Catabolism and biotechnological applications of cholesterol degrading bacteria

Aerobic degradation of cholesterol

Although the bacterial catabolism of cholesterol has not been fully elucidated in any of the bacterial strains degrading sterols, the metabolic pathway can be postulated by combining the biochemical and genetic studies carried out in different organisms (Martin, 1977; Schoemer and Martin, 1980; Owen et al., 1983; Kieslich, 1985; Sedlaczek and Smith, 1988; Szentirmai, 1990; Yam et al., 2010; García et al., 2011; Griffin et al., 2011; Ouellet et al., 2011) (Fig. 2). Experiments performed with ¹⁴C‐radiolabelled cholesterol showed that carbon from the sterol ring system was preferentially converted to CO₂ and used for energy generation via tricarboxylic acid cycle, whereas the side‐chain is assimilated into the mycobacterial lipids (Cox et al., 1999). To facilitate the comprehension of the catabolic process it can be divided into four major steps as presented below.

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Figure 2

Proposed pathway for cholesterol degradation under aerobic conditions. Cholest‐4‐en‐3‐one or any of the subsequent metabolites from degradation of the side‐chain up to (and including) AD may undergo a dehydrogenation reaction to introduce a double bond in the position 1, leading to compound cholest‐1,4‐diene‐3‐one in the case of cholest‐4‐en‐3‐one, or to the corresponding 1,2‐dehydro derivatives for other molecules. The side‐chain degradation of this compounds will be identical to that of the cholest‐4‐en‐3‐one to the common intermediate 9α‐hydroxyandrosta‐1,4‐diene‐3,17‐dione. The microorganisms from which enzymes implicated in different steps are indicated by numbers. Numbers in brackets are assigned arbitrarily to facilitate the compound identification in the text.

Transformation of cholesterol into cholestenone

It is generally assumed that the first reaction in the aerobic metabolism of cholesterol is its oxidation to cholestenone through two sequential reactions. That is, the oxidation of cholesterol to cholest‐5‐en‐3‐one [Fig. 2 (2)] followed by its isomerization to cholestenone [Fig. 2 (3)]. The enzymes responsible for this activity are cholesterol oxidases (ChOx), or 3β‐hydroxysteroid dehydrogenase/isomerases (HSDs).

ChOx is a monomeric flavoenzyme that catalyses the oxidation and isomerization of cholesterol to cholestenone with a concomitant reduction of FAD. Regeneration of the oxidized cofactor is achieved by the reduction of O₂ to hydrogen peroxide. The enzyme is extracellular and occurs as secreted and/or cell‐surface‐associated forms, depending on the microorganism and growth conditions (Kreit and Sampson, 2009). The ChOx from Streptomyces sp. SA‐COO (Yue et al., 1999; Lario et al., 2003) and the BOC1 form of ChOx from Brevibacterium sterolicum (Vrielink et al., 1991; Li et al., 1993) have been classified as type I oxidases and belong to the glucose‐methanol‐choline (GMC) oxidoreductase family of flavoenzymes where the FAD cofactor is non‐covalently bound. However, the BOC2 form of ChOx from B. sterolicum (Coulombe et al., 2001) has been classified as a type II oxidase belonging to the family of vanillyl‐alcohol oxidase enzymes, which contain a covalently bound FAD molecule (Motteran et al., 2001). Both classes of ChOxs share the same catalytic activity but show significant differences in their redox and kinetic properties. In general, they exhibit a broad range of steroid specificities and can oxidize a number of hydroxysterols, including sterols, steroid hormones and bile acids, but the presence of a 3β‐hydroxyl group is an important requirement for activity (Pollegioni et al., 1999). Remarkably, some ChOxs, like those from Burkholderia cepacia ST‐200, Pseudomonas spp. and Chromobacterium sp. DS‐1, oxidize cholesterol mainly to 6β‐hydroperoxy‐cholest‐4‐en‐3‐one and hydrogen peroxide instead of cholestenone (Doukyu and Aono, 1999; Doukyu et al., 2008; Doukyu, 2009) but this intermediate has not been associated so far to any cholesterol degradation pathway. Recently, a cholesterol inducible ChoX encoding gene (choG) has been identified in Rhodococcus sp. strain CECT3014 within a gene cluster involved in the metabolism of cholesterol (Fernández de Las Heras et al., 2011).

