Microbial Statins: Production Process and Potential for New Substances

Although most of the newer statins are synthetic, microbial statins are of interest because its production skips several synthetic steps, providing a high value-added molecule from low-cost substrates in an eco-friendly process.

ML-236B, known as compactin or mevastatin, was the first breakthrough in efforts to find a hypolipidemic agent by Akira Endo (1976). By 1980, Merck had discovered lovastatin which has been shown to be chemically very similar to mevastatin, differing by one methyl group (absent in mevastatin). Endo also had discovered the same compound, lovastatin (Brown and Goldstein 2004). Soon after the development of lovastatin, several screening efforts led to the development of similar molecules.

Fig. 13.4 Microbial statins biosynthetic pathway

The biosynthetic pathway for microbial statins starts with the condensation of acetyl and malonyl-CoA (Auclair et al. 2001; Barrios-gonzález and Miranda 2010; Komagata et al. 1989) and proceeds through the mevalonate pathway (Fig. 13.4).

Lovastatin

13.4.1.1 General

Lovastatin is a fungal secondary metabolite discovered in the seventies and introduced in the American market in the 1980s as Mevacor by Merck. It was the first statin to be approved by FDA, and it was formerly called as mevinolin or monacolin K. Lovastatin is administered as β-hydroxy lactone which over the time converts in vivo to the respective hydroxy acid form, partly similar to HMG-CoA. The hydroxyl acid form is a weak acid (pKa = 4.31) with a molar mass of 422.55 while its lactone form has a higher (pKa = 13.5) (Bizukojc et al. 2007; Brown and Goldstein 2004; Lisec et al. 2012; Seenivas et al. 2008; Seraman et al. 2010).

13.4.1.2 Current and Potential Uses of Lovastatin

Besides its cholesterol-lowering properties, lovastatin has been reported as a potential therapeutic agent for the treatment of various types of cancer. Recent in vitro studies have shown that lovastatin inhibits proliferation of anaplastic thyroid cancer cells through upregulation of p27 by interfering with the Rho/ROCK (serine/ threonine kinase Rho kinase)-mediated pathwaythis pathway has been suggested to be involved in the regulation of cancer cell motility. Other studies have shown endothelial protection function of lovastatin in the presence of hyperglycemia. Endothelial dysfunction, such as decreased endothelium-dependent vasorelaxation, plays a key role in the pathogenesis of diabetic vascular disease. Lovastatin was able to improve mesenteric responses to acetylcholine (Gajendragadkar et al. 2009; Zhong et al. 2011). Oxidative stress has been linked to the cause of many human diseases, such as heart failure, coronary artery and chronic kidney disease, and neurodegenerative disturbances. It arises from an imbalance between an excessive generation of reactive oxygen species, reactive nitrogen species, and insufficiency of antioxidant agents. Oral administration of lovastatin has been demonstrated to reduce oxidative stress and change the activities of antioxidant enzymes (Kumar et al. 2011).

13.4.1.3 Production Process

Potential Producers

Lovastatin is produced by a variety of filamentous fungi. Some of the important microbial sources are Monascus sp., Penicillium sp., and Aspergillus sp. Species were found to be the most significant producers of lovastatin, such as Monascus purpureus, Monascus ruber, Aspergillus terreus, and Aspergillus flavipes. Table 13.2 lists some species evaluated or developed for lovastatin production. Although several species produce low amounts of lovastatin, they are shown in order to illustrate the genera variability.

New rapid screening methods were developed to find new potential producers based on the activity of lovastatin against the yeast Candida albicans. In this method, the diameter of the inhibition zones (obtained on plates of Candida albicans) correlated linearly with the quantity of lovastatin impregnated in the paper disc (Bizukojc et al. 2007; Seraman et al. 2010; Vilches Ferrón et al. 2005). Other method for detecting lovastatin-producing strain is based on PCR for specific genes related to lovastatin synthesis, detecting suitable strains more quickly and effectively (Kim et al. 2011).

Table 13.2 Lovastatin production (in mg/L of fermented broth) by selected strains

Microorganism

Strain

mg/L

Reference

A. terreus

ATCC 20542

100

Porcel et al. (2008)

Penicillium citrinum

MTCC 1256

589

Ahmad et al. (2010)

Aspergillus terreus

Isolate

400

Szakács et al. (1998)

Aspergillus terreus

DRCC 122 (uv mutant)

2,200

Kumar et al. (2000)

Aspergillus terreus

Isolate

400

Samiee et al. (2003)

Monascus pilosus

MK-1 (a mutant strain)

725

Miyake et al. (2006)

Monascus purpureus

MTCC 369

351

Sayyad et al. (2007)

Biospora sp.

