Key role of alternative oxidase in lovastatin solid-state fermentation.

Lovastatin is a commercially important secondary metabolite produced by Aspergillus terreus, either by solid-state fermentation or by submerged fermentation. In a previous work, we showed that reactive oxygen species (ROS) accumulation in idiophase positively regulates lovastatin biosynthetic genes. In addition, it has been found that lovastatin-specific production decreases with aeration in solid-state fermentation (SSF). To study this phenomenon, we determined ROS accumulation during lovastatin SSF, under high and low aeration conditions. Paradoxically, high aeration caused lower ROS accumulation, and this was the underlying reason of the aeration effect on lovastatin production. Looking for a mechanism that is lowering ROS production under those conditions, we studied alternative respiration. The alternative oxidase provides an alternative route for electrons passing through the electron transport chain to reduce oxygen. Here, we showed that an alternative oxidase (AOX) is expressed in SSF, and only during idiophase. It was shown that higher aeration induces higher alternative respiration (AOX activity), and this is a mechanism that limits ROS generation and keeps them within healthy limits and adequate signaling limits for lovastatin production. Indeed, the aox gene was induced in idiophase, i.e., at the time of ROS accumulation. Moreover, exogenous ROS (H2O2), added to lovastatin solid-state fermentation, induced higher AOX activity. This suggests that high O2 availability in SSF generates dangerously high ROS, so alternative respiration is induced in SSF, indirectly favoring lovastatin production. Conversely, alternative respiration was not detected in lovastatin-submerged fermentation (SmF), although exogenous ROS also induced relatively low AOX activity in SmF.

Lovastatin production: From molecular basis to industrial process optimization.

Lovastatin, composed of secondary metabolites produced by filamentous fungi, is the most frequently used drug for hypercholesterolemia treatment due to the fact that lovastatin is a competitive inhibitor of HMG-CoA reductase. Moreover, recent studies have shown several important applications for lovastatin including antimicrobial agents and treatments for cancers and bone diseases. Studies regarding the lovastatin biosynthetic pathway have also demonstrated that lovastatin is synthesized from two-chain reactions using acetate and malonyl-CoA as a substrate. It is also known that there are two key enzymes involved in the biosynthetic pathway called polyketide synthases (PKS). Those are characterized as multifunctional enzymes and are encoded by specific genes organized in clusters on the fungal genome. Since it is a secondary metabolite, cultivation process optimization for lovastatin biosynthesis has included nitrogen limitation and non-fermentable carbon sources such as lactose and glycerol. Additionally, the influences of temperature, pH, agitation/aeration, and particle and inoculum size on lovastatin production have been also described. Although many reviews have been published covering different aspects of lovastatin production, this review brings, for the first time, complete information about the genetic basis for lovastatin production, detection and quantification, strain screening and cultivation process optimization. Moreover, this review covers all the information available from patent databases covering each protected aspect during lovastatin bio-production.

Response surface methodology for optimization of production of lovastatin by solid state fermentation

Lovastatin, an inhibitor of HMG-CoA reductase, was produced by solid state fermentation (SSF) using a strain of Aspergillus terreus UV 1718. Different solid substrates and various combinations thereof were evaluated for lovastatin production. Wheat bran supported the maximum production (1458 ± 46 µg g-1 DFM) of lovastatin. Response surface methodology (RSM) was applied to optimize the medium constituents. A 2(4) full-factorial central composite design (CCD) was chosen to explain the combined effects of the four medium constituents, viz. moisture content, particle size of the substrate, di-potassium hydrogen phosphate and trace ion solution concentration. Maximum lovastatin production of 2969 µg g-1 DFM was predicted by the quadratic model which was verified experimentally to be 3004 ± 25 µg g-1 DFM. Further RSM optimized medium supplemented with mycological, peptone supported highest yield of 3723.4±49 µg g-1 DFM. Yield of lovastatin increased 2.6 fold as with compared to un-optimized media.

Lovastatin biosynthetic genes of Aspergillus terreus are expressed differentially in solid-state and in liquid submerged fermentation.