The 3β‐hydroxy‐Δ5‐steroid HSD belongs to a family of enzymes that catalyse the conversion of 3β‐hydroxy‐5‐ene‐steroids to 3‐oxo‐4‐ene‐steroids through consecutive oxidation and isomerization reactions, using NAD or NADP as cofactors. Gene database analysis revealed the existence of a protein superfamily that in addition to the bacterial HSDs includes mammalian 3β‐hydroxysteroid dehydrogenases, plant dihydroflavonol reductases, bacterial UDP‐galactose‐4‐epimerases and viral 3β‐hydroxysteroid dehydrogenase (Baker and Blasco, 1992). All of them present a glycine‐rich sequence (Gly‐X‐X‐Gly‐X‐X‐Gly) similar to the Rossman fold sequence and an atypical active site motif (Tyr‐X‐X‐X‐Lys). The NAD(P)‐dependent HSD from Nocardia sp. has been well characterized and appears to be responsible for the transformation of cholesterol to cholestenone in this cholesterol degrader organism (Horinouchi et al., 1991). Recent genetic and biochemical studies have also demonstrated the direct involvement of a NAD‐dependent HSD in mycobacterial cholesterol catabolism (Yang et al., 2007; Griffin et al., 2011; Thomas et al., 2011; Uhía et al., 2011a). In fact, it has been demonstrated that the ChOx analogue (ChoD) found in Mycobacteria does not seem to play a role in cholesterol degradation (Griffin et al., 2011; Uhía et al., 2011a).

Cholesterol side‐chain degradation

It has been assumed that simultaneously and independently of the first oxidative process, the long aliphatic chain of cholesterol is removed via a process similar to the β‐oxidation of fatty acids, leading to the formation of a steroid C22‐oic acid intermediate with the concomitant release of two molecules of propionyl‐CoA and one acetyl‐CoA (Fig. 2). This process has been elucidated at the biochemical level in strains of Mycobacterium and Nocardia and proceeds differently for cholesterol and β‐sitosterol (Sih, 1968; Martin, 1976; Wilbrink et al., 2011).

Recent studies have pointed out that the Cyp125 cytochrome from Rhodococcus jostii RHA1 (ro04679 coding gene) and M. tuberculosis (Rv3545c coding gene) initiates the side‐chain degradation of cholesterol (Capyk et al., 2009a; Rosłoniec et al., 2009). This cytochrome belongs to the P450 family and catalyses the hydroxylation at carbon C‐26 yielding 26‐hydroxycholest‐4‐en‐3‐one. Cyp125 from M. tuberculosis CDC1551 was proved to be involved not only in the hydroxylation of cholesterol but also in its further oxidation to cholest‐4‐en‐3‐one‐27 oic acid (Ouellet et al., 2010). The crystallographic structure of Cyp125 from M. tuberculosis has been recently determined (McLean et al., 2009).

Driscoll and colleagues (2010) and Johnston and colleagues (2010) have demonstrated that Cyp142 is a cytochrome that is able to replace the activity of Cyp125 in M. tuberculosis H37Rv, explaining why a Cyp125 deletion mutant of this strain is still able to grow using cholesterol as a carbon source. Furthermore, Δcyp125 mutant strains of Mycobacterium bovis and M. bovis BCG (Driscoll et al., 2010) and M. tuberculosis CDC1551 (Johnston et al., 2010) could not grow on cholesterol because they have a defective cyp142 gene. In addition, its crystal structure was very similar to that of Cyp125 (Driscoll et al., 2010). Although Griffin and colleagues (2011) have identified Cyp125 as the sole monooxygenase significantly required for growth on cholesterol the involvement of Cyp142 cannot be completely discarded.

