Elsevier

Journal of Biotechnology

Volume 239, 10 December 2016, Pages 39-46
Journal of Biotechnology

Whole-cell biosensor of cellobiose and application to wood decay detection

https://doi.org/10.1016/j.jbiotec.2016.10.003Get rights and content

Highlights

  • The whole-cell biosensor specifically detects cellobiose.

  • The biosensor is a fast and powerful tool to detect fungal wood degradation.

  • Colorimetric detection is based on a catechol dioxygenase reporter gene under control of the engineered Streptomyces CebR master regulator.

Abstract

Fungal biodegradation of wood is one of the main threats regarding its use as a material. So far, the detection of this decaying process is empirically assessed by loss of mass, when the fungal attack is advanced and woody structure already damaged. Being able to detect fungal attack on wood in earlier steps is thus of special interest for the wood economy. In this aim, we designed here a new diagnostic tool for wood degradation detection based on the bacterial whole-cell biosensor technology. It was designed in diverting the soil bacteria Streptomyces CebR sensor system devoted to cellobiose detection, a cellulolytic degradation by-product emitted by lignolytic fungi since the onset of wood decaying process. The conserved regulation scheme of the CebR system among Streptomyces allowed constructing a molecular tool easily transferable in different strains or species and enabling the screen for optimal host strains for cellobiose detection. Assays are performed in microplates using one-day culture lysates. Diagnostic is performed within one hour by a spectrophotometric measuring of the cathecol deshydrogenase activity. The selected biosensor was able to detect specifically cellobiose at concentrations similar to those measured in decaying wood and in a spruce leachate attacked by a lignolytic fungus, indicating a high potential of applicability to detect ongoing wood decay process.

Introduction

Thanks to its intrinsic mechanical properties, wood is used since the Neolithic era as a material for construction, furnishing, heating or wood-derived products such as paper. Nowadays, there is also a regain of interest for this material as it is renewable and a source of biomass for conversion into bio-ethanol (Wang et al., 2013). As a material, wood is highly resistant to the environmental conditions such as rain, sun or other abiotic stresses and the main threat regarding its use comes mostly from the attack by lignolytic organisms. This wood bio-degradation represents a major economic problem. For instance, in the US only, it costs more than 5 billion USD each year to homeowners and almost 10% of the annual product of forest is used to replace the degraded products (Schultz and Nicholas, 2008). Being able to detect the early wood bio-degradation is thus crucial. During storage, it would avoid to use contaminated wood for construction. When the wood is already used as a material it would enable to apply early curative measures before any significant loss of mechanical properties. However, to our knowledge, there is no tool available to achieve this goal and wood degradation is so far only assessed by resistance testing and visual expertise only relevant and operating on wood decayed at an advanced level.

Wood bio-degradation in forest ecosystems is mainly the fact of diverse fungi (Rajala et al., 2012) classified according to the type of decay they cause (e.g. white-rots, brown-rots, soft-rots, see for review (Schwarze et al., 2000)). If rot-fungi have distinct specificities in term of ecology or degradation strategies, they nevertheless all degrade and use as a carbon source the cellulose that represents 40%–50% of the plant dry weight (Howard et al., 2004). Two main degradation strategies exist during the wood degradation by fungi: enzymatic pathways involving cellulases or oxidative mechanisms using Fenton reaction and polysaccharide monooxygenases (Lynd et al., 2002, Phillips et al., 2011). However, a canonical degradation pathway is common for all of them where the cellulose fiber is shortened into simpler forms: the cellobiose (two β-1,4 linked glucose units) or sometimes into cellodextrines (generally from three to six β-1,4-linked glucose units) (Langston et al., 2011, Lynd et al., 2002). These cellulose degradation by-products will be hydrolyzed later on into glucose by the action of β-glucosidases (Langston et al., 2011). Thus, the initial release and presence of cellobiose and cellodextrines is a common denominator and can be considered as a signature of the attack of wood by rot-fungi.

The Streptomyces are filamentous spore forming soil dwelling bacteria that are generally not able to degrade native wood, but are considered as essential to recycle biomass polymers in environment thanks to their large-enzymatic arsenal able to degrade wood by-products (Bontemps et al., 2013, Bruce et al., 2010). The detection of these compounds and the activation of the enzymatic pool to degrade them has been linked to the regulator CebR of the LacI family in Streptomyces griseus (Marushima et al., 2009), Streptomyces reticuli (Schlösser et al., 2000), Streptomyces sp. ActE (Takasuka et al., 2013). Since its characterization by Schlösser et al. (Schlösser et al., 2000), and Marushima et al. (Marushima et al., 2009), it is known that the CebR transcriptional repressor prevents gene expression from binding a conserved 22 bp hairpin motif (cebR-box) found in the transcriptional region of its targets. In presence of inducer molecules such as cellobiose or in some cases cellodextrines or cellulose, the CebR repression is alleviated and enables the expression of the controlled genes. So far, the complete CebR regulome is not well-known. However, a transcriptomic analysis of the Streptomyces sp. ActE (Sirex) (a symbiotic strain that helps the pine-boring woodwasp Sirex noctilio to deconstruct wood biomass) has shown that, in presence of wood derived compounds, the most up-regulated genes were under the control of the CebR-system. These genes were mostly involved in the uptake (ABC transporter system) or in the production of cellulolytic and hemicellulolytic enzymes (β-glucosidases, cellulases, cellobiohydrolases, mannosidases) (Takasuka et al., 2013). Moreover, CebR could induce other functions like the pathogenic factors of S. scabies in presence of cellobiose (Francis et al., 2015).

Since cellobiose is a key product of cellulose hydrolysis and indirectly indicates fungal wood degradation, we designed and developed a cebR-box based biosensor expressed by Streptomyces in order to detect the presence of cellobiose, demonstrated its sensibility and specificity and showed its applicability in a case of wood degradation detection.

Section snippets

Plasmids, strains and media

Plasmids and strains used in this work are presented in Table 1. Escherichia coli strains were grown in LB medium (Kieser et al., 2000) at 37 °C and Streptomyces at 30 °C either on solid SFM medium [20 g mannitol, 20 g soy flour and 20 g bacto-agar per liter] or in liquid modified HT medium (HT*) [1 g yeast extract, 1 g beef extract, 5 g mannitol, 2 g bacto-tryptone and 0.02 g COCL2 per liter, pH = 7.3]. For the biosensor tests, cellobiose (Alfa Aesar, Karlsruhe, Germany) and cellodextrines (Elicityl,

In silico analysis to assess the potential of Streptomyces as biosensor hosts

CebR prevents gene transcription in binding a specific sequence called the cebR-box and this repression is alleviated by the recognition of the inducer molecules, i.e. the cellobiose (Fig. 1A). The CebR sensor system has been previously found and reported as a highly conserved regulatory system in some Streptomyces species (Marushima et al., 2009, Schlösser et al., 2000, Takasuka et al., 2013), notably in term of sequence identity of the cebR-box. The concept of the biosensor was to build a

Discussion

The aim of this study was to create a biosensor dedicated to cellobiose detection. We based its construction on a microbial cellobiose sensor: the Streptomyces CebR system. Since, its characterization (Marushima et al., 2009, Schlösser et al., 2000), it is known that the CebR transcriptional repressor prevents gene expression from binding a conserved cebR-box. In presence of cellobiose it releases its action and enables the expression of the repressed genes such as genes coding cellulolytic

Conflict of interest

The authors declare no financial or commercial conflict of interest.

Acknowledgement

This work was funded by the Région Lorraine and the French National Research Agency through the Laboratory of Excellence ARBRE (ANR-11- LABX-000-01).

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