Relación del estrés oxidante con la biosíntesis de lovastatina por Aspergillus terreus en fermentación sólida y líquida
This work represents the first attempt to establish a relationship between lovastatin biosynthesis and oxidative stress (EOX), through generation of Reactive Oxygen Species (ROS). In addition, an effort was made to establish the possible mechanism by which ROS can regulate the biosynthesis of this s...
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| Format: | doctoral thesis |
| Status: | Published version |
| Publication Date: | 2013 |
| Country: | México |
| Institution: | Universidad Autónoma Metropolitana |
| Repository: | Repositorio Institucional de la UAM Iztapalapa |
| Language: | Spanish |
| OAI Identifier: | oai:bindani.izt.uam.mx:q811kj79x |
| Online Access: | https://doi.org/10.24275/uami.q811kj79x |
| Access Level: | Open access |
| Keyword: | info:eu-repo/classification/LEM/Lovastatina -- Biosíntesis info:eu-repo/classification/LEM/Fermentación en estado sólido info:eu-repo/classification/LEM/Oxidative stress info:eu-repo/classification/LEM/Biotecnología info:eu-repo/classification/LEM/Solid-state fermentation info:eu-repo/classification/LEM/Biotechnology info:eu-repo/classification/LEM/Fermentación en estado líquido info:eu-repo/classification/LEM/Estrés oxidativo info:eu-repo/classification/LEM/Aspergillus terreus info:eu-repo/classification/LEM/Cholesterol Biosynthesis info:eu-repo/classification/cti/6 |
| Summary: | This work represents the first attempt to establish a relationship between lovastatin biosynthesis and oxidative stress (EOX), through generation of Reactive Oxygen Species (ROS). In addition, an effort was made to establish the possible mechanism by which ROS can regulate the biosynthesis of this secondary metabolite produced by the fungus Aspergillus terreus. Lovastatin has great commercial and pharmaceutical importance due to its anticholesterolamic properties. Commonly, lovastatin is produced by Submerged Fermentation (SmF), but Solid-State Fermentation (SSF) has become in an alternative process for industrial production. It is known that lovastatin biosynthesis is regulated by catabolic repression; however, studies in SSF have made apparent that there are other mechanisms of regulation, since higher lovastatin production is obtained by mycelium growing in SSF, which conducted to the name “Physiology of Solid Medium”. It is currently known that ROS plays an important role in cellular processes and also in fungal differentiation. We considered that the change from trophophase to idiophase is a “metabolic” differentiation. Studies by Miranda et al., (2008) showed that during the trophophase (fast growth phase) sod1 gen (antioxidant enzyme) was strongly expressed, in what we called initially an Oxidative Stress Period (PEO), that occurs just before the start of lovastatin idiophase (production phase). These results conducted to hypothesize the existences of PEO where high quantities of ROS accumulate just before the onset of idiophase. Under this assumption we decided to characterize the redox state in both phases (tropho- and idiophase), and in both culture systems: SSF and SmF, through the measurement of ROS and redox balance (measured by the ratio of GSH/GSSG) kinetics during lovastatin fermentations. However, results showed an important build up of ROS (at the start of lovastatin biosynthesis) that was maintained throughout idiophase. This suggested a relationship between ROS accumulation and biosynthesis of lovastatin on both production systems. Furthermore, we found that the level of ROS was 10 times higher in SmF in relation to the levels of ROS in SSF. Another difference found, between SSF and SmF, was that in idiophase ROS levels were constant in the first system, unlike what was found in SmF where ROS levels showed important fluctuations during culture development. These results suggest that the low and controlled ROS levels, as found in SSF are adequate to achieve good and clear idiophase, particularly good lovastatin production. In both culture systems, the GSH/GSSG ratio indicated that mycelium in trophophase had a more reducing intracellular environment than in idiophase. Interestingly, the GSH/GSSG ratio was four times greater in SSF compared with SmF, which is consistent with smaller quantities of ROS found in SSF. Moreover, the GSH/GSSG ratio indicated that in trophophase there was a more reducing environment than in idiophase. Also, that the GSH/GSSG ratio was four times greater in SSF than SmF during the idiophase, which is coincident whit the fact that was found ROS low quantities than SSF. These findings contradict the existence of a PEO during trophophase, because ROS levels were relatively low during this stage, but there is indeed a coincidence between a start of ROS accumulation and the lovastatin biosynthesis. These results suggest that the high expression of sod1 gen helps the cell contain ROS, maintaining an adequate redox state (reducing) to carry out the cellular functions of development. Results also suggest that the high ROS concentrations in idiophase is a consequence of Ajtyap1 and sod1 down regulation, and that ROS are important for signaling lovastatin biosynthetic genes. We used exogenous antioxidants and oxidants to establish a relationship between ROS and lovastatin production, and to observe their influence on the metabolite biosynthesis. The use of NAC antioxidant (N-acetylcysteine) significantly reduced lovastatin biosynthesis in both culture systems. In SmF, a NAC concentration of ≥10 mM reduced near of 62 % (p˂ 0.05) the lovastatin production level. In this culture we observed a significantly reduction on ROS accumulation levels in idiophase near of 71% (p˂ 0.05). We conducted a linear regression analysis (α≤0.05) to establish a relation between ROS and lovastatin production. |
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