After the formation of the carboxylic acid intermediate, the cholesterol side‐chain is shortened by a β‐oxidation‐like process initiated by an ATP‐dependent sterol/steroid CoA ligase catalysing the CoA activation of the C27 carboxylic acid intermediate (Sih, 1968; Sih et al., 1968). Subsequent cleavage of the side‐chain to 17‐ketosteroid takes place stepwise in three consecutive cycles and two molecules of propionyl‐CoA and one molecule of acetyl‐CoA are formed from the side‐chain (Fig. 3). This process involves two initial steps of typical β‐oxidation with the participation of one or two different sets of four enzymes, i.e. acyl‐CoA dehydrogenases, enoyl‐CoA‐hydratases, hydroxyacyl‐CoA dehydrogenases and thiolases. These steps will release one propionyl‐CoA and one acetyl‐CoA. The additional propionyl‐CoA cannot be released by a conventional β‐oxidation due to the presence of the cyclopentane ring D. Although the two following steps would proceed by the action of an acyl‐CoA dehydrogenase and an enoyl‐CoA‐hydratase, as occurs in a typical β‐oxidation, they will render a tertiary alcohol, which is not susceptible to dehydrogenation to form the keto group. Therefore, the last step will require the participation of a hydroxyacyl‐CoA lyase similar to other CoA‐lyases (e.g. malyl‐CoA lyase, citramalyl‐CoA lyase or 3‐hydroxy‐3‐methylglutaryl‐CoA lyase) that will render androst‐4‐ene‐3,17‐dione (AD) and propionyl‐CoA. A similar mechanism has been proposed for the metabolism of cyclohexaneacetate in Arthrobacter sp. CA1 (Ougham and Trudgill, 1982). A number of genes related to β‐oxidation have been recently identified in M. tuberculosis by transposon mutagenesis and suggested to be involved in cholesterol side‐chain degradation (Griffin et al., 2011). Some of these genes were located inside the large cholesterol degradation gene cluster (hsd4A, ltp2, fadE29, fadE28, fadA5, fadE30, fadE32, fadE33, fadE34 and hsd4B), but others were found outside of the cluster (fadE5, echA9, fadD36 and fadE25).

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Figure 3

Proposed β‐oxidation‐like reactions for cholesterol side‐chain degradation. The Fad proteins have been assigned according to the nomenclature of the E. coli genes involved in the β‐oxidation of fatty acids. LiuE is the name assigned to 3‐hydroxy‐3‐methylglutaryl‐coenzyme A lyases.

Our knowledge of genes involved in microbial degradation of cholesterol side‐chain is still very limited, but a catabolic gene cluster was identified in R. jostii RHA1 and Mycobacterium smegmatis, encoding several enzymes predicted to be involved in the β‐oxidation process (van der Geize et al., 2007; Uhía et al., 2012) (Fig. 4). Recently, the fadD19 gene product of this cluster has been identified as a steroid‐CoA ligase with an essential role in the degradation of C‐24 branched‐chain‐sterols (Wilbrink et al., 2011). In addition, the fadA5 (Rv3546) gene from M. tuberculosis H37Rv has been proposed to encode a β‐ketoacyl‐CoA thiolase that functions in cholesterol side‐chain β‐oxidation (Nesbitt et al., 2010). This gene is upregulated by cholesterol and repressed by KstR (see below), and is required for utilization of cholesterol as a sole carbon source in vitro and for full virulence of M. tuberculosis in the chronic stage of mouse lung infection (Nesbitt et al., 2010).

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Figure 4

Organization of the main gene clusters implied or suggested to be involved in the degradation of cholesterol in M. smegmatis mc²155. The identity number for each MSMEG gene is indicated within the arrows. The name of some genes of interest is written above them. Numbers below genes indicate the number of bp between adjacent genes; numbers in brackets indicate separation and numbers in parentheses indicate overlap. Numbers above diagonal lines indicate the genomic position in kb. Orange: mce cluster. Green: genes suggested and/or proved to participate in the side‐chain degradation. Blue: genes suggested and/or proved to participate in the central or lower catabolic pathway. Yellow: genes coding the transcriptional repressors KstR and KstR2. Genes surrounded by a dashed line are controlled by KstR2, the rest of the genes showed in this figure are controlled by KstR (except for MSMEG_5905, 5909, 5910, 5912, 5916, 5917, 5924, 5926, 5928, 5936, 5938, 6005, 6006, 6007, 6010, 6034, which could not be proved to be controlled by any of both repressors) (Kendall et al., 2007; 2010; Uhía et al., 2012).

