Combinatorial pathway balancing provides biosynthetic access to 2-fluoro-cis,cis-muconate in engineered Pseudomonas putida

Wirth, Nicolas T.; Nikel, Pablo I.

doi:10.1016/j.checat.2021.09.002

Cited by 26 publications

(9 citation statements)

References 122 publications

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“…E. coli DH5α λpir was employed for cloning purposes. Chemically-competent E. coli cells were prepared and transformed with plasmids according to well-established protocols [46][47][48][49][50][51]. P. putida was rendered electro-competent by treatment Choi et al [52].…”

Section: Cloning Procedures and Plasmid Constructionmentioning

confidence: 99%

A synthetic C2 auxotroph of Pseudomonas putida for evolutionary engineering of alternative sugar catabolic routes

Wirth

Gurdo

Krink

et al. 2022

Preprint

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Acetyl-coenzyme A (AcCoA) is a metabolic hub in virtually all living cells, serving as both a key precursor of essential biomass components and a metabolic sink for catabolic pathways of a large variety of substrates. Owing to this dual role, tight growth-production coupling schemes can be implemented around the AcCoA node. Inspired by this concept, a synthetic C2 auxotrophy was implemented in the platform bacterium Pseudomonas putida through an in silico-guided engineering approach. A growth-coupling strategy, driven by AcCoA demand, allowed for direct selection of an alternative sugar assimilation route-the phosphoketolase (PKT) shunt from bifidobacteria. Adaptive laboratory evolution forced the synthetic auxotroph to integrate the PKT shunt to restore C2 prototrophy. Large-scale structural chromosome rearrangements were identified as possible mechanisms for adjusting the network-wide proteome profile, resulting in improved PKT-dependent growth phenotypes. 13C-based metabolic flux analysis revealed an even split between the native Entner-Doudoroff and the synthetic PKT pathway for glucose processing, leading to enhanced carbon conservation. These results demonstrate that the P. putida metabolism can be radically rewired to incorporate a synthetic C2 metabolism, creating novel network connectivities and highlighting the importance of unconventional engineering strategies to support efficient microbial production.

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Section: Cloning Procedures and Plasmid Constructionmentioning

confidence: 99%

A synthetic C2 auxotroph of Pseudomonas putida for evolutionary engineering of alternative sugar catabolic routes

Wirth

Gurdo

Krink

et al. 2022

Preprint

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“…To the freeze‐dried sample was added a MeOD/D 2 O solution (adjusted according to the sample size), and the resulting suspension was subjected to sonication to ensure dissolution of all fluorometabolites in the sample. After centrifugation (to remove any precipitate), 19 F‐NMR experiments were recorded at 298 K on a Bruker AVANCE III HD instrument with either a SmartProbe BBFO + (for proton coupled experiments) or a TCI CryoProbe (for proton decoupled experiments), using CFCl 3 as an external reference (Calero et al ., 2020 ; Wirth and Nikel, 2021 ).…”

Section: Methodsmentioning

confidence: 99%

Oligomerization engineering of the fluorinase enzyme leads to an active trimer that supports synthesis of fluorometabolites in vitro

Kittilä

Calero

Fredslund

et al. 2022

Microbial Biotechnology

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Summary The fluorinase enzyme represents the only biological mechanism capable of forming stable C–F bonds characterized in nature thus far, offering a biotechnological route to the biosynthesis of value‐added organofluorines. The fluorinase is known to operate in a hexameric form, but the consequence(s) of the oligomerization status on the enzyme activity and its catalytic properties remain largely unknown. In this work, this aspect was explored by rationally engineering trimeric fluorinase variants that retained the same catalytic rate as the wild‐type enzyme. These results ruled out hexamerization as a requisite for the fluorination activity. The Michaelis constant ( K M ) for S ‐adenosyl‐ l ‐methionine, one of the substrates of the fluorinase, increased by two orders of magnitude upon hexamer disruption. Such a shift in S ‐adenosyl‐ l ‐methionine affinity points to a long‐range effect of hexamerization on substrate binding – likely decreasing substrate dissociation and release from the active site. A practical application of trimeric fluorinase is illustrated by establishing in vitro fluorometabolite synthesis in a bacterial cell‐free system.

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“…Several microorganisms are reported to produce cis-cis muconic acid as an intermediate in lignin biodegradation ( Wang et al, 2020 ). However, recently, metabolically engineered microbes for the selective conversion of lignin into cis-cis muconic acid has gained essence ( Pyne et al, 2018 ; Choi et al, 2020 ; Aravind et al, 2021 ; Wirth and Nikel, 2021 ). Most of the microbes convert lignin into cis-cis muconic acid by using glucose as the additional growth substrate ( Becker et al, 2018 ).…”

Section: Methods Of Lignin Depolymerizationmentioning

confidence: 99%

Endophytes in Lignin Valorization: A Novel Approach

Mattoo

Nonzom

2022

Front. Bioeng. Biotechnol.

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Lignin, one of the essential components of lignocellulosic biomass, comprises an abundant renewable aromatic resource on the planet earth. Although 15%––40% of lignocellulose pertains to lignin, its annual valorization rate is less than 2% which raises the concern to harness and/or develop effective technologies for its valorization. The basic hindrance lies in the structural heterogeneity, complexity, and stability of lignin that collectively makes it difficult to depolymerize and yield common products. Recently, microbial delignification, an eco-friendly and cheaper technique, has attracted the attention due to the diverse metabolisms of microbes that can channelize multiple lignin-based products into specific target compounds. Also, endophytes, a fascinating group of microbes residing asymptomatically within the plant tissues, exhibit marvellous lignin deconstruction potential. Apart from novel sources for potent and stable ligninases, endophytes share immense ability of depolymerizing lignin into desired valuable products. Despite their efficacy, ligninolytic studies on endophytes are meagre with incomplete understanding of the pathways involved at the molecular level. In the recent years, improvement of thermochemical methods has received much attention, however, we lagged in exploring the novel microbial groups for their delignification efficiency and optimization of this ability. This review summarizes the currently available knowledge about endophytic delignification potential with special emphasis on underlying mechanism of biological funnelling for the production of valuable products. It also highlights the recent advancements in developing the most intriguing methods to depolymerize lignin. Comparative account of thermochemical and biological techniques is accentuated with special emphasis on biological/microbial degradation. Exploring potent biological agents for delignification and focussing on the basic challenges in enhancing lignin valorization and overcoming them could make this renewable resource a promising tool to accomplish Sustainable Development Goals (SDG’s) which are supposed to be achieved by 2030.

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Combinatorial pathway balancing provides biosynthetic access to 2-fluoro-cis,cis-muconate in engineered Pseudomonas putida

Cited by 26 publications

References 122 publications

A synthetic C2 auxotroph of Pseudomonas putida for evolutionary engineering of alternative sugar catabolic routes

A synthetic C2 auxotroph of Pseudomonas putida for evolutionary engineering of alternative sugar catabolic routes

Oligomerization engineering of the fluorinase enzyme leads to an active trimer that supports synthesis of fluorometabolites in vitro

Endophytes in Lignin Valorization: A Novel Approach

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