Comparación de la actividad de la vía señalización AMPc-PKA en la producción de Lovastatina por Aspergillus terreus en fermentación sólida y fermentación líquida
In this thesis was studied the functioning of the FadA-AMPc-PKA pathway linked to sporulation and the secondary metabolism of Aspergillus terreus. For this, some parameters indicative of the functioning of the c´AMP-PKA pathway were taken into account, during the production of lovastatin in in solid...
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| Formato: | tesis doctoral |
| Estado: | Versión publicada |
| Fecha de publicación: | 2017 |
| País: | México |
| Recursos: | Universidad Autónoma Metropolitana |
| Repositorio: | Repositorio Institucional de la UAM Iztapalapa |
| Idioma: | español |
| OAI Identifier: | oai:bindani.izt.uam.mx:r781wg01v |
| Acesso em linha: | https://doi.org/10.24275/uami.r781wg01v |
| Access Level: | acceso abierto |
| Palavra-chave: | info:eu-repo/classification/LEM/Fermentación info:eu-repo/classification/LEM/Fermentación en estado sólido info:eu-repo/classification/LEM/Solid fermentation info:eu-repo/classification/LEM/Aspergillus -- Investigación -- Biotecnología info:eu-repo/classification/LEM/Aspergillus terreus -- Research -- Biotecnology info:eu-repo/classification/LEM/Fermentation info:eu-repo/classification/cti/6 |
| Resumo: | In this thesis was studied the functioning of the FadA-AMPc-PKA pathway linked to sporulation and the secondary metabolism of Aspergillus terreus. For this, some parameters indicative of the functioning of the c´AMP-PKA pathway were taken into account, during the production of lovastatin in in solid-state fermentation (SSF) and submerged fermentation (SmF). The first level studied was the expression pattern of the genes of the main components: fadA (α subunit of the G protein), laeA (global regulator of secondary metabolism) and its putative downstream targets: the specific transcriptional factor of the clúster biosynthetic of lovastatin, lovE, a polyketide synthase, lovF, and the main transcription factor of conidiation, brlA. In addition, the stress resistance genes atfB and msnA were also analyzed. The panorama was complemented with the profiles of intracellular concentration of c´AMP and the profile of PKA activity during SFF and SmF. In the transcription pattern of fadA, which perhaps reflects the activation of the pathway (separation and phosphorylation of this subunit), differential expression between SFF and SmF was found, suggesting that environmental stimuli in FS are perceived through this signaling pathway. In SFF, had a relatively low expression profile (except at 21 h) and more or less constant (slightly lower in idiofase). In contrast, in SmF, fadA was high in trophophase (18 h) and low from 21 h until idiofase. This expression profile of fadA was opposite to the c´AMP concentration pattern and with the PKA activity pattern in both culture systems. A direct relation of these two variables with fadA was expected, however, similarity was only found between the profiles of c´AMP and PKA. In SSF, both the intracellular concentration of c´AMP and the PKA activity showed high levels in trophophase, decreasing at 21 h and being slightly constant in idiofase, indicating a negative regulation on lovE/lovF (probably through LaeA). However, in SmF, a completly opposite behavior was observed: low in trophophase and increased in idiofase (slightly increased PKA activity, similar to the level in SSF), these results being controversial. On the other hand, the expression results of the brlA gene also indicate that the conidiation is negatively regulated by PKA (in SSF, in SmF there was no sporulation). In relation to the transcription factors (FT´s) of stress response, msnA showed a transcription pattern somewhat inverse to the activity of PKA (more expression in idiofase). In contrast, atfB showed a more intense transcription in trophophase, perhaps regulated positively by PKA, although it could have influence of the SAPK MAPKinase pathway. As for the laeA gene, it was found that its expression profile, both in SSF and in SmF, is opposite to the expression of lovE and lovF (and synthesis of lovastatin). That is, it is expressed strongly in trophophase and slightly in idiofase. This was unexpected, so the expression profile does not seem to be a good indicator of LaeA activity. The results suggest that the expression of laeA is present from the trophophase, but is activated by a post-transcriptional or posttranslational regulation, in idiofase. This, probably due to a negative regulation by PKA, particularly in SSF. On the other hand, transformants that overexpressed the laeA gene (SE::laeA) were characterized (under any of the 2 strategies used: with own promoter and with a constitutive promoter). The results showed that both strategies were very good methods of molecular genetic improvement, especially for SSF. In SE::laeA overproducing strains of lovastatin were generated especially for SSF, and in less abundance for SmF. A greater sporulation was also observed in SSF, confirming LaeA as a regulator of secondary metabolism and a positive regulator in sporulation. The best transformants for SSF (T9laeA and T2laeAcons) and for SmF (T1laeA and T5laeAcons) showed an extension or prolongation of their production stage, this being an important factor in the increase of production. In SSF, the transformant with constitutive promoter, T2laeAcons, produced 30.6 mg of lovastatin/gss (104% increase). In SmF, the transformant with its own promoter, T1laeA, produced 0.89 mg /mL (102% increase). |
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