It is worth to mention that cholesterol catabolism would follow an obligatory order for side‐chain and ring structure modifications depending of the microorganism. In this sense, while the Δcyp125 mutants of BCG (Bacillus Calmette‐Guerin, an inactivated form of M. bovis) and Rhodococcus rhodochrous RG32 transformed cholesterol to cholestenone, the corresponding mutant of R. jostii RHA1 did not (Capyk et al., 2009a). Furthermore, ring degradation cannot proceed with substrates having a full‐length aliphatic side‐chain in the BCG mutant as it is not able to grow or further transform cholestenone, while R. jostii RHA1 mutant grows in this compound (Capyk et al., 2009a).

The combination of these two first enzymatic steps, i.e. the oxidation of cholesterol together with the degradation of its lateral chain, renders the central intermediate AD [Fig. 2 (5A)] that is further metabolized by the central catabolic pathway.

Central catabolic pathway

Once the aliphatic chain has been oxidized, cholesterol catabolism appears to follow the common pathway described for C19 steroids. Briefly, 3‐ketosteroid‐Δ¹‐dehydrogenases, as KsdD in M. smegmatis (Brzostek et al., 2005), KstD in R. erythropolis (van der Geize et al., 2000; 2001; 2002a) and M. tuberculosis (Knol et al., 2008) that are equivalent to TesH of Comamonas testosteroni (Horinouchi et al., 2003a) transform AD into androsta‐1,4‐diene‐3,17‐dione (ADD) [Fig. 2 (5B) and Fig. 4]. Later on, a 9α‐hydroxylation takes place catalysed by KshAB in R. erythropolis (van der Geize et al., 2001; 2002b; 2008a), KshAB (Rv3526/Rv3571) in M. tuberculosis H37Rv (Capyk et al., 2009b), and KstH (KshA‐like) in M. smegmatis (Fig. 4) (Andor et al., 2006), followed by a non‐enzymatic transformation of the 9α‐hydroxyandrosta‐1,4‐diene‐3,17‐dione [Fig. 2 (6B)] to 3‐hydroxy‐9,10‐secoandrosta‐1,3,5 (10)‐trien‐9,17‐dione (3‐HSA) [Fig. 2 (7)]. The crystal structure of KshA of M. tuberculosis H37Rv has been recently elucidated revealing that it is a trimer (Capyk et al., 2009b). Five kshA homologues have been identified in R. rhodochrous DSM43269 showing different substrate specificity (Petrusma et al., 2011).

Subsequently 3‐HSA is hydroxylated by enzymes similar to the two‐component oxygenase TesA1A2 from C. testosteroni (Horinouchi et al., 2004) leading to the production of 3,4‐dihydroxy‐9,10‐secoandrosta‐1,3,5(10)‐trien‐9,17‐dione (3,4‐DHSA) [Fig. 2 (8)]. Mycobacterium tuberculosis and R. jostii RHA1 have a similar enzyme named HsaAB (Dresen et al., 2010). The crystal structure of HsaA from M. tuberculosis revealed that the enzyme is very similar to p‐hydroxyphenylacetate hydroxylase (Dresen et al., 2010). The last catechol derivative is cleaved by a meta extradiol dioxygenase similar to TesB in C. testosteroni (Horinouchi et al., 2001), which was named HsaC in R. jostii RHA1 (van der Geize et al., 2007) or M. tuberculosis (Yam et al., 2009) yielding 4,5,9,10‐diseco‐3‐hydroxy‐5,9,17‐trioxoandrosta‐1(10),2‐diene‐4‐oic acid (4,9‐DSHA) [Fig. 2 (9)]. This compound is then hydrolysed by enzymes similar to TesD from C. testosteroni (Horinouchi et al., 2003b) named HsaD in M. tuberculosis (Lack et al., 2008; 2010) or R. jostii RHA1 (van der Geize et al., 2007) yielding 2‐hydroxyhexa‐2,4‐dienoic [Fig. 2 (10)] and 9,17‐dioxo‐1,2,3,4,10,19‐hexanorandrostan‐5‐oic (DOHNAA) acids [Fig. 2 (11)].

