Unraveling 1,4-Butanediol Metabolism in Pseudomonas putida KT2440

Li, Wing‐Jin; Narancic, Tanja; Kenny, Shane T.; Niehoff, Paul-Joachim; O’Connor, Kevin E.; Blank, Lars M.; Wierckx, Nick

doi:10.3389/fmicb.2020.00382

Cited by 49 publications

(50 citation statements)

References 79 publications

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“…When PP_2047 is nonfunctional, 4-hydroxycrotonyl-CoA likely accumulates as a deadend metabolite resulting in decreased fitness. Li et al also showed that deletion mutants of PP_2046 are unable to grow on 1,4-butanediol (30). Our data suggests that this is because PP_2049 appears to be the main alcohol dehydrogenase acting on either 1,4-butanediol or 4-hydroxybutyrate, and is in the operon under the control of PP_2046.…”

Section: Short Chain Diol Catabolismsupporting

confidence: 63%

“…1,4-butanediol catabolism has been previously studied; based on the results of expression data and adaptive laboratory evolution, Li et al proposed three potential catabolic pathways for 1,4-butanediol, including a beta-oxidation pathway (Figure 8) (30). Their evolved strains had mutations in the LysR activator PP_2046 that resulted in overexpression of the beta-oxidation operon PP_2047-51 (30). Interestingly, we found that when grown on 1,4 butanediol, transposon mutants of the acyl-CoA dehydrogenase PP_2048 had significant fitness benefits and no CoAligase mutants showed significant fitness defects.…”

Section: Short Chain Diol Catabolismmentioning

confidence: 99%

“…Much work has focused on the unique biochemistry and regulation of two pyrroloquinoline quinone (PQQ)dependent alcohol dehydrogenases (ADH), pedE and pedH, which exhibit broad substrate specificity for both alcohols and aldehydes (25,26). Specific work has also investigated the suitability of P. putida for the production of ethanol (27) and the genetic basis for its ability to catabolize butanol and 1,4-butanediol (28)(29)(30). P. putida is also known for its ability to tolerate solvents and alcohols, making it an attractive host for their industrial production (31,32).…”

Section: Importance Introductionmentioning

confidence: 99%

“…A recent surge in omics-level data has revealed much about the metabolism of P. putida, with adaptive evolution (30), proteomics (10,28,29), and 13 C flux analysis (37-39) yielding valuable insights. An approach that has proven to be particularly powerful is Random Barcode Transposon Sequencing (RB-TnSeq) (40,41).…”

Section: Importance Introductionmentioning

confidence: 99%

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Fatty Acid and Alcohol Metabolism in Pseudomonas putida: Functional Analysis Using Random Barcode Transposon Sequencing

Thompson

Incha

Pearson

et al. 2020

Appl Environ Microbiol

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With its ability to catabolize a wide variety of carbon sources and a growing engineering toolkit, Pseudomonas putida KT2440 is emerging as an important chassis organism for metabolic engineering. Despite advances in our understanding of this organism, many gaps remain in our knowledge of the genetic basis of its metabolic capabilities. These gaps are particularly noticeable in our understanding of both fatty acid and alcohol catabolism, where many paralogs putatively coding for similar enzymes co-exist making biochemical assignment via sequence homology difficult. To rapidly assign function to the enzymes responsible for these metabolisms, we leveraged Random Barcode Transposon Sequencing (RB-TnSeq). Global fitness analyses of transposon libraries grown on 13 fatty acids and 10 alcohols produced strong phenotypes for hundreds of genes. Fitness data from mutant pools grown on varying chain length fatty acids indicated specific enzyme substrate preferences, and enabled us to hypothesize that DUF1302/DUF1329 family proteins potentially function as esterases. From the data we also postulate catabolic routes for the two biogasoline molecules isoprenol and isopentanol, which are catabolized via leucine metabolism after initial oxidation and activation with CoA. Because fatty acids and alcohols may serve as both feedstocks or final products of metabolic engineering efforts, the fitness data presented here will help guide future genomic modifications towards higher titers, rates, and yields. IMPORTANCE To engineer novel metabolic pathways into P. putida, a comprehensive understanding of the genetic basis of its versatile metabolism is essential. Here we provide functional evidence for the putative roles of hundreds of genes involved in the fatty acid and alcohol metabolism of this bacterium. These data provide a framework facilitating precise genetic changes to prevent product degradation and channel the flux of specific pathway intermediates as desired.

