Engineering glucose metabolism for enhanced muconic acid production in Pseudomonas putida KT2440

Bentley, Gayle J.; Narayanan, Niju; Jha, Ramesh K.; Salvachúa, Davinia; Elmore, Joshua R.; Peabody, George L.; Black, Brenna A.; Ramirez, Kelsey J.; Capite, Annette De; Michener, William E.; Werner, Allison Z.; Klingeman, Dawn M.; Schindel, Heidi S.; Nelson, Robert S.; Foust, Lindsey; Guss, Adam M.; Dale, Taraka; Johnson, Christopher W.; Beckham, Gregg T.

doi:10.1016/j.ymben.2020.01.001

Cited by 81 publications

(98 citation statements)

References 56 publications

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“…For example, gacS was mutated in all TALE and ALE experiments ( Supplementary Data File 1 ). The GacS/GacA two-component regulatory system is implicated in biofilm formation ( Martinez-Gil et al., 2014 ) and flagellar transport ( Kim et al., 2014 ), suggesting alternate regulation of these functions may be generally beneficial in well-mixed planktonic cultivations, as has been previously reported ( Bentley et al., 2020 ). Genes related to flagellar transport ( fleQ and fliQ ( Blanco-Romero et al., 2018 )) were also mutated in several TALE/ALE conditions ( Fig.…”

Section: Discussionmentioning

confidence: 65%

Adaptive laboratory evolution of Pseudomonas putida KT2440 improves p-coumaric and ferulic acid catabolism and tolerance

Mohamed

Werner

Salvachúa

et al. 2020

Metabolic Engineering Communications

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Pseudomonas putida KT2440 is a promising bacterial chassis for the conversion of lignin-derived aromatic compound mixtures to biofuels and bioproducts. Despite the inherent robustness of this strain, further improvements to aromatic catabolism and toxicity tolerance of P. putida will be required to achieve industrial relevance. Here, tolerance adaptive laboratory evolution (TALE) was employed with increasing concentrations of the hydroxycinnamic acids p -coumaric acid ( p CA) and ferulic acid (FA) individually and in combination ( p CA + FA). The TALE experiments led to evolved P. putida strains with increased tolerance to the targeted acids as compared to wild type. Specifically, a 37 h decrease in lag phase in 20 g/L p CA and a 2.4-fold increase in growth rate in 30 g/L FA was observed. Whole genome sequencing of intermediate and endpoint evolved P. putida populations revealed several expected and non-intuitive genetic targets underlying these aromatic catabolic and toxicity tolerance enhancements. PP_3350 and ttgB were among the most frequently mutated genes, and the beneficial contributions of these mutations were verified via gene knockouts. Deletion of PP_3350, encoding a hypothetical protein, recapitulated improved toxicity tolerance to high concentrations of p CA, but not an improved growth rate in high concentrations of FA. Deletion of ttgB, part of the TtgABC efflux pump, severely inhibited growth in p CA + FA TALE-derived strains but did not affect growth in p CA + FA in a wild type background, suggesting epistatic interactions. Genes involved in flagellar movement and transcriptional regulation were often mutated in the TALE experiments on multiple substrates, reinforcing ideas of a minimal and deregulated cell as optimal for domesticated growth. Overall, this work demonstrates increased tolerance towards and growth rate at the expense of hydroxycinnamic acids and presents new targets for improving P. putida for microbial lignin valorization.

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Section: Discussionmentioning

confidence: 65%

Adaptive laboratory evolution of Pseudomonas putida KT2440 improves p-coumaric and ferulic acid catabolism and tolerance

Mohamed

Werner

Salvachúa

et al. 2020

Metabolic Engineering Communications

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“…However, growth and productivity were drastically reduced compared to CJ442. [31,49] Growth was enhanced in an evolutionary approach followed by the application of a cis,cis-muconate-responsive biosensor to allow high-throughput fluorescence-activated cell sorting for mutants that showed sufficient cis,cis-muconate production. Analysis of resulting mutants indicated several central metabolic regulators including HexR, GntZ, and GacS, whose deletion further increased strain growth and cis,cis-muconate production performance.…”

