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Metabolic and regulatory rearrangements underlying efficient D-xylose utilization in engineered Pseudomonas putida S12.

ABSTRACT: Previously, an efficient D-xylose utilizing Pseudomonas putida S12 strain was obtained by introducing the D-xylose isomerase pathway from Escherichia coli, followed by evolutionary selection. In the present study, systemic changes associated with the evolved phenotype were identified by transcriptomics, enzyme activity analysis, and inverse engineering. A key element in improving the initially poor D-xylose utilization was the redistribution of 6-phospho-D-gluconate (6-PG) between the Entner-Doudoroff pathway and the oxidative pentose phosphate (PP) pathway. This redistribution increased the availability of 6-PG for oxidative decarboxylation to D-ribose-5-phosphate, which is essential for the utilization of D-xylose via the nonoxidative PP pathway. The metabolic redistribution of 6-PG was procured by modified HexR regulation, which in addition appeared to control periplasmic sugar oxidation. Because the absence of periplasmic D-xylonate formation was previously demonstrated to be essential for achieving a high biomass yield on D-xylose, the aberrant HexR control appeared to underlie both the improved growth rate and biomass yield of the evolved D-xylose utilizing P. putida strain. The increased oxidative PP pathway activity furthermore resulted in an elevated NADH/NAD(+) ratio that caused the metabolic flux to be redirected from the TCA cycle to the glyoxylate shunt, which was also activated transcriptionally. Clearly, these findings may serve as an important case in point to engineer and improve the utilization of non-natural carbon sources in a wide range of industrial microorganisms.

SUBMITTER: Meijnen JP

PROVIDER: S-EPMC3340264 | biostudies-literature | 2012 Apr

REPOSITORIES: biostudies-literature

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Metabolic and regulatory rearrangements underlying efficient D-xylose utilization in engineered Pseudomonas putida S12.

Meijnen Jean-Paul JP de Winde Johannes H JH Ruijssenaars Harald J HJ

The Journal of biological chemistry 20120313 18

Previously, an efficient D-xylose utilizing Pseudomonas putida S12 strain was obtained by introducing the D-xylose isomerase pathway from Escherichia coli, followed by evolutionary selection. In the present study, systemic changes associated with the evolved phenotype were identified by transcriptomics, enzyme activity analysis, and inverse engineering. A key element in improving the initially poor D-xylose utilization was the redistribution of 6-phospho-D-gluconate (6-PG) between the Entner-Dou ...[more]

PMID: 22416130

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Project description:Pseudomonas putida S12 is inherently solvent tolerant and constitutes a promising platform for biobased production of aromatic compounds and biopolymers. The megaplasmid pTTS12 of P. putida S12 carries several gene clusters involved in solvent tolerance, and the removal of this megaplasmid caused a significant reduction in solvent tolerance. In this study, we succeeded in restoring solvent tolerance in plasmid-cured P. putida S12 using adaptive laboratory evolution (ALE), underscoring the innate solvent tolerance of this strain. Whole-genome sequencing identified several single nucleotide polymorphisms (SNPs) and a mobile element insertion enabling ALE-derived strains to survive and sustain growth in the presence of a high toluene concentration (10% [vol/vol]). We identified mutations in an RND efflux pump regulator, arpR, that resulted in constitutive upregulation of the multifunctional efflux pump ArpABC. SNPs were also found in the intergenic region and subunits of ATP synthase, RNA polymerase subunit β', a global two-component regulatory system (GacA/GacS), and a putative AraC family transcriptional regulator, Afr. Transcriptomic analysis further revealed a constitutive downregulation of energy-consuming activities in ALE-derived strains, such as flagellar assembly, FoF1 ATP synthase, and membrane transport proteins. In summary, constitutive expression of a solvent extrusion pump in combination with high metabolic flexibility enabled the restoration of the solvent tolerance trait in P. putida S12 lacking its megaplasmid.IMPORTANCE Sustainable production of high-value chemicals can be achieved by bacterial biocatalysis. However, bioproduction of biopolymers and aromatic compounds may exert stress on the microbial production host and limit the resulting yield. Having a solvent tolerance trait is highly advantageous for microbial hosts used in the biobased production of aromatics. The presence of a megaplasmid has been linked to the solvent tolerance trait of Pseudomonas putida; however, the extent of innate, intrinsic solvent tolerance in this bacterium remained unclear. Using adaptive laboratory evolution, we successfully adapted the plasmid-cured P. putida S12 strain to regain its solvent tolerance. Through these adapted strains, we began to clarify the causes, origins, limitations, and trade-offs of the intrinsic solvent tolerance in P. putida This work sheds light on the possible genetic engineering targets to enhance solvent tolerance in Pseudomonas putida as well as other bacteria.

Dataset Information

Metabolic and regulatory rearrangements underlying efficient D-xylose utilization in engineered Pseudomonas putida S12.

Publications

Metabolic and regulatory rearrangements underlying efficient D-xylose utilization in engineered Pseudomonas putida S12.

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