Biotechnological Production of Statins: Metabolic Aspects and Genetic Approaches.

Statins are drugs used for people with abnormal lipid levels (hyperlipidemia) and are among the best-selling medications in the United States. Thus, the aspects related to the production of these drugs are of extreme importance for the pharmaceutical industry. Herein, we provide a non-exhaustive review of fungal species used to produce statin and highlighted the major factors affecting the efficacy of this process. The current biotechnological approaches and the advances of a metabolic engineer to improve statins production are also emphasized. The biotechnological production of the main statins (lovastatin, pravastatin and simvastatin) uses different species of filamentous fungi, for example Aspergillus terreus. The statins production is influenced by different types of nutrients available in the medium such as the carbon and nitrogen sources, and several researches have focused their efforts to find the optimal cultivation conditions. Enzymes belonging to Lov class, play essential roles in statin production and have been targeted to genetic manipulations in order to improve the efficiency for Lovastatin and Simvastatin production. For instance, Escherichia coli strains expressing the LovD have been successfully used for lovastatin production. Other examples include the use of iRNA targeting LovF of A. terreus. Therefore, fungi are important allies in the fight against hyperlipidemias. Although many studies have been conducted, investigations on bioprocess optimization (using both native or genetic- modified strains) still necessary.

Protein engineering of CYP105s for their industrial uses.

Cytochrome P450 enzymes belonging to the CYP105 family are predominantly found in bacteria belonging to the phylum Actinobacteria and the order Actinomycetales. In this review, we focused on the protein engineering of P450s belonging to the CYP105 family for industrial use. Two Arg substitutions to Ala of CYP105A1 enhanced its vitamin D3 25- and 1α-hydroxylation activities by 400 and 100-fold, respectively. The coupling efficiency between product formation and NADPH oxidation was largely improved by the R84A mutation. The quintuple mutant Q87W/T115A/H132L/R194W/G294D of CYP105AB3 showed a 20-fold higher activity than the wild-type enzyme. Amino acids at positions 87 and 191 were located at the substrate entrance channel, and that at position 294 was located close to the heme group. Semi-rational engineering of CYP105A3 selected the best performing mutant, T85F/T119S/V194N/N363Y, for producing pravastatin. The T119S and N363Y mutations synergistically had remarkable effects on the interaction between CYP105A3 and putidaredoxin. Although wild-type CYP105AS1 hydroxylated compactin to 6-epi-pravastatin, the quintuple mutant I95T/Q127R/A180V/L236I/A265N converted almost all compactin to pravastatin. Five amino acid substitutions by two rounds of mutagenesis almost completely changed the stereo-selectivity of CYP105AS1. These results strongly suggest that the protein engineering of CYP105 enzymes greatly increase their industrial utility. This article is part of a Special Issue entitled: Cytochrome P450 biodiversity and biotechnology, edited by Erika Plettner, Gianfranco Gilardi, Luet Wong, Vlada Urlacher, Jared Goldstone.

Single-step fermentative production of the cholesterol-lowering drug pravastatin via reprogramming of Penicillium chrysogenum.

The cholesterol-lowering blockbuster drug pravastatin can be produced by stereoselective hydroxylation of the natural product compactin. We report here the metabolic reprogramming of the antibiotics producer Penicillium chrysogenum toward an industrial pravastatin production process. Following the successful introduction of the compactin pathway into the ß-lactam-negative P. chrysogenum DS50662, a new cytochrome P450 (P450 or CYP) from Amycolatopsis orientalis (CYP105AS1) was isolated to catalyze the final compactin hydroxylation step. Structural and biochemical characterization of the WT CYP105AS1 reveals that this CYP is an efficient compactin hydroxylase, but that predominant compactin binding modes lead mainly to the ineffective epimer 6-epi-pravastatin. To avoid costly fractionation of the epimer, the enzyme was evolved to invert stereoselectivity, producing the pharmacologically active pravastatin form. Crystal structures of the optimized mutant P450(Prava) bound to compactin demonstrate how the selected combination of mutations enhance compactin binding and enable positioning of the substrate for stereo-specific oxidation. Expression of P450(Prava) fused to a redox partner in compactin-producing P. chrysogenum yielded more than 6 g/L pravastatin at a pilot production scale, providing an effective new route to industrial scale production of an important drug.

Evaluation of a method using high performance liquid chromatography with ultraviolet detection for the determination of statins in macromycetes of the genus Pleurotus cultivated by fermentation processes.

The applicability of high-performance liquid chromatography with ultraviolet light (HPLC-UV) for the determination of the presence of statins in macromycetes of the genus Pleurotus was analyzed. The fungi were obtained by liquid-state fermentation (LSF) using unconventional sources of carbon as substrates and solid-state fermentation (SSF) employing agro industrial wastes. Five statins were used as standards: lovastatin and simvastatin in the lactone form (LOVL and SIML), their corresponding hydro-acidic forms (LOVH and SIMH) and pravastatin (PRA). The following measures were evaluated: the linearity, accuracy and precision, detection limit (DL) and quantification limit (QL). The results demonstrated HPLC-UV to be an effective tool for detecting the presence of statins in extracts of LSF and SSF products. Likewise, it was hypothesized that the strains that were used for the study do not produce statins. This finding highlights the importance of continuing to evaluate other strains of the same genus by using techniques such as HPLC to first separate sufficient quantities of the compounds that were detected using the standard technique but that did not match the retention time (tR) of any of the standards used.

