Article

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

ABSTRACT

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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    • "onas putida S12 . A laboratory evolution approach was used to generate a P . putida S12 strain with an increased biomass yield . The strain , however , failed to overcome diauxic growth . Introduction of the transporter genes xylFGH under the tac promoter failed to improve xylose metabolism , indicating that transport was not the limiting factor ( Meijnen et al . , 2008 ) . xylAB genes from Streptomyces lividans TK23 , when expressed under the tac promoter in the actinobacteria Rhodococcus opacus PD630 and Rhodococcus jostii RHA1 , yielded efficient co - fermenting strains ( Xiong et al . , 2012 ) . Heterologous expression of the Glf transporter from Z . mobilis CP4 , which had been modified by error -"
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    ABSTRACT: Wood sugars such as xylose can be used as an inexpensive carbon source for biotechnological applications. The model cyanobacterium Synechocystis sp. PCC 6803 lacks the ability to catabolize wood sugars as an energy source. Here, we generated four Synechocystis strains that heterologously expressed XylAB enzymes, which mediate xylose catabolism, either in combination with or without one of three xylose transporters, namely XylE, GalP, or Glf. Except for glf, which is derived from the bacterium Zymomonas mobilis ZM4, the heterologous genes were sourced from Escherichia coli K-12. All of the recombinant strains were able to utilize xylose in the absence of catabolite repression. When xylose was the lone source of organic carbon, strains possessing the XylE and Glf transporters were most efficient in terms of dry biomass production and xylose consumption and the strain lacking a heterologous transporter was the least efficient. However, in the presence of a xylose-glucose mixed sugar source, the strains exhibited similar levels of growth and xylose consumption. This study demonstrates that various bacterial xylose transporters can boost xylose catabolism in transgenic Synechocystis strains, and paves the way for the sustainable production of bio-compounds and green fuels from lignocellulosic biomass.
    Full-text · Article · Dec 2015 · Frontiers in Microbiology
    • "onas putida S12 . A laboratory evolution approach was used to generate a P . putida S12 strain with an increased biomass yield . The strain , however , failed to overcome diauxic growth . Introduction of the transporter genes xylFGH under the tac promoter failed to improve xylose metabolism , indicating that transport was not the limiting factor ( Meijnen et al . , 2008 ) . xylAB genes from Streptomyces lividans TK23 , when expressed under the tac promoter in the actinobacteria Rhodococcus opacus PD630 and Rhodococcus jostii RHA1 , yielded efficient co - fermenting strains ( Xiong et al . , 2012 ) . Heterologous expression of the Glf transporter from Z . mobilis CP4 , which had been modified by error -"

    No preview · Article · Oct 2015 · Frontiers in Microbiology
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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.
    Full-text · Article · Jun 2015 · Metabolic Engineering
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