TY - JOUR
T1 - Computer-aided design and implementation of efficient biosynthetic pathways to produce high added-value products derived from tyrosine in Escherichia coli
AU - Ferreira, Sofia
AU - Balola, Alexandra
AU - Sveshnikova, Anastasia
AU - Hatzimanikatis, Vassily
AU - Vilaça, Paulo
AU - Maia, Paulo
AU - Carreira, Rafael
AU - Stoney, Ruth
AU - Carbonell, Pablo
AU - Souza, Caio Silva
AU - Correia, João
AU - Lousa, Diana
AU - Soares, Cláudio M.
AU - Rocha, Isabel
N1 - Funding Information:
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\u2019s Horizon 2020 research and innovation programme under grant agreement 814408 SHIKIFACTORY100\u2013Modular cell factories for the production of 100 compounds from the Shikimate pathway. This work was supported by FCT\u2013Funda\u00E7\u00E3o para a Ci\u00EAncia 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).
Publisher Copyright:
Copyright © 2024 Ferreira, Balola, Sveshnikova, Hatzimanikatis, Vilaça, Maia, Carreira, Stoney, Carbonell, Souza, Correia, Lousa, Soares and Rocha.
PY - 2024/6/24
Y1 - 2024/6/24
N2 - 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.
AB - 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.
KW - bioprocesses
KW - biosynthetic pathways
KW - computational tools
KW - dopamine
KW - Escherichia coli
KW - L-DOPA
KW - pathway design
KW - retrobiosynthesis algorithms
UR - http://www.scopus.com/inward/record.url?scp=85197689699&partnerID=8YFLogxK
U2 - 10.3389/fbioe.2024.1360740
DO - 10.3389/fbioe.2024.1360740
M3 - Article
AN - SCOPUS:85197689699
SN - 2296-4185
VL - 12
JO - Frontiers in Bioengineering and Biotechnology
JF - Frontiers in Bioengineering and Biotechnology
M1 - 1360740
ER -