Construction of a novel expression vector in Pseudonocardia autotrophica and its application to efficient biotransformation of compactin to pravastatin, a specific HMG-CoA reductase inhibitor.

The novel plasmid vector (pTAOR4-Rev) suitable for gene expression in actinomycete strains of Pseudonocardia autotrophica was constructed from 2 P. autotrophica genetic elements, the novel replication origin and the acetone-inducible promoter. The replication origin was isolated from the endogenous plasmid of strain DSM 43082 and the acetone-inducible promoter was determined by analysis of the upstream region of an acetaldehyde dehydrogenase gene homologue in strain NBRC 12743. P. autotrophica strains transformed with pTAOR4-P450, carrying a gene for cytochrome P450 monooxygenase, expressed P450 from the acetone-inducible promoter, as verified by SDS-PAGE and spectral analysis. The biotransformation test of acetone-induced resting cells prepared from a strain of P. autotrophica carrying pTAOR4 that harbors a compactin (CP)-hydroxylating P450 gene revealed 3.3-fold increased production of pravastatin (PV), a drug for hypercholesterolemia. Biotransformation of CP by the same strain in batch culture yielded PV accumulation of 14.3 g/l after 100 h. The expression vector pTAOR4-Rev and its function-enhancing derivatives provide a versatile approach to industrial biotransformation by Pseudonocardia strains, which can be good hosts for P450 monooxygenase expression.

Biotechnological production and applications of statins.

Statins are a group of extremely successful drugs that lower cholesterol levels in blood; decreasing the risk of heath attack or stroke. In recent years, statins have also been reported to have other biological activities and numerous potential therapeutic uses. Natural statins are lovastatin and compactin, while pravastatin is derived from the latter by biotransformation. Simvastatin, the second leading statin in the market, is a lovastatin semisynthetic derivative. Lovastatin is mainly produced by Aspergillus terreus strains, and compactin by Penicillium citrinum. Lovastatin and compactin are produced industrially by liquid submerged fermentation, but can also be produced by the emerging technology of solid-state fermentation, that displays some advantages. Advances in the biochemistry and genetics of lovastatin have allowed the development of new methods for the production of simvastatin. This lovastatin derivative can be efficiently synthesized from monacolin J (lovastatin without the side chain) by a process that uses the Aspergillus terreus enzyme acyltransferase LovD. In a different approach, A. terreus was engineered, using combinational biosynthesis on gene lovF, so that the resulting hybrid polyketide synthase is able to in vivo synthesize 2,2-dimethylbutyrate (the side chain of simvastatin). The resulting transformant strains can produce simvastatin (instead of lovastatin) by direct fermentation.

Screening of compactin-resistant microorganisms capable of converting compactin to pravastatin.

A simple method of using compactin for effective screening of microbial strains with high hydroxylation activity at the 6beta position of compactin was developed. Agar plates containing different carbon sources and 500 microg compactin mL(-1) were used to screen the microorganisms that can convert compactin to pravastatin. About 100 compactin-resistant strains were isolated from the Basal agar containing 7% (w/v) mannitol as a carbon source, in which two bacteria, Pseudomocardia autotrophica BCRC 12444 and Streptomyces griseolus BCRC 13677, capable of converting compactin to pravastatin with the yield of 20 and 32% (w/w), respectively, were found. High-performance liquid chromatography using C-18 column and two sequential mobile phases, 30% and 50% (v/v) acetonitrile, was also established to simultaneously determine the concentration of compactin and pravastatin in the culture broth. As such, about 2% of target microorganisms could be obtained from the screening program.

Bioconversion of compactin into pravastatin by Streptomyces sp.

Streptomyces sp. Y-110, isolated from soil, modified compactin to pravastatin, a therapeutic agent for hypercholesterolemia. In a batch culture, the highest production of pravastatin was 340 mg l(-1) from 750 mg compactin l(-1) in 24 h. By intermittent feeding of compactin into the culture medium, both the compactin concentration and its conversion increased to 2000 mg l(-1) and 1000 mg pravastatin l(-1), respectively, with the conversion rate of 10 mg l(-1) h(-1). Continuous feeding of compactin increased production of pravastatin to 15 mg l(-1) h(-1).

[Tracing the past half century of my microbial transformation studies].

In the middle of 1950's, microbial transformation technology was introduced into the field of synthetic chemistry as a new methodology. There was a sudden interest in research on the problems of producing steroid hormones by microbial transformation. At that time, the first project entitled "The Study for Microbial Transformation of Steroids", the "Tsuda Project", was established in the Institute of Applied Microbiology (IAM), University of Tokyo, in the spring 1956, in which I took part. This paper summarizes a number of results of our microbial transformation reactions not only in the synthesis of steroidal compounds, but also more broadly for other organic compounds, such as pravastatin, etc. The results are divided into five categories: 1) Microbial transformation of steroids, 2) Correlation between isolation sources of Pseudomonas spp. and their transformation activities, 3) Fermentation Production of prednisolone by Bacillus pulvifaciens SANK 71760, 4) Microbial transformation of siccanin, and 5) Development and fermentation production of pravastatin. About 30 years later, almost at the end of my microbial transformation studies, I had the opportunity to find some microbial strains having superior hydroxylation ability of ML-236BNa to pravastatin. Fortunately, Streptomyces carbophilus SANK 62585 was finally selected as a potent microbial converter with the formation of a lesser amount of by-products. With the view of industrial production of pravastatin, many studies and improvements were made to the culturing conditions to obtain productivity available commercially.

Crystallization and preliminary X-ray diffraction analysis of cytochrome P450sca-2 from Streptomyces carbophilus involved in production of pravastatin sodium, a tissue-selective inhibitor of HMG-CoA reductase.

Cytochrome P450sca-2 from Streptomyces carbophilus catalyzes the hydroxylation of ML-236B sodium salt to pravastatin sodium, a tissue-selective inhibitor of 3-hydroxy-3-methylglutaryl-coenzymeA (HMG-CoA) reductase. HMG-CoA reductase is a key enzyme in cholesterol biosynthesis. Crystals of the protein were obtained by the vapour-diffusion method, using ammonium sulfate as a precipitant. The crystals belong to the trigonal space group P3121 (or its enantiomorph, P3221) with unit-cell dimensions a = 103.5, c = 79.8 A. Assuming the presence of one molecule in the asymmetric unit, the calculated value of Vm is 2.68 A3 Da-1. A native data set was collected to a resolution of 2.2 A.

Molecular approaches for production of pravastatin, a HMG-CoA reductase inhibitor: transcriptional regulation of the cytochrome p450sca gene from Streptomyces carbophilus by ML-236B sodium salt and phenobarbital.

We have characterized the transcriptional regulation of ML-236B.Na and phenobarbital-inducible cytochrome P450sca-2 (CytP450sca-2) from Streptomyces carbophilus, an industrial pravastatin-producing strain. ML-236B.Na and phenobarbital enhanced the expression of the cytP450sca-2 gene in S. carbophilus. The cytP450sca-2 gene was also ML-236B.Na-inductive in S. lividans. Analysis of various deletion and mutation of the 5'-flanking region of the cytP450sca-2 gene revealed that the 1-kb region was required for ML-236B.Na-dependent CytP450sca-2 induction. We have found a putative ORF in the 5'-flanking region that encodes a protein of 174 amino acid residues containing a helix-turn-helix DNA-binding motif. A gel mobility shift assay showed that the protein was bound by an imperfect palindromic sequence between -46bp and -24bp in the 5'-flanking region, and ML-236B.Na was found to inhibit its binding. These findings suggest that induction of cytP450sca-2 is negatively regulated at the transcriptional level and that the protein encoded by the putative ORF is possibly functional as a repressor of the cytP450sca-2 gene.

[Research and development of pravastatin].

The attempts to find a potent inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase which catalyzes the rate limiting step of cholesterol biosynthesis were started from 1971. The first potent inhibitor, ML-236B (compactin), was found from the culture broth of Penicillium citrinum. Among many derivatives of ML-236B, pravastatin sodium (hereafter refer to pravastatin) was finally selected because of its potency and tissue selectivity. Since pravastatin has a hydroxyl group at 6 beta position in the skeleton of decaline of ML-236B, the microbial hydroxylation was adopted for the production of pravastatin. Streptomyces carbophilus was finally chosen as a potent converter with the formation of a lesser amount of by-products. For the sake of industrial production of pravastatin, many devices and improvements were performed for selecting high potent strains and for culturing conditions both with ML-236B and pravastatin. Pravastatin strongly inhibited the sterol synthesis in freshly isolated rat hepatocytes, but only weakly inhibited in the cells from nonhepatic tissues. This selective inhibition of pravastatin in sterol synthesis was further confirmed by ex vivo and in vivo experiments by using rats and mice. Pravastatin markedly reduced serum cholesterol levels in dogs, monkeys and rabbits, including Watanabe heritable hyperlipidemic (WHHL) rabbits, an animal model for familial hypercholesterolemia. Pravastatin showed the preventive effect on the development of coronary atherosclerosis and xanthoma in young WHHL rabbits in consequence of maintaining the serum cholesterol levels low. In the clinical trials, pravastatin significantly reduced serum cholesterol and low density lipoprotein cholesterol levels, whereas inversely increased high density lipoprotein cholesterol levels.