A Case of Adaptive Laboratory Evolution (ALE): Biodegradation of Furfural by Pseudomonas pseudoalcaligenes CECT 5344.

M Isabel Igeño,Rafael Blasco,Daniel Macias

doi:10.3390/genes10070499

Abstract

Pseudomonas pseudoalcaligenes CECT 5344 is a bacterium able to assimilate cyanide as a nitrogen source at alkaline pH. Genome sequencing of this strain allowed the detection of genes related to the utilization of furfurals as a carbon and energy source. Furfural and 5-(hydroxymethyl) furfural (HMF) are byproducts of sugars production during the hydrolysis of lignocellulosic biomass. Since they inhibit the yeast fermentation to obtain bioethanol from sugars, the biodegradation of these compounds has attracted certain scientific interest. P. pseudoalcaligenes was able to use furfuryl alcohol, furfural and furoic acid as carbon sources, but after a lag period of several days. Once adapted, the evolved strain (R1D) did not show any more prolonged lag phases. The transcriptomic analysis (RNA-seq) of R1D revealed a non-conservative punctual mutation (L261R) in BN5_2307, a member of the AraC family of activators, modifying the charge of the HTH region of the protein. The inactivation of the mutated gene in the evolved strain by double recombination reverted to the original phenotype. Although the bacterium did not assimilate HMF, it transformed it into value-added building blocks for the chemical industry. These results could be used to improve the production of cost-effective second-generation biofuels from agricultural wastes.

Highlights

The development of renewable resources is one of the key actions to palliate climate change, which is largely a consequence of the world’s dependence on petrol
Furfural concentrations higher than 5 mM increased the lag phase, suggesting that this compound is toxic at a high concentration
The tolerance of P. pseudoalcaligenes CECT 5344 to furfural is relatively high if compared to C. basilensis HMF14 [16]. This toxic effect was not observed for furoic acid (FA), and furfuryl alcohol (FFA) was not toxic up to a concentration of 20 mM

Summary

Introduction

The development of renewable resources is one of the key actions to palliate climate change, which is largely a consequence of the world’s dependence on petrol. Contamination of the environment is an inevitable consequence of human development. These are global problems that need international agreements [1]. Biotechnology can offer solutions to these challenges, such as the production of bioethanol as a substitute to gasoline [2]. Biotechnology can offer alternatives to the physical-chemical treatment of contaminating compounds, either by avoiding their production, or by mitigating their impact once it has occurred. The biodegradation of pollutants is, in general, a technology that has good social acceptance [3]. Pseudomonas pseudoalcaligenes CECT 5344 was isolated from sludge of Guadalquivir River, and it is able to use cyanide as the only source of nitrogen [4]

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Results

Conclusion