Engineering Pseudomonas putida S12 for Efficient Utilization of D-Xylose and L-Arabinose

TNO-Quality of Life, Julianalaan 67, 2628 BC Delft, The Netherlands.
Applied and Environmental Microbiology (Impact Factor: 3.67). 07/2008; 74(16):5031-7. DOI: 10.1128/AEM.00924-08
Source: PubMed


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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Available from: Jean-Paul Meijnen,
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    • "Inside cells D-xylose is converted by xylose isomerase (XI, encoded by xylA) to D-xylulose, which is subsequently phosphorylated to xylulose-5-phosphate (X5P) by xylulokinase (XK, encoded by xylB), where it enters the pentose phosphate pathway (PPP). Genetic engineering by introduction of heterologous pentose metabolic pathway genes has enabled xylose utilization in heterotrophic strains (Fan et al., 2011; Hahn- Hagerdal et al., 2007; Jeffries, 2006; Meijnen et al., 2008; Rogers et al., 2007; Toivari et al., 2001; van Maris et al., 2007; Zhang et al., 1995). Recently this approach has also been successful in the cyanobacterium, Synechococcus elongates PCC 7942 (McEwen et al., 2013), although improved biofuel productivity was not reported. "
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    ABSTRACT: Hydrolysis of plant biomass generates a mixture of simple sugars that is particularly rich in glucose and xylose. Fermentation of the released sugars emits CO2 as byproduct due to metabolic inefficiencies. Therefore, the ability of a microbe to simultaneously convert biomass sugars and photosynthetically fix CO2 into target products is very desirable. In this work, the cyanobacterium, Synechocystis 6803, was engineered to grow on xylose in addition to glucose. Both the xylA (xylose isomerase) and xylB (xylulokinase) genes from E. coli were required to confer xylose utilization, but a xylose-specific transporter was not required. Introduction of xylAB into an ethylene-producing strain increased the rate of ethylene production in the presence of xylose. Additionally, introduction of xylAB into a glycogen-synthesis mutant enhanced production of keto acids. Isotopic tracer studies found that nearly half of the carbon in the excreted keto acids was derived from the engineered xylose metabolism, while the remainder was derived from CO2 fixation. Copyright © 2015. Published by Elsevier Inc.
    Metabolic Engineering 06/2015; 30. DOI:10.1016/j.ymben.2015.06.002 · 6.77 Impact Factor
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    • "Until now, several Saccharomyces cerevisiae strains capable of fermenting xylose efficiently have been successfully obtained by adaptive laboratory evolution (Scalcinati et al. 2012; Shen et al. 2012; Sonderegger and Sauer 2003; Zhou et al. 2012). Pseudomonas putida was also subjected to laboratory evolution and a strain that could efficiently utilize xylose and arabinose was obtained (Meijnen et al. 2008). "
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    ABSTRACT: Efficient utilization of xylose by bacteria is essential for production of fuels and chemicals from lignocellulosic biomass. In this study, Bacillus subtilis 168 was subjected to laboratory adaptive evolution, and a mutant E72, which could grow on xylose with a maximum specific growth rate of 0.445 h−1, was obtained. By whole-genome sequencing, 16 mutations were identified in strain E72. Through further analysis, three of them, which were in the coding regions of genes araR, sinR, and comP, were identified as the beneficial mutations. The reconstructed strain 168ARSRCP harboring these three mutations exhibited similar growth capacity on xylose to the evolved strain E72, and the average xylose consumption rate of this strain is 0.530 g/l/h, much higher than that of E72 (0.392 g/l/h). Furthermore, genes acoA and bdhAwere deleted and the final strain could utilize xylose to produce acetoin at 71 % of the maximum theoretical yield. These results suggested that this strain could be used as a potential platform for production of fuels and chemicals from lignocellulosic biomass.
    Applied Microbiology and Biotechnology 10/2014; DOI:10.1007/s00253-014-6131-7 · 3.34 Impact Factor
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    • "Pseudomonas putida KT2440 was used as a model organism owing to its numerous qualities as an expression host, such as safety (Bagdasarian et al. 1981; Nakazawa and Yokota 1973), fast growth, a fully sequenced genome (Nelson et al. 2002) and high stress tolerance (Martins Dos Santos et al. 2004). Together with simple nutrient demand, the potential to regenerate redox cofactors at a high rate (Blank et al. 2008) and its amenability to genetic manipulation, P. putida is an ideal host for heterologous gene expression (Meijnen et al. 2008). With the advance of genome-wide pathway modeling (Puchałka et al. 2008) and 'omics techniques, the way for systemswide engineering strategies was paved to turn P. putida into a flexible cell factory chassis (Yuste et al. 2006). "
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    ABSTRACT: Population heterogeneity occurring in industrial microbial bioprocesses is regarded as a putative effector causing performance loss in large scale. While the existence of subpopulations is a commonly accepted fact, their appearance and impact on process performance still remains rather unclear. During cell cycling, distinct subpopulations differing in cell division state and DNA content appear which contribute individually to the efficiency of the bioprocess. To identify stressed or impaired subpopulations, we analyzed the interplay of growth rate, cell cycle and phenotypic profile of subpopulations by using flow cytometry and cell sorting in conjunction with mass spectrometry based global proteomics. Adjusting distinct growth rates in chemostats with the model strain Pseudomonas putida KT2440, cells were differentiated by DNA content reflecting different cell cycle stages. The proteome of separated subpopulations at given growth rates was found to be highly similar, while different growth rates caused major changes of the protein inventory with respect to e.g. carbon storage, motility, lipid metabolism and the translational machinery. In conclusion, cells in various cell cycle stages at the same growth rate were found to have similar to identical proteome profiles showing no significant population heterogeneity on the proteome level. In contrast, the growth rate clearly determines the protein composition and therefore the metabolic strategy of the cells.
    AMB Express 09/2014; 4(71): DOI:10.1186/s13568-014-0071-6
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