uhía and B. Galán Table 1. Sequenced genomes of described organisms able to growth in cholesterol as a sole source of carbon and energy. Organismsa GenBank Chromosome (nt) Igrb Hsab Mceb Gordonia neofelifaecis NZ_AEUD00000000 4 257 286 Yes* Yes Yes Mycobacterium avium NC_008595 5 475 491 Yes Yes Yes Mycobacterium bovis NC_008769 4 374 522 Yes Yes Yes Mycobacterium smegmatis NC_008596 6 988 209 Yes Yes Yes Mycobacterium tuberculosis NC_000962 4 411 532 Yes Yes Yes Rhodococcus equi NC_014659 5 043 170 Yes* Yes Yes Rhodococcus erythropolis NC_012490 6 516 310 Yes* Yes Yes Rhodococcus jostii NC_008268 7 804 765 Yes* Yes Yes Streptomyces venezuelae FR845719.1 8 226 158 Yes* No Yes Streptomyces viridochromogenes NZ_ACEZ00000000 8 548 109 Uncomplete* No Yes a. Only one representative organism has been included in the table. b. The presence of the igr, hsa and mce genes has been analysed. The asterisk (*) means that the Cyp125 encoding gene is not present in the locus. among others, were reported to accomplish partial or mer and Martin, 1980; Owen et al., 1983; Kieslich, 1985; complete cholesterol degradation (Whitmarsh, 1964; Sedlaczek and Smith, 1988; Szentirmai, 1990; Yam et al., Brown and Peterson, 1966; Arima et al., 1969; Nagasawa 2010; García et al., 2011; Griffin et al., 2011; Ouellet et al., 1969; Chipley et al., 1975; Martin, 1977; Ferreira et al., 2011) (Fig. 2). Experiments performed with 14C- and Tracey, 1984; Drzyzga et al., 2009; 2011; Fernández radiolabelled cholesterol showed that carbon from the de Las Heras et al., 2009; Ge et al., 2011). Nevertheless, sterol ring system was preferentially converted to CO2 and in spite of the large number of described bacteria able to used for energy generation via tricarboxylic acid cycle, use cholesterol as a sole source of carbon and energy whereas the side-chain is assimilated into the mycobac- only the genomes of few of them have been completely terial lipids (Cox et al., 1999). To facilitate the comprehen- sequenced (Table 1). When we analysed in the genome sion of the catabolic process it can be divided into four of these microorganisms the presence of some of the major steps as presented below. most representative gene loci involved in cholesterol metabolism [e.g. igr (side-chain degradation), hsa (central Transformation of cholesterol into cholestenone pathway) and mce (transport)], we have observed that some of them do not contain these genes. This suggests It is generally assumed that the first reaction in the aerobic that although these bacteria could transform cholesterol metabolism of cholesterol is its oxidation to cholestenone they may not be able to completely mineralize this com- through two sequential reactions. That is, the oxidation of pound. The relevance of these genes in cholesterol cholesterol to cholest-5-en-3-one [Fig. 2 (2)] followed catabolism has been also confirmed recently by high- by its isomerization to cholestenone [Fig. 2 (3)]. The density mutagenesis and deep-sequencing analysis in enzymes responsible for this activity are cholesterol M. tuberculosis (Griffin et al., 2011). Finally, it is worth to oxidases (ChOx), or 3b-hydroxysteroid dehydrogenase/ mention that most of these genes are randomly located in isomerases (HSDs). different
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