Lower catabolic pathway

The lower metabolic steps of the cholesterol catabolic pathway should degrade the 2‐hydroxyhexa‐2,4‐dienoic and DOHNAA acids leading to metabolites able to enter the central metabolic pathways (Kieslich, 1985), but the enzymes involved in this downward pathway have not been yet investigated in detail.

Most likely the catabolism of the 2‐hydroxyhexa‐2,4‐dienoic acid involves genes similar to the tesE, tesF and tesG testosterone catabolic genes from C. testosteroni (Horinouchi et al., 2005). In this sense, the 2‐hydroxyhexa‐2,4‐dienoic acid can be metabolized to 4‐hydroxy‐2‐oxo‐hexanoic acid by the TesE‐like hydratase [Fig. 2 (12)] that eventually will be transformed by the action of TesG‐like aldolase into pyruvic acid and propionaldehyde that is further transformed into propionic acid by the action of TesF‐like aldehyde dehydrogenase (Horinouchi et al., 2005). Although it has been suggested that TesF could render propionyl‐CoA, the similarity of TesF with CoA acylating aldehyde dehydrogenases is very low and therefore, the most probably end‐product should be propionic acid.

DOHNAA would be further transformed to succinic acid and it is assumed that the first step in the degradation of this compound could be the elimination of a propionyl moiety, to produce 9,17‐dioxo‐1,2,3,4,5,6,10,19‐octanorandrostan‐7‐oic acid [Fig. 2 (13)] through a typical β‐oxidation (Kieslich, 1985). This reaction is carried out in two steps, i.e. the activation of DOHNAA by ATP and coenzyme A, followed by a reduction of DOHNAA‐CoA by an NADPH‐dependent dehydrogenase (Miclo and Germain, 1990). The enzymes specifically involved in these catabolic steps have not been precisely identified nor characterized so far.

Biosensors for cholesterol analysis in biological samples

The increase of cardiovascular disorders mainly due to hypercholesterolemia make the quantification of cholesterol levels an important clinical target and therefore several biosensors have been developed for this purpose (Arya et al., 2008; Jubete et al., 2009; Solanki et al., 2011). Biosensors for cholesterol are also useful in the food industry to maintain quality control (Bragagnolo and Rodriguez‐Amaya, 2002; Hwang et al., 2003; Jubete et al., 2009). Some of these cholesterol biosensors are based on cholesterol degrading enzymes (Table 2).

ChOxs are widely utilized for the enzymatic analyses of cholesterol. The most common source of ChOxs are from Streptomyces hygroscopicus, B. sterolicum and Pseudomonas fluorescens (Arya et al., 2008; Nien et al., 2009; Singh et al., 2009). Because 70% of cholesterol exists in ester form and 30% as free form in blood samples, cholesterol esterases are also included in biosensors together with ChOxs (Arya et al., 2008; Shih et al., 2009). In addition, horseradish peroxidase has been used in the fabrication of photometry based biosensors (Arya et al., 2008). This enzyme can be also coupled to 4‐aminoantipyrine and phenol to determine the hydrogen peroxide released in the ChOx reaction, a method routinely used in clinical laboratories (Pollegioni et al., 2009). To avoid the interference of blood pigments in colorimetric assays some electrochemical methods based on oxygen consumption by ChOx reaction have been also developed (Cheillan et al., 1989; Pollegioni et al., 2009).

Microbial ChDHs, which catalyse the conversion of 3β‐hydroxy‐5‐ene‐steroids to 3‐oxo‐4‐ene‐steroids using NAD or NADP as cofactors, are also utilized in cholesterol biosensors (Ishige et al., 2009; Wallace‐Davis et al., 2011).

Several authors have also shown that the P450scc cytochrome, an isoform of the P450 (CYP) cytochrome superfamily, which catalyses the side‐chain cleavage of cholesterol to pregnenolone in the presence of molecular oxygen and NADPH‐ferrihaemoprotein reductase (Carrara et al., 2008), can be utilized for determination of cholesterol with high sensitivity (Antonini et al., 2004; Paternolli et al., 2004; Shumyantseva et al., 2004; 2005). The highly specific conversion of steroid hormones by cythocromes makes feasible a specific substrate determination in complex media by using CYP based sensors (Bistolasa et al., 2005)

Biotransformations

The synthesis of the complex steroid molecules requires very specific reactions, and the utilization of microorganisms should enable the performance of reactions of high regio‐ and stereo‐selectivity. Moreover, the mild conditions required for bioconversions as well as the more ecological processes compared with chemical synthesis make the high yield biological production an interesting field to develop.

