A rapid method for the purification of methanol dehydrogenase from Methylobacterium extorquens

Liu, Qinfeng; Kirchhoff, Jon R.; Faehnle, C.R.; Viola, Ronald E.; Hudson, Richard

doi:10.1016/j.pep.2005.07.014

Cited by 19 publications

(27 citation statements)

References 22 publications

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“…Pyrroloquinoline quinone-dependent D-glucose dehydrogenase activity was determined as described by Liu et al (13) by spectrophotometrically measuring the decrease in absorbance of 2,6-dichlorphenolindophenol (DCPIP) at 600 nm. The assay was performed at 30°C in a 1-ml (total volume) mixture.…”

Section: Methodsmentioning

confidence: 99%

Establishment of Oxidative d -Xylose Metabolism in Pseudomonas putida S12

Meijnen

Winde

Ruijssenaars

2009

Appl Environ Microbiol

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The oxidative D-xylose catabolic pathway of Caulobacter crescentus, encoded by the xylXABCD operon, was expressed in the gram-negative bacterium Pseudomonas putida S12. This engineered transformant strain was able to grow on D-xylose as a sole carbon source with a biomass yield of 53% (based on g [dry weight] g D-xylose ؊1 ) and a maximum growth rate of 0.21 h ؊1 . Remarkably, most of the genes of the xylXABCD operon appeared to be dispensable for growth on D-xylose. Only the xylD gene, encoding D-xylonate dehydratase, proved to be essential for establishing an oxidative D-xylose catabolic pathway in P. putida S12. The growth performance on D-xylose was, however, greatly improved by coexpression of xylXA, encoding 2-keto-3-deoxy-Dxylonate dehydratase and ␣-ketoglutaric semialdehyde dehydrogenase, respectively. The endogenous periplasmic glucose dehydrogenase (Gcd) of P. putida S12 was found to play a key role in efficient oxidative D-xylose utilization. Gcd activity not only contributes to D-xylose oxidation but also prevents the intracellular accumulation of toxic catabolic intermediates which delays or even eliminates growth on D-xylose.

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Section: Methodsmentioning

confidence: 99%

Establishment of Oxidative d -Xylose Metabolism in Pseudomonas putida S12

Meijnen

Winde

Ruijssenaars

2009

Appl Environ Microbiol

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“…Therefore, gene expression in M. extorquens AM1 has almost exclusively been driven by the strong promoter region of the mxaF gene (PmxaF) that encodes the large subunit of methanol dehydrogenase, which represents up to 9% of the total soluble protein in M. extorquens AM1 under methylotrophic growth conditions. 25 Only very recently has the similarly strong promoter PR from the rhizobial phage 16−3 (PR, 16−3) been introduced for gene expression in M. extorquens AM1. 26 Inducibility of gene expression was achieved by combining these promoters with regulatory elements, such as the CymR or TetR repressor.…”

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confidence: 99%

A Set of Versatile Brick Vectors and Promoters for the Assembly, Expression, and Integration of Synthetic Operons in Methylobacterium extorquens AM1 and Other Alphaproteobacteria

et al. 2014

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The discipline of synthetic biology requires standardized tools and genetic elements to construct novel functionalities in microorganisms; yet, many model systems still lack such tools. Here, we describe a novel set of vectors that allows the convenient construction of synthetic operons in Methylobacterium extorquens AM1, an important alphaproteobacterial model organism for methylotrophy and a promising platform organism for methanol-based biotechnology. In addition, we provide a set of constitutive alphaproteobacterial promoters of different strengths that were characterized in detail by two approaches: on the single-cell scale and on the cell population level. Finally, we describe a straightforward strategy to deliver synthetic constructs to the genome of M. extorquens AM1 and other Alphaproteobacteria. This study defines a new standard to systematically characterize genetic parts for their use in M. extorquens AM1 by using single-cell fluorescence microscopy and opens the toolbox for synthetic biological applications in M. extorquens AM1 and other alphaproteobacterial model systems.

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“…The pyrroloquinoline quinone (PQQ)-dependent Gcd activity was determined as described by Liu et al (16). The activity of Gcd was determined spectrophotometrically by measuring the decrease in the absorbance of 2,6-dichlorophenolindophenol (DCPIP) at 600 nm.…”

mentioning

confidence: 99%

Engineering Pseudomonas putida S12 for Efficient Utilization of d -Xylose and l -Arabinose

Meijnen

Winde

Ruijssenaars

2008

Appl Environ Microbiol

102

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The solvent-tolerant bacterium Pseudomonas putida S12 was engineered to utilize xylose as a substrate by expressing xylose isomerase (XylA) and xylulokinase (XylB) from Escherichia coli. The initial yield on xylose was low (9% [g CDW g substrate ؊1 ], where CDW is cell dry weight), and the growth rate was poor (0.01 h ؊1 ). The main cause of the low yield was the oxidation of xylose into the dead-end product xylonate by endogenous glucose dehydrogenase (Gcd). Subjecting the XylAB-expressing P. putida S12 to laboratory evolution yielded a strain that efficiently utilized xylose (yield, 52% [g CDW g xylose ؊1 ]) at a considerably improved growth rate (0.35 h ؊1 ). The high yield could be attributed in part to Gcd inactivity, whereas the improved growth rate may be connected to alterations in the primary metabolism. Surprisingly, without any further engineering, the evolved D-xylose-utilizing strain metabolized L-arabinose as efficiently as D-xylose. Furthermore, despite the loss of Gcd activity, the ability to utilize glucose was not affected. Thus, a P. putida S12-derived strain was obtained that efficiently utilizes the three main sugars present in lignocellulosic hydrolysate: glucose, xylose, and arabinose. This strain will form the basis for a platform host for the efficient production of biochemicals from renewable feedstock.

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A rapid method for the purification of methanol dehydrogenase from Methylobacterium extorquens

Cited by 19 publications

References 22 publications

Establishment of Oxidative d -Xylose Metabolism in Pseudomonas putida S12

Establishment of Oxidative d -Xylose Metabolism in Pseudomonas putida S12

A Set of Versatile Brick Vectors and Promoters for the Assembly, Expression, and Integration of Synthetic Operons in Methylobacterium extorquens AM1 and Other Alphaproteobacteria

Engineering Pseudomonas putida S12 for Efficient Utilization of d -Xylose and l -Arabinose

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