Metabolic engineering of Pseudomonas taiwanensis VLB120 with minimal genomic modifications for high-yield phenol production

Wynands, Benedikt; Lenzen, Christoph; Otto, Maike; Koch, Falk; Blank, Lars M.; Wierckx, Nick

doi:10.1016/j.ymben.2018.03.011

Cited by 81 publications

(127 citation statements)

References 70 publications

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“…For P. putida transformations either conjugational transfer or electroporation were performed as described by Wynands et al . (). Knockout strains were obtained using the pEMG system described by Martínez‐García and de Lorenzo () with a modified protocol described by Wynands et al .…”

Section: Methodsmentioning

confidence: 97%

“…Knockout strains were obtained using the pEMG system described by Martínez‐García and de Lorenzo () with a modified protocol described by Wynands et al . (). Plasmid inserts and gene deletions were confirmed by Sanger sequencing performed by Eurofins Genomics (Ebersberg, Germany).…”

Section: Methodsmentioning

confidence: 97%

“…For the transformation of DNA assemblies and purified plasmids into competent E. coli a heat shock protocol was performed (Hanahan, 1983). For P. putida transformations either conjugational transfer or electroporation were performed as described by Wynands et al (2018). Knockout strains were obtained using the pEMG system described by Martínez-García and de Lorenzo (2011) with a modified protocol described by Wynands et al (2018).…”

Section: Dna Methodsmentioning

confidence: 99%

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Laboratory evolution reveals the metabolic and regulatory basis of ethylene glycol metabolism byPseudomonas putidaKT2440

Jayakody

Franden

et al. 2019

Environmental Microbiology

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Summary Pollution from ethylene glycol, and plastics containing this monomer, represent a significant environmental problem. The investigation of its microbial metabolism therefore provides insights into the environmental fate of this pollutant and also enables its utilization as a carbon source for microbial biotechnology. Here, we reveal the genomic and metabolic basis of ethylene glycol metabolism in Pseudomonas putida KT2440. Although this strain cannot grow on ethylene glycol as sole carbon source, it can be used to generate growth‐enhancing reducing equivalents upon co‐feeding with acetate. Mutants that utilize ethylene glycol as sole carbon source were isolated through adaptive laboratory evolution. Genomic analysis of these mutants revealed a central role of the transcriptional regulator GclR, which represses the glyoxylate carboligase pathway as part of a larger metabolic context of purine and allantoin metabolism. Secondary mutations in a transcriptional regulator encoded by PP_2046 and a porin encoded by PP_2662 further improved growth on ethylene glycol in evolved strains, likely by balancing fluxes through the initial oxidations of ethylene glycol to glyoxylate. With this knowledge, we reverse engineered an ethylene glycol utilizing strain and thus revealed the metabolic and regulatory basis that are essential for efficient ethylene glycol metabolism in P. putida KT2440.

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Section: Methodsmentioning

confidence: 97%

Section: Methodsmentioning

confidence: 97%

Section: Dna Methodsmentioning

confidence: 99%

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Laboratory evolution reveals the metabolic and regulatory basis of ethylene glycol metabolism byPseudomonas putidaKT2440

Jayakody

Franden

et al. 2019

Environmental Microbiology

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“…Examples of production of added‐value aromatic molecules in P . putida are illustrated by cinnamic acid (Nijkamp et al ., ) and phenol (Wierckx et al ., ; Wynands et al ., ) – achieved, among other manipulations, by introducing a phenylalanine‐ammonia lyase from Rhodosporidium toruloides and the tyrosine phenol lyase from Pantoea agglomerans . Other strains producing derivatives of these aromatic compounds were developed, producing, for example p ‐hydroxystyrene (Verhoef et al ., ), p ‐hydroxybenzoate (Yu et al ., ), anthranilate (Kuepper et al ., ), vanillate (Graf and Altenbuchner, ) and p ‐coumaric acid (Calero et al ., ).…”

Section: Bacterial Species Adopted As a Chassis: From Historical Exammentioning

confidence: 99%

Chasing bacterial chassis for metabolic engineering: a perspective review from classical to non‐traditional microorganisms