The importance of microbial biotransformations in the production of steroid drugs and hormones became a reality when the 11α‐hydroxylation of progesterone by a Rhizopus species was patented in 1952 (Hogg, 1992). Since then, microbial reactions for the transformation of steroids have proliferated and specific transformation steps have been incorporated into numerous partial syntheses of steroids with therapeutic use and commercial value (Mahato and Garai, 1997). Although some of these bioconversions are well‐established, increasing the efficiency of the existing processes as well as identifying new potentially useful bioconversions, are two current objectives of the steroid industry (Fernandes et al., 2003).

Microbial bioconversion has been focused mainly in steroid hydroxylation, Δ¹‐dehydrogenation and sterol side‐chain cleavage that associated to chemical synthesis steps, have enhanced the large‐scale production of natural and modified steroid analogues. The use of whole cells instead of enzymes is preferred as the production costs are lower and it is possible to perform multi‐steps conversions with a single biocatalyst (Bortolini et al., 1997). The manufactured steroid compounds have a wide range of therapeutic applications as anti‐inflammatory, immunosuppressive, diuretic, anabolic, contraceptive agents, breast and prostate cancer and anti‐obesity agents, among others. The therapeutic action of steroid hormones has been traditionally associated to their binding to the respective intracellular receptors, which act as transcription factors in the regulation of gene expression. Some steroid molecules are also called neurosteroids due to their role as memory enhancers, inducers of endocrine response to stress, anxiolytic agents, anticonvulsants, antidepressives and neuroprotective effect (Fernandes et al., 2003).

Many different steroids have been used for biotransformations, but specifically cholesterol obtained from animal fats and oils, such as lard, beef tallow, milk fat or fish oil has been also employed as starting raw material. It has been biotransformed into different compounds such as 17‐ketosterols, AD, 9α‐OH‐AD and ADD (Fernandes et al., 2003). Microorganisms capable of oxidizing cholesterol have a serious draw‐back for steroid synthesis, as they do not only degrade the side‐chain but usually also cause the complete undesirable fission of the steroid skeleton (Kieslich, 1980). Some of the microorganisms capable of cleavage the side‐chain of cholesterol and used by their potential interest as biocatalysts are R. coralline, Arthrobacter simplex, R. equi, Mycobacterium fortuitum, R. erythropolis, M. neoaurum or Micrococcus roseus (Liu and Lee, 1992; Shi et al., 1992; Ahmed and Johri, 1993; Ahmed et al., 1993; Lee et al., 1993; Smith et al., 1993; Srivastava and Patil, 1994; Liu et al., 1994a; Mahato and Garai, 1997; van der Geize and Dijkhuizen, 2004; Sripalakit et al., 2006; Molchanova et al., 2007). Mycobacterium sp. NRRL B‐3805 (Liu et al., 1994b; Liu and Lo, 1997) and Lactobacillus bulgaricus (Kumar et al., 2001) have been used to produce testosterone from cholesterol using a single strain. Production of ADD from cholesterol using two‐step microbial transformation has been reported (Lee et al., 1993). First, cholesterol is converted to cholestenone by the inducible ChOx of A. simplex, and second, cholestenone is transformed into ADD by Mycobacterium sp. (Mahato and Garai, 1997). Some of the microorganisms used to produce cholestenone from cholesterol are Mycobacterium sp. (Smith et al., 1993), R. equi (Ahmed and Johri, 1993), Escherichia (Panchishina, 1992), Nocardia rubra (Osipowicz et al., 1992), A. simplex (Liu et al., 1994a), R. erythropolis (Jadoun and Bar, 1993), R. equi (Myamato and Toyoda, 1994), Agrobacterium sp. M4 (Mahato and Garai, 1997; Yazdi et al., 2000).

Microbial transformation of steroids can also be an environmentally beneficial process. Dias and colleagues (2002) studied the composition of different industrial waste materials for their steroid/cholesterol content in order to find out if these industrial by‐products were suitable as steroid sources for the microbiological production of AD and ADD. They were used as a substrate for microbial degradation by a Mycobacterium sp. strain and proved to be easily converted to AD and ADD. Thus, a profitable application of these wastes could be the production of sterol‐rich fractions, reducing the pollution loads.

