Journal
MOLECULES
Volume 26, Issue 18, Pages -Publisher
MDPI
DOI: 10.3390/molecules26185625
Keywords
cellulose; 2; 3-butanediol; Bacillus licheniformis; Bacillus velezensis; cellulase; heterologous expression; signal peptide; RT-PCR; pBE-S shuttle vector
Funding
- National Scientific Fund, Ministry of Education and Science, Republic of Bulgