Abstract
To adapt to various ecological niches, the members of genus Bacillus display a wide spectrum of glycoside hydrolases (GH) responsible for the hydrolysis of cellulose and lignocellulose. Being abundant and renewable, cellulose-containing plant biomass may be applied as a substrate in second-generation biotechnologies for the production of platform chemicals. The present study aims to enhance the natural cellulase activity of two promising 2,3-butanediol (2,3-BD) producers, Bacillus licheniformis 24 and B. velezensis 5RB, by cloning and heterologous expression of cel8A and cel48S genes of Acetivibrio thermocellus. In B. licheniformis, the endocellulase Cel8A (GH8) was cloned to supplement the action of CelA (GH9), while in B. velezensis, the cellobiohydrolase Cel48S (GH48) successfully complemented the activity of endo-cellulase EglS (GH5). The expression of the natural and heterologous cellulase genes in both hosts was demonstrated by reverse-transcription PCR. The secretion of clostridial cellulases was additionally enhanced by enzyme fusion to the subtilisin-like signal peptide, reaching a significant increase in the cellulase activity of the cell-free supernatants. The results presented are the first to reveal the possibility of genetic complementation for enhancement of cellulase activity in bacilli, thus opening the prospect for genetic improvement of strains with an important biotechnological application.
Highlights
Cellulose is the most abundant but least degradable polymer in nature
Two strains isolated by our team, Bacillus licheniformis 24 and B. velezensis 5RB, have been pointed out as promising for 2,3-BD production by conversion of plant biomass, as they utilize the sugars in lignocellulose content: glucose, cellobiose, galactose, mannose, xylose, and arabinose, and display some cellulase activity [19]
Plasmid DNA from the selected E. coli clones was isolated on a large scale and used for the transformation of B. licheniformis 24 and B. velezensis 5RB hosts
Summary
Cellulose is the most abundant but least degradable polymer in nature. As the major component of plant biomass and agro-industrial waste, the hydrolyzed form of cellulose is engaged as a substrate in microbial processes for the production of industrial enzymes [1,2], biofuels [3,4], biosurfactants [5], and organic chemicals such as lactic acid, succinic acid, 2,3-butanediol (2,3-BD) [6,7,8,9,10], and many others. The natural producers of 2,3-BD do not exhibit cellulase activity sufficient for the direct conversion of cellulose fraction to the target product. This necessitates costly preliminary steps of chemical or enzymatic hydrolysis of cellulose-containing substrates because the decomposition of biomass to soluble sugars is the most important and critical stage in its biochemical conversion into fuels and chemicals [11,12]. Two strains isolated by our team, Bacillus licheniformis 24 and B. velezensis 5RB, have been pointed out as promising for 2,3-BD production by conversion of plant biomass, as they utilize the sugars in lignocellulose content: glucose, cellobiose, galactose, mannose, xylose, and arabinose, and display some cellulase activity [19]
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