Abstract:
|
[EN] Developing efficient bioprocesses requires selecting the best biosynthetic pathways, which can be challenging and time-consuming due to the vast amount of data available in databases and literature. The extension of ...[+]
[EN] Developing efficient bioprocesses requires selecting the best biosynthetic pathways, which can be challenging and time-consuming due to the vast amount of data available in databases and literature. The extension of the shikimate pathway for the biosynthesis of commercially attractive molecules often involves promiscuous enzymes or lacks well-established routes. To address these challenges, we developed a computational workflow integrating enumeration/retrosynthesis algorithms, a toolbox for pathway analysis, enzyme selection tools, and a gene discovery pipeline, supported by manual curation and literature review. Our focus has been on implementing biosynthetic pathways for tyrosine-derived compounds, specifically L-3,4-dihydroxyphenylalanine (L-DOPA) and dopamine, with significant applications in health and nutrition. We selected one pathway to produce L-DOPA and two different pathways for dopamine-one already described in the literature and a novel pathway. Our goal was either to identify the most suitable gene candidates for expression in Escherichia coli for the known pathways or to discover innovative pathways. Although not all implemented pathways resulted in the accumulation of target compounds, in our shake-flask experiments we achieved a maximum L-DOPA titer of 0.71 g/L and dopamine titers of 0.29 and 0.21 g/L for known and novel pathways, respectively. In the case of L-DOPA, we utilized, for the first time, a mutant version of tyrosinase from Ralstonia solanacearum. Production of dopamine via the known biosynthesis route was accomplished by coupling the L-DOPA pathway with the expression of DOPA decarboxylase from Pseudomonas putida, resulting in a unique biosynthetic pathway never reported in literature before. In the context of the novel pathway, dopamine was produced using tyramine as the intermediate compound. To achieve this, tyrosine was initially converted into tyramine by expressing TDC from Levilactobacillus brevis, which, in turn, was converted into dopamine through the action of the enzyme encoded by ppoMP from Mucuna pruriens. This marks the first time that an alternative biosynthetic pathway for dopamine has been validated in microbes. These findings underscore the effectiveness of our computational workflow in facilitating pathway enumeration and selection, offering the potential to uncover novel biosynthetic routes, thus paving the way for other target compounds of biotechnological interest.
[-]
|
Project ID:
|
info:eu-repo/grantAgreement/EC/H2020/814408/EU/Modular cell factories for the production of 100 compounds from the shikimate pathway/
info:eu-repo/grantAgreement/FCT/6817 - DCRRNI ID/LA%2FP%2F0087%2F2020/PT/Life Sciences for a Healthy and Sustainable Future/
info:eu-repo/grantAgreement/FCT/6817 - DCRRNI ID/UIDB%2F04612%2F2020/PT/Molecular, Structural and Cellular Microbiology ¿ Instituto de Tecnologia Química e Biológica António Xavier/
info:eu-repo/grantAgreement/FCT/6817 - DCRRNI ID/UIDP%2F04612%2F2020/PT/Molecular, Structural and Cellular Microbiology ¿ Instituto de Tecnologia Química e Biológica António Xavier/
|
Thanks:
|
The author(s) declare that financial support was received for the research, authorship, and/or publication of this article. This project has received funding from the European Union's Horizon 2020 research and innovation ...[+]
The author(s) declare that financial support was received for the research, authorship, and/or publication of this article. This project has received funding from the European Union's Horizon 2020 research and innovation programme under grant agreement 814408 SHIKIFACTORY100-Modular cell factories for the production of 100 compounds from the Shikimate pathway. This work was supported by FCT-Fundacao para a Ciencia e a Tecnologia, I.P., through MOSTMICRO-ITQB R&D Unit (UIDB/04612/2020, UIDP/04612/2020) and LS4FUTURE Associated Laboratory (LA/P/0087/2020).r The author(s) declare that financial support was received for the research, authorship, and/or publication of this article. This project has received funding from the European Union's Horizon 2020 research and innovation programme under grant agreement 814408 SHIKIFACTORY100-Modular cell factories for the production of 100 compounds from the Shikimate pathway. This work was supported by FCT-Fundac & atilde;o para a Ciencia e a Tecnologia, I.P., through MOSTMICRO-ITQB R&D Unit (UIDB/04612/2020, UIDP/04612/2020) and LS4FUTURE Associated Laboratory (LA/P/0087/2020).
[-]
|