Probiotics

Probiotics are living microorganisms, which upon ingestion in certain numbers exert health benefits on the host beyond inherent basic nutrition (Guarner and Schaafsma, 1998). Although many in vitro and in vivo studies have shown that the administration of probiotics reduces serum/plasma total cholesterol, LDL‐cholesterol and triglycerides or increases HDL‐cholesterol, their hypocholesterolemic effects remain controversial (Ooi and Liong, 2010). The species with cholesterol‐lowering effects studied include genera as Lactobacillus, Lactococcus, Enterococcus, Streptococcus or Bifidobacterium (Pereira and Gibson, 2002).

Probiotics have been suggested to reduce cholesterol via various mechanisms that do not necessarily imply the transformation of cholesterol by the probiotic strains, e.g. enzymatic deconjugation of bile acids by bile‐salt hydrolases of probiotics (Lambert et al., 2008), assimilation of cholesterol (Pereira and Gibson, 2002), co‐precipitation of cholesterol with deconjugated bile (Liong and Shah, 2006), cholesterol binding to cell walls of probiotics (Liong and Shah, 2005), incorporation of cholesterol into the cellular membranes of probiotics during growth (Lye et al., 2010a), production of short‐chain fatty acids upon fermentation by probiotics in the presence of prebiotics (De Preter et al., 2007) and conversion into coprostanol (Lye et al., 2010b).

As stated above, the most frequent transformation of cholesterol in the intestinal anoxic habitats is a biogenic reduction of its double bond yielding coprostanol, which is directly excreted in faeces (Li et al., 1995; Harder and Probian, 1997). Many intestinal fermenting bacteria are able to catalyse this reaction (Groh et al., 1993; Freier et al., 1994) decreasing the amount of absorbed cholesterol and leading to a reduced concentration in the physiological cholesterol pool (Ooi and Liong, 2010). Lye and colleagues (2010b) evaluated the conversion of cholesterol to coprostanol by strains of lactobacilli such as L. acidophilus, L. bulgaricus and Lactobacillus casei detecting both intracellular and extracellular cholesterol reductases in these strains, indicating possible intracellular and extracellular conversions of cholesterol to coprostanol. Cholesterol reductase has been also directly administered to humans to convert cholesterol to coprostanol in the small intestines to reach a bloodstream cholesterol‐lowering effect (Ooi and Liong, 2010).

One of the best‐studied mechanisms proposed for the cholesterol‐lowering effects of probiotics is the enzymatic deconjugation of bile acids by bile salt hydrolases (Begley et al., 2006). Bile, a water‐soluble end‐product of cholesterol in the liver that consists of cholesterol, phospholipids, conjugated bile acids, bile pigments and electrolytes, is released into the duodenum upon ingestion of food. When it is deconjugated by bile salt hydrolases‐producing strains, bile acids are less soluble, being no absorbed by intestines and eliminated in the faeces. Cholesterol is then used to synthesize new bile acids as a homeostatic response, resulting in a hypocholesterolemic effect of probiotics (Begley et al., 2006; Ooi and Liong, 2010).

The cholesterol‐lowering effects are also attributed to the ability of probiotic strains to bind cholesterol, a process that is growth‐dependent and strain‐specific. However, although growing cells remove more cholesterol than dead cells, the heat‐killed cells can still remove cholesterol from media, indicating that some cholesterol is bound to the cellular surface (Usman and Hosono, 1999). During the probiotic growth, the presence of cholesterol increases the concentration of saturated and unsaturated fatty acids, leading to increased membrane strength and subsequently higher cellular resistance towards lysis (Lye et al., 2010a; Ooi and Liong, 2010).

We thank Dr E. Díaz and Dr M.A. Prieto for the critical reading of the manuscript and helpful discussions. This work was supported by grants from the Ministry of Science and Innovation (BFU2006‐15214‐C03‐01, BFU2009‐11545‐C03‐03), and an I3P predoctoral fellowship from the Consejo Superior de Investigaciones Científicas.