Tuning a high performing multiplexed-CRISPRi Pseudomonas putida strain to further enhance indigoidine production

Czajka, Jeffrey J.; Banerjee, Deepanwita; Eng, Thomas; Menasalvas, Javier; Yan, Chunsheng; Munoz, Nathalie Munoz; Poirier, Brenton C.; Kim, Young-Mo; Baker, Scott; Tang, Yinjie J.; Mukhopadhyay, Aindrila

doi:10.1016/j.mec.2022.e00206

Cited by 17 publications

(5 citation statements)

References 71 publications

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“…In-frame genomic deletions and promoter substitutions were generated exactly as described in (45). All recombineering 90-mer ssDNA oligos and gRNA targeting sequences were synthesized by IDT (Integrated DNA Technologies, Redwood City, CA) and are described in Supplementary Table 3 .…”

Section: Methodsmentioning

confidence: 99%

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Bottlenecks in the Implementation of Genome Scale Metabolic Model Based Designs for Bioproduction from Aromatic Carbon Sources

Banerjee,

Menasalvas,

Chen

et al. 2024

Preprint

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Genome scale metabolic models (GSMM) are commonly used to identify gene deletion sets that result in growth coupling, pairing product formation with substrate utilization. While such approaches can improve strain performance beyond levels typically accessible using targeted strain engineering approaches, sustainable feedstocks often pose a challenge for GSMM-based methods due to incomplete underlying metabolic data. Specifically, we address a four-gene deletion design for the lignin-derived non-sugar carbon source, para-coumarate, that proved challenging to implement. We examine the performance of the fully implemented design for p-coumarate to glutamine, a useful biomanufacturing intermediate. In this study glutamine is then converted to indigoidine, an alternative sustainable pigment and a model heterologous product. Through omics, promoter-variation and growth characterization of a fully implemented gene deletion design, we provide evidence that aromatic catabolism in the completed design is rate-limited by fumarate hydratase activity in the citrate cycle and required careful optimization of the final fumarate hydratase protein (PP_0897) expression to achieve growth and production. A metabolic cross-feeding experiment with the completed design strain also revealed an unanticipated nutrient requirement suggesting additional functions for the fumarate hydratase protein. A double sensitivity analysis confirmed a strict requirement for fumarate hydratase activity in the strain where all genes in the growth coupling design have been implemented. While a complete implementation of the design was achieved, this study highlights the challenge of precisely inactivating metabolic reactions encoded by under-characterized proteins especially in the context of multi-gene edits.

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Section: Methodsmentioning

confidence: 99%

“…Strains used in this study were propagated using conventional laboratory cultivation protocols for P. putida KT2440 (44)(45)(46). Engineered strains were grown in a modified M9 minimal medium as previously described (10), and p-CA (Sigma-Aldrich, Product No.…”

Section: Cultivation Of Pseudomonas Putidamentioning

confidence: 99%

Bottlenecks in the Implementation of Genome Scale Metabolic Model Based Designs for Bioproduction from Aromatic Carbon Sources

Banerjee,

Menasalvas,

Chen

et al. 2024

Preprint

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“…ssDNA recombineering was performed in K. michiganensis with slight modifications from our existing recombineering protocol used in Pseudomonas putida (Czajka et al 2022) . The strain was transformed with a plasmid containing a 3-methyl benzoate (3MB; m-Toluic Acid; Sigma Aldrich Catalog No.…”

Section: Methodsmentioning

confidence: 99%

Development of Modular Expression and Genome Editing Across Phylogenetically Distinct Diazotrophs

Kulakowski,

Rivier,

Kuo

et al. 2024

Preprint

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Diazotrophic bacteria can reduce atmospheric nitrogen into ammonia enabling bioavailability of the essential element. Many diazotrophs associate closely with plant roots increasing nitrogen availability, acting as plant growth promoters. These associations have the potential to reduce the need for costly synthetic fertilizers if they could be engineered for agricultural applications. However, despite the importance of diazotrophic bacteria, genetic tools are poorly developed in a limited number of species, in turn narrowing the crops and root microbiomes that can be targeted. Here we report optimized protocols and plasmids to manipulate phylogenetically diverse diazotrophs with the goal of enabling synthetic biology and genetic engineering. Three broad-host-range plasmids can be used across multiple diazotrophs, with the identification of one specific plasmid (containing origin of replication RK2 and a kanamycin resistance marker) showing the highest degree of compatibility across bacteria tested. We then demonstrated modular expression testing seven promoters and eleven ribosomal binding sites using proxy fluorescent proteins. Finally, we tested four small molecule inducible systems to report expression in three diazotrophs and demonstrate genome editing in Klebsiella michiganensis M5al.

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“…Construction of targeted genomic mutants in P. putida via allelic exchange or recombineering. Generation of in-frame precise deletions in P. putida was implemented primarily using a recombineering/CRISPR counterselection protocol (Czajka et al, 2022). In brief, 90 bp ssDNA oligos were designed to bind 45 bp upstream and downstream precisely targeting the start and stop codons of the desired gene for removal.…”

Section: Reagents and Bacterial Cultivation Conditionsmentioning

confidence: 99%

Ensemble and Iterative Engineering for Maximized Bioconversion to the Blue Pigment, Indigoidine from Non-Canonical Sustainable Carbon Sources

Eng

Banerjee

Menasalvas

et al. 2023

Preprint

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While many heterologous molecules can be produced at trace concentrations via microbial bioconversion processes, maximizing their titers, rates, and yields from lignin-derived carbon streams remains challenging. Growth coupling can not only increase titers and yields but also shift the production period from stationary phase to growth phase. These methods for designing growth-coupling strains however require multi-gene edits for implementation which may be perceived as impractical. Here, we computationally evaluated 4,114 potential solutions for growth couplingpara-coumarate to indigoidine production and prototype two cut sets inPseudomonas putidaKT2440. We used adaptive laboratory evolution (ALE) on the initial triple deletion strain to restore growth onp-CA. Using X-ray tomography on this post-ALE strain we revealed increased cell density and decreased cell volume. Proteomics identified upregulated peroxidases that mitigate reactive oxygen species formation. Nine iterative stepwise modifications further informed by model-guided and rational approaches realized a growth coupled strain that produced 7.3 g/L indigoidine at 77% MTY inpara-coumarate minimal media. These ensemble strategies provide a blueprint for producing target molecules at high product titers, rates, and yields.

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Tuning a high performing multiplexed-CRISPRi Pseudomonas putida strain to further enhance indigoidine production

Cited by 17 publications

References 71 publications

Bottlenecks in the Implementation of Genome Scale Metabolic Model Based Designs for Bioproduction from Aromatic Carbon Sources

Bottlenecks in the Implementation of Genome Scale Metabolic Model Based Designs for Bioproduction from Aromatic Carbon Sources

Development of Modular Expression and Genome Editing Across Phylogenetically Distinct Diazotrophs

Ensemble and Iterative Engineering for Maximized Bioconversion to the Blue Pigment, Indigoidine from Non-Canonical Sustainable Carbon Sources

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