Isolate

13

Osman et al. (2011)

Cylindrocarpon radicicola

Isolate

7.1

Osman et al. (2011)

Penicillium spinulosum

Isolate

15.8

Osman et al. (2011)

Trichoderma viride

Isolate

36

Osman et al. (2011)

Mycelia sterilia

Isolate

15.3

Osman et al. (2011)

A. terreus

DSM 13596

310

Benedetti et al. (2002)

Fermentation

Submerged fermentation and solid-state fermentation (SSF) have been used for lovastatin production. Large-scale processes were developed using Aspergillus terreus in submerged fermentation. Enhanced strategies, such as the use of antibiotics, cultivation in fed-batch mode, and medium development, led to higher yields (Jia et al. 2010; Seenivas et al. 2008; Porcel et al. 2008).

Solid-state fermentation is a potential alternative to produce lovastatin which generates less effluent and uses less power. SSF uses various solid substrates, such as besan flour, barley, sago, and long-grain rice (all these substrates yield high lovastatin production, >110 mg/g dry substrate); mixed solids can also be used to formulate economical substrates for commercial production (Subhagar et al. 2009; Valera et al. 2005). The inocula for these processes may be either a liquid culture or a spore suspension; after inoculation, the fermenters are maintained at a temperature, pH, and aeration rate which are characteristics of each strain for several days. Typical values are 28 °C, pH 6.5, 1.5 vvm, and 7 days for Aspergillus terreus strains.

Culture Medium Characteristics

As with any fermentation product, the culture medium has a significant effect on the rate of production and yield of lovastatin. The type of carbon source (e.g., fructose, lactose, glycerol), nitrogen source (e.g., soybean meal, corn steep liquor, yeast extract), and the C:N mass ratio used in the medium influenced production of lovastatin and microbial biomass by A. terreus. The results have shown that the presence of excess carbon (slowly metabolizable carbon source) under nitrogen limitation greatly enhanced the rate of production of lovastatin. Nitrogen limitation diverts more carbon to lovastatin metabolic pathways (Casas López et al. 2003). Most liquid culture media use glucose as the main carbon source, but some authors suggest that this sugar strongly represses lovastatin synthesis (Miyake et al. 2006), which explains why continuous or fed-batch processes enhance the yield. In addition to glucose, several culture media also use starches and complex mixed sources, such as oatmeal, soybean meal, peptones, and yeast extract in the culture medium.

A variety of mineral nutrients is also added to the culture media, usually K2HPO4, MgSO4, and ammonia, urea, or nitrates. Microelements are seldom added, being provided by the complex nutrients used.

Studies have shown that the supplementation of the culture medium with B-group vitamins enhances lovastatin synthesis by Aspergillus terreus. It is probable that the synthesis of lovastatin requires a high throughput of coenzymes, thus the application of its precursors in the form of B-group vitamins would give a positive effect on lovastatin production (Bizukojc et al. 2007). The impact of other supplements, such as linoleic acid, has demonstrated that micromolar concentrations of this fatty acid enhance lovastatin yield. Possibly, early supplementation of linoleic acid anticipates the production of oxylipins, thus mimicking the critical cell mass necessary for the onset of lovastatin production (Sorrentino et al. 2010). Vegetable oils stand out as a promising substrate as an additional carbon source for lovastatin production (Sripalakit et al. 2011).

Downstream Processing of Lovastatin

As lovastatin has a very low polarity, the concentration from the culture broth may be carried out by liquid-liquid extraction. However, there is a substantial portion of intracellular lovastatin (Benedetti et al. 2002), which may not be easily extracted. In addition, the molecule may be oxidized if not properly processed, leading to hard to separate impurities—antioxidants or inert atmospheres should be used. After extraction and concentration, the statin is usually purified by crystallization, although chromatography and ion exchange steps may also be used. The final compound should have a purity of at least 99.5 %. Figure 13.5 illustrates a possible lovastatin downstream process.

13.4.1.4 Simvastatin Production (Derivatization of Lovastatin)

The natural product lovastatin can be derived in its analog semisynthetic simvastatin. Substitution of the α-methylbutyrate side chain with α-dimethylbutyrate is most effective in treating hypercholesterolemia while lowering undesirable side effects. An alternative method for the simvastatin synthesis is a selective enzymatic deacylation of lovastatin. Due to the effectiveness of simvastatin, numerous multistep syntheses from lovastatin to simvastatin have been described in the patent literature. For example, a process for preparing simvastatin from lovastatin or mevinolinic acid in salt form comprises treating either starting material with cyclopropyl or butyl amine. In another process, lovastatin was hydrolyzed to acid form and then isolated in the form of amine salt like cyclopropyl or t-octylpropyl

Fig. 13.5 Lovastatin downstream processing relying on crystallization operations

amines. The salts isolated were directly methylated without any protection or deprotection of hydroxyl groups. Then, simvastatin ammonium salts were converted to simvastatin by conventional methods of lactonization (Kumar et al. 1998; Vaid and Narula 2006).

 
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