Abstract
With the aim to develop biocatalysts for enhanced hydrolysis of (hemi)cellulose into monosaccharides, random diversity by directed evolution was introduced in the gene coding for the endo-β-1,4-glucanase from Streptomyces sp. G12 which had been recombinantly expressed in Escherichia coli and named rCelStrep. The main objectives were therefore to set up a complete strategy for creation and automated screening of rCelStrep evolved direct mutants and to apply it to generate and screen a library of 10,000 random mutants to select the most active variants. The diversity was introduced in the gene by error-prone polymerase chain reaction. A primary qualitative screening on solid plates containing carboxymethylcellulose as the substrate allowed selecting 2200 active clones that were then subjected to a secondary quantitative screening towards AZO-CMC for the selection of 76 improved variants that were cultured in flasks and characterized. Five rCelStrep mutants exhibiting the highest hydrolytic activities than the wild-type enzyme were further characterized and applied to the bioconversion of the pretreated Arundo donax lignocellulosic biomass. It is worth of noting that one of the five tested mutants exhibited a 30% improvement in bioconversion yields compared to the wild-type enzyme, despite the absence of the carbohydrate binding module domain in this variant. Homology models of the three-dimensional structures of the catalytic and binding modules of rCelStrep were obtained and localization of mutations on these models allowed us to speculate on the structure–function relationships of the mutants.
Highlights
Construction and screening of rCelStrep error‐prone PCR library In the present study, an automated workstation including the robot colony picker QPIX 450 (Molecular Devices, LLC, CA, USA) and the robot BioMek NXP (Beckman Coulter, CA, USA) was adopted for the library screening and an ad hoc strategy of automated highthroughput screening (HTS) for variants endowed with higher cellulolytic activity developed (Additional file 1: Fig. S1)
G12 recombinantly expressed in E. coli BL21(DE3) and named rCelStrep (Amore et al 2012), this enzyme was subjected to random biodiversity generation applying an errorprone PCR mutagenesis approach on the corresponding gene
Directed evolution has been shown to be a potent technology for the improvement of enzymes that are potentially important for industrial applications, there are several limitations that need to be solved, mainly related to the lack of rapid and generalized highthroughput screening (HTS) systems
Summary
There is an increasing demand for both enhanced bioconversion routes and novel cellulases with higher hydrolytic efficiencies and improved stability properties: great efforts have being employed for developing improved bioconversion conditions and reactor configurations (Liguori et al 2016) and discovering novel enzymes and engineering the existing ones (Wang et al 2012a) In this context, directed evolution is a process that more closely resembles natural protein evolution and is advantageous compared with rational design because it does not require a detailed knowledge of the target system (Tizei et al 2016). This variant was used in lignocellulosic biomasses (Arundo donax, corn cobs and brewer’s spent grains) conversion (Marcolongo et al 2014), improving the sugars yields in comparison to the wild type enzyme
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