Model-guided dynamic control of essential metabolic nodes boosts acetyl-coenzyme A–dependent bioproduction in rewired Pseudomonas putida

Kozaeva, Ekaterina; Volkova, Svetlana; Matos, Marta R. A.; Mezzina, Mariela P.; Wulff, Tune; Volke, Daniel C.; Nielsen, Lars K.; Nikel, Pablo I.

doi:10.1016/j.ymben.2021.07.014

Cited by 49 publications

(36 citation statements)

References 117 publications

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“…Moreover, Pseudomonas tolerates high levels of abiotic stress, including oxidative damage and solvents, underscoring their potential as biotechnological hosts for large-scale fermentations 20 . Pseudomonas putida is endowed with a rich and versatile metabolism 21 that renders this species an ideal platform for metabolic engineering towards the production of chemicals 22 – 25 . The biochemical repertoire of P .…”

Section: Introductionmentioning

confidence: 99%

Modular (de)construction of complex bacterial phenotypes by CRISPR/nCas9-assisted, multiplex cytidine base-editing

et al. 2022

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CRISPR/Cas technologies constitute a powerful tool for genome engineering, yet their use in non-traditional bacteria depends on host factors or exogenous recombinases, which limits both efficiency and throughput. Here we mitigate these practical constraints by developing a widely-applicable genome engineering toolset for Gram-negative bacteria. The challenge is addressed by tailoring a CRISPR base editor that enables single-nucleotide resolution manipulations (C·G → T·A) with >90% efficiency. Furthermore, incorporating Cas6-mediated processing of guide RNAs in a streamlined protocol for plasmid assembly supports multiplex base editing with >85% efficiency. The toolset is adopted to construct and deconstruct complex phenotypes in the soil bacterium Pseudomonas putida. Single-step engineering of an aromatic-compound production phenotype and multi-step deconstruction of the intricate redox metabolism illustrate the versatility of multiplex base editing afforded by our toolbox. Hence, this approach overcomes typical limitations of previous technologies and empowers engineering programs in Gram-negative bacteria that were out of reach thus far.

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Section: Introductionmentioning

confidence: 99%

Modular (de)construction of complex bacterial phenotypes by CRISPR/nCas9-assisted, multiplex cytidine base-editing

et al. 2022

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“…Importantly, once modules have been indentified, currency metabolites (redox and co-factors) may be reincorporated to the respective reactions if desired. For example, a kinetic model of a module would typically include cofactors as fixed concentration external metabolites [43,44]. It is for the sole purpose of identifying modules that currency metabolites are reversibly removed.…”

Section: Discussionmentioning

confidence: 99%

The topology of genome-scale metabolic reconstructions unravels independent modules and high network flexibility

et al. 2022

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The topology of metabolic networks is recognisably modular with modules weakly connected apart from sharing a pool of currency metabolites. Here, we defined modules as sets of reversible reactions isolated from the rest of metabolism by irreversible reactions except for the exchange of currency metabolites. Our approach identifies topologically independent modules under specific conditions associated with different metabolic functions. As case studies, the E.coli iJO1366 and Human Recon 2.2 genome-scale metabolic models were split in 103 and 321 modules respectively, displaying significant correlation patterns in expression data. Finally, we addressed a fundamental question about the metabolic flexibility conferred by reversible reactions: “Of all Directed Topologies (DTs) defined by fixing directions to all reversible reactions, how many are capable of carrying flux through all reactions?”. Enumeration of the DTs for iJO1366 model was performed using an efficient depth-first search algorithm, rejecting infeasible DTs based on mass-imbalanced and loopy flux patterns. We found the direction of 79% of reversible reactions must be defined before all directions in the network can be fixed, granting a high degree of flexibility.

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“…These tools can be used to tune endogenous metabolism and/or heterologous metabolic pathways ( Peng et al, 2018 ; Banerjee et al, 2020 ). CRISPRi/a tools are most often combined with other metabolic engineering techniques, such as the deletion, overexpression, or mutation of select genes and optimization of medium, to further increase titers of the desired product ( Park et al, 2019 ; Dietsch et al, 2021 ; Kozaeva et al, 2021 ).…”

Section: Applications Of Crispri/a In Non-model Bacteriamentioning

confidence: 99%

CRISPR-Based Approaches for Gene Regulation in Non-Model Bacteria

Call

Andrews

2022

Front. Genome Ed.

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CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) have become ubiquitous approaches to control gene expression in bacteria due to their simple design and effectiveness. By regulating transcription of a target gene(s), CRISPRi/a can dynamically engineer cellular metabolism, implement transcriptional regulation circuitry, or elucidate genotype-phenotype relationships from smaller targeted libraries up to whole genome-wide libraries. While CRISPRi/a has been primarily established in the model bacteria Escherichia coli and Bacillus subtilis, a growing numbering of studies have demonstrated the extension of these tools to other species of bacteria (here broadly referred to as non-model bacteria). In this mini-review, we discuss the challenges that contribute to the slower creation of CRISPRi/a tools in diverse, non-model bacteria and summarize the current state of these approaches across bacterial phyla. We find that despite the potential difficulties in establishing novel CRISPRi/a in non-model microbes, over 190 recent examples across eight bacterial phyla have been reported in the literature. Most studies have focused on tool development or used these CRISPRi/a approaches to interrogate gene function, with fewer examples applying CRISPRi/a gene regulation for metabolic engineering or high-throughput screens and selections. To date, most CRISPRi/a reports have been developed for common strains of non-model bacterial species, suggesting barriers remain to establish these genetic tools in undomesticated bacteria. More efficient and generalizable methods will help realize the immense potential of programmable CRISPR-based transcriptional control in diverse bacteria.

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Model-guided dynamic control of essential metabolic nodes boosts acetyl-coenzyme A–dependent bioproduction in rewired Pseudomonas putida

Cited by 49 publications

References 117 publications

Modular (de)construction of complex bacterial phenotypes by CRISPR/nCas9-assisted, multiplex cytidine base-editing

Modular (de)construction of complex bacterial phenotypes by CRISPR/nCas9-assisted, multiplex cytidine base-editing

The topology of genome-scale metabolic reconstructions unravels independent modules and high network flexibility

CRISPR-Based Approaches for Gene Regulation in Non-Model Bacteria

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