Section: Increasing the Phosphoenolpyruvate Supplymentioning

confidence: 99%

Pseudomonas as Versatile Aromatics Cell Factory

Schwanemann

Otto

Wierckx

et al. 2020

Biotechnology Journal

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Aromatics and their derivatives are valuable chemicals with a plethora of important applications and thus play an integral role in modern society. Their current production relies mostly on the exploitation of petroleum resources. Independency from dwindling fossil resources and rising environmental concerns are major driving forces for the transition towards the production of sustainable aromatics from renewable feedstocks or waste streams. Whole‐cell biocatalysis is a promising strategy that allows the valorization of highly abundant, low‐cost substrates. In the last decades, extensive efforts are undertaken to allow the production of a wide spectrum of different aromatics and derivatives using microbes as biocatalysts. Pseudomonads are intriguing hosts for biocatalysis, as they display unique characteristics beneficial for the production of aromatics, including a distinct tolerance and versatile metabolism. This review highlights biotechnological applications of Pseudomonas as host for the production of aromatics and derived compounds. This includes their de novo biosynthesis from renewable resources, biotransformations in single‐ and biphasic fermentation setups, metabolic funneling of lignin‐derived aromatics, and the upcycling of aromatic monomers from plastic waste streams. Additionally, this review provides insights into unique features of Pseudomonads that make them exceptional hosts for aromatics biotechnology and discusses engineering strategies.

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“…A common strategy for minimizing, or even overcoming, this undesired effect on the production host is to conduct adaptive laboratory evolution (ALE) 91 . ALE works best when the desired flux can be selected such that derived mutations for improved growth are not exclusive with desired pathway flux, and ALE can fail if growth is improved at the 24 expense of product titer 92 . ALE often requires months of cell passaging to enable mutations to be acquired, especially when the acquisition of multiple mutations are required 91 .…”

Section: Discussionmentioning

confidence: 99%

Repurposing the yeast peroxisome to compartmentalize a toxic enzyme enables improved (S)-reticuline production

Grewal¹,

Samson²,

Baker

et al. 2020

Preprint

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Eukaryotic cells compartmentalize metabolic pathways in organelles to achieve optimal reaction conditions and avoid crosstalk with other factors in the cytosol. Increasingly, engineers are researching ways in which synthetic compartmentalization could be used to address challenges in metabolic engineering. Here, we identified that norcoclaurine synthase (NCS), the enzyme which catalyzes the first committed reaction in benzylisoquinoline alkaloid (BIA) biosynthesis, is toxic when expressed cytosolically in Saccharomyces cerevisiae and, consequently, restricts (S)-reticuline production. We developed a compartmentalization strategy that alleviates NCS toxicity while promoting increased (S)-reticuline titer, achieved through efficient targeting of toxic NCS to the peroxisome while, crucially, taking advantage of the free flow of metabolite substrates and product across the peroxisome membrane. We identified that peroxisome protein capacity in S. cerevisiae becomes a limiting factor for further improvement of BIA production and demonstrate that expression of engineered transcription factors can mimic the oleate response for larger peroxisomes, further increasing BIA titer without the requirement for peroxisome induction with fatty acids. This work specifically addresses the challenges associated with toxic NCS expression and, more broadly, highlights the potential for engineering organelles with desired characteristics for metabolic engineering.

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Engineering glucose metabolism for enhanced muconic acid production in Pseudomonas putida KT2440

Cited by 81 publications

References 56 publications

Adaptive laboratory evolution of Pseudomonas putida KT2440 improves p-coumaric and ferulic acid catabolism and tolerance

Adaptive laboratory evolution of Pseudomonas putida KT2440 improves p-coumaric and ferulic acid catabolism and tolerance

Pseudomonas as Versatile Aromatics Cell Factory

Repurposing the yeast peroxisome to compartmentalize a toxic enzyme enables improved (S)-reticuline production

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