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Section: Short Chain Diol Catabolismsupporting

confidence: 63%

Section: Short Chain Diol Catabolismmentioning

confidence: 99%

Section: Importance Introductionmentioning

confidence: 99%

Section: Importance Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Fatty Acid and Alcohol Metabolism in Pseudomonas putida: Functional Analysis Using Random Barcode Transposon Sequencing

Thompson

Incha

Pearson

et al. 2020

Appl Environ Microbiol

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“…Recently, shotgun proteomics identified the oxoprolinase-encoding oplBA genes, responsible for the undesired hydrolysis of valerolactam P. putida, leading to a more efficient production process (Thompson et al 2019). Besides, application of proteomics analysis and genome resequencing enhanced the understanding of the plastic monomer 1,4-butanediol metabolism in P. putida KT2440 (Li et al 2020).…”

Section: Transcriptomics and Proteomicsmentioning

confidence: 99%

Industrial biotechnology of Pseudomonas putida: advances and prospects

Weimer

Kohlstedt

Volke

et al. 2020

Appl Microbiol Biotechnol

172

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Pseudomonas putida is a Gram-negative, rod-shaped bacterium that can be encountered in diverse ecological habitats. This ubiquity is traced to its remarkably versatile metabolism, adapted to withstand physicochemical stress, and the capacity to thrive in harsh environments. Owing to these characteristics, there is a growing interest in this microbe for industrial use, and the corresponding research has made rapid progress in recent years. Hereby, strong drivers are the exploitation of cheap renewable feedstocks and waste streams to produce value-added chemicals and the steady progress in genetic strain engineering and systems biology understanding of this bacterium. Here, we summarize the recent advances and prospects in genetic engineering, systems and synthetic biology, and applications of P. putida as a cell factory. Key points • Pseudomonas putida advances to a global industrial cell factory. • Novel tools enable system-wide understanding and streamlined genomic engineering. • Applications of P. putida range from bioeconomy chemicals to biosynthetic drugs. Electronic supplementary material The online version of this article (10.1007/s00253-020-10811-9) contains supplementary material, which is available to authorized users.

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Pseudomonas putida KT2440 endures temporary oxygen limitations

Demling

Ankenbauer

Klein

et al. 2021

Biotech & Bioengineering

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The obligate aerobic nature of Pseudomonas putida, one of the most prominent whole-cell biocatalysts emerging for industrial bioprocesses, questions its ability to be cultivated in large-scale bioreactors, which exhibit zones of low dissolved oxygen tension. P. putida KT2440 was repeatedly subjected to temporary oxygen limitations in scale-down approaches to assess the effect on growth and an exemplary production of rhamnolipids. At those conditions, the growth and production of P. putida KT2440 were decelerated compared to well-aerated reference cultivations, but remarkably, final biomass and rhamnolipid titers were similar. The robust growth behavior was confirmed across different cultivation systems, media compositions, and laboratories, even when P. putida KT2440 was repeatedly exposed to dual carbon and oxygen starvation. Quantification of the nucleotides ATP, ADP, and AMP revealed a decrease of intracellular ATP concentrations with increasing duration of oxygen starvation, which can, however, be restored when re-supplied with oxygen.Only small changes in the proteome were detected when cells encountered oscillations in dissolved oxygen tensions. Concluding, P. putida KT2440 appears to be able to cope with repeated oxygen limitations as they occur in large-scale bioreactors, affirming its outstanding suitability as a whole-cell biocatalyst for industrialscale bioprocesses.

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Unraveling 1,4-Butanediol Metabolism in Pseudomonas putida KT2440

Cited by 49 publications

References 79 publications

Fatty Acid and Alcohol Metabolism in Pseudomonas putida: Functional Analysis Using Random Barcode Transposon Sequencing

Fatty Acid and Alcohol Metabolism in Pseudomonas putida: Functional Analysis Using Random Barcode Transposon Sequencing

Industrial biotechnology of Pseudomonas putida: advances and prospects

Pseudomonas putida KT2440 endures temporary oxygen limitations

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