Molecular studies were performed to establish the causes of the superior lovastatin productivity of a novel solid-state fermentation (SSF) process, in relation with liquid submerged fermentation (SmF; 20 mg/g vs. 0.65 mg/ml). In SSF, biosynthetic genes lovE and lovF transcripts accumulated to high levels from day 1 to day 7. In this period, lovE transcript showed 4.6-fold higher accumulation levels (transcription) than the highest level detected in SmF (day 5). lovF transcript showed two-fold higher expression than the highest point in SmF. In SmF, the expression was only detected clearly on day 5 and, showing a 50% decrease, on day 7. These results show that the higher lovastatin production in SSF is related to a more intense transcription of these biosynthetic genes. A strong expression of gldB gene in lovastatin SSF indicated that Aspergillus terreus senses osmotic stress during the course of SSF, but not in SmF. However, when a liquid medium of identical concentration was used in SmF, lovastatin production decreased in SSF.

Cholesterol potentiates beta-amyloid-induced toxicity in human neuroblastoma cells: involvement of oxidative stress.

Alterations in brain cholesterol concentration and metabolism seem to be involved in Alzheimer's disease (AD). In fact, several experimental studies have reported that modification of cholesterol content can influence the expression of the amyloid precursor protein (APP) and amyloid beta peptide (Abeta) production. However, it remains to be determined if changes in neuronal cholesterol content may influence the toxicity of Abeta peptides and the mechanism involved. Aged mice, AD patients and neurons exposed to Abeta, show a significant increase in membrane-associated oxidative stress. Since Abeta is able to promote oxidative stress directly by catalytically producing H(2)O(2) from cholesterol, the present work analyzed the effect of high cholesterol incorporated into human neuroblastoma cells in Abeta-mediated neurotoxicity and the role of reactive oxygen species (ROS) generation. Neuronal viability was studied also in the presence of 24S-hydroxycholesterol, the main cholesterol metabolite in brain, as well as the potential protective role of the lipophilic statin, lovastatin.

[Comparison between lovastatin and gemfibrozil in treatment of primary hyperlipidemias]. / Comparação entre a lovastatina e o gemfibrozil no tratamento de hiperlipidemias primárias.

PURPOSE: To compare the effects of lovastatin and gemfibrozil in patients with primary hyperlipidemias. PATIENTS AND METHODS: Forty patients with cholesterolemia over 200 mg/dl and triglyceridemia not higher than 350 mg/dl, excluded secondary causes, were selected. Twenty patients received lovastatin and 20 gemfibrozil. In order to establish the lipid profile, blood samples were taken after 2 months without medication, after 4 weeks of diet and placebo and after 6 and 12 weeks of active treatment. Biochemical profile was determined before and after the treatment with active drug. RESULTS: Thirty nine patients completed the study. Total and LDL-cholesterol were significantly reduced (p less than 0.05) by both drugs but lovastatin had greater effect. Only gemfibrozil reduced triglycerides significantly. Neither drug had significant effects on HDL-cholesterol. The tolerance was satisfactory; only one patient (using gemfibrozil) needed to stop the treatment due to gastrointestinal side effects. The biochemical profile did not present any significant alteration. CONCLUSION: Both drugs produced useful effects on the lipid profile. Lovastatin produced greater reductions of total and LDL-cholesterol, while gemfibrozil was more active reducing triglycerides. Neither drug changed significantly the HDL-cholesterol.

Comparação entre lovastatina e o gemfibrozil no tratamento de hiperlipidemias primárias / Comparison between lovastatin and gemfibrozil in treatment of primary hyperlipidemias

Purpose - To compare the effects of lovastatin and gemfibrozil in patients with primary hyperlipidemias. Patients and Methods - Forty patients with cholesterolemia over 200 mgldl and triglyceridemia not higher than 350 mp/dl, excluded secondary causes, were selected. Twenty patients received lovastatin and 20 gemfibrozil. In order to establish the lipid profile, blood samples were taken after 2 months without medication, after 4 weeks of diet and placebo and after 6 and 12 weeks active treatment. Biochemic profile was determined before and after the treatment with active drug. Results - Thirty nine patients completed the study. Total and LDL-cholesterol were significantly reduced (p < 0.05) by both drugs but lovastatin had greater effect. Only gemfibrozil reduced triglycerides significantly. Neither drug had significant effects on HDL-cholesterol. The tolerance was satisfactory; only one patient (using gemfibrozil) needed to stop the treatment due to gastrointestinal side effects. The biochemic profïle did not present any significant alteration. Conclusion - Both drugs produced useful effects on the lipid profile. Lovastatin produced greater reductions of total and LDL-cholesterol, while gemfibrozil was more active reducing triglycerides. Neither drug changed significantly the HDL-cholesterol