Calero

Nikel

2018

Microbial Biotechnology

236

184

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The last few years have witnessed an unprecedented increase in the number of novel bacterial species that hold potential to be used for metabolic engineering. Historically, however, only a handful of bacteria have attained the acceptance and widespread use that are needed to fulfil the needs of industrial bioproduction - and only for the synthesis of very few, structurally simple compounds. One of the reasons for this unfortunate circumstance has been the dearth of tools for targeted genome engineering of bacterial chassis, and, nowadays, synthetic biology is significantly helping to bridge such knowledge gap. Against this background, in this review, we discuss the state of the art in the rational design and construction of robust bacterial chassis for metabolic engineering, presenting key examples of bacterial species that have secured a place in industrial bioproduction. The emergence of novel bacterial chassis is also considered at the light of the unique properties of their physiology and metabolism, and the practical applications in which they are expected to outperform other microbial platforms. Emerging opportunities, essential strategies to enable successful development of industrial phenotypes, and major challenges in the field of bacterial chassis development are also discussed, outlining the solutions that contemporary synthetic biology-guided metabolic engineering offers to tackle these issues.

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“…Microbial production of aromatic chemicals has largely been enabled via pathway engineering, generally consisting of either: (i) the functional reconstruction of naturally‐occurring but non‐native (often plant) pathways; or (ii) the bottom‐up construction of novel pathways comprised of individual enzymes derived from a diversity of heterologous sources. Recent examples include the successful engineering of microbes capable of the de novo production of, in the first case, flavonoids (usually consisting of two phenyl groups and a heterocyclic ring), stilbenes (ethylene moiety with two phenyl groups), and coumarins (containing a 1,2‐benzopyrone backbone), and, in the second case, numerous aromatic aldehydes, alcohols, and acids, styrenics, and phenolics . In most cases, these heterologous pathways stem from natively produced aromatic chemicals such as the aromatic amino acids (i.e.…”

Section: Modular Engineering Strategies For Optimizing Pathway Flux Amentioning

confidence: 99%

Emerging tools, enabling technologies, and future opportunities for the bioproduction of aromatic chemicals

Machas

Kurgan

Jha

et al. 2018

J of Chemical Tech & Biotech

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Aromatic compounds, which are traditionally derived from petroleum feedstocks, represent a diverse class of molecules with a wide range of industrial and commercial applications. Significant progress has been made to alternatively and sustainably produce many aromatics from renewable substrates using microbial biocatalysts. While the construction of both natural and non‐natural pathways has expanded the number and diversity of aromatic bioproducts, pathway modularization in both single‐ and multi‐strain systems continues to support the enhancement of key production metrics towards economically‐viable levels. Emerging tools for implementing more precise metabolic control (e.g. CRISPRi, sRNA) as well as the engineering of novel high‐throughput screening platforms utilizing in vivo aromatic biosensors, meanwhile, continue to facilitate further optimization of both pathways and hosts. While product toxicity persists as a key challenge limiting the production of many aromatics, various successful strategies have been demonstrated towards improving tolerance, including via membrane and efflux pump engineering as well as by exploiting alternative production hosts. Finally, as a further step towards sustainable and economical aromatic bioproduction, non‐model substrates including lignin‐derived compounds continue to emerge as viable feedstocks. This review highlights recent and notable achievements related to such efforts while offering future outlooks towards engineering microbial cell factories for aromatic production. © 2018 Society of Chemical Industry

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Metabolic engineering of Pseudomonas taiwanensis VLB120 with minimal genomic modifications for high-yield phenol production

Cited by 81 publications

References 70 publications

Laboratory evolution reveals the metabolic and regulatory basis of ethylene glycol metabolism byPseudomonas putidaKT2440

Laboratory evolution reveals the metabolic and regulatory basis of ethylene glycol metabolism byPseudomonas putidaKT2440

Chasing bacterial chassis for metabolic engineering: a perspective review from classical to non‐traditional microorganisms

Emerging tools, enabling technologies, and future opportunities for the bioproduction of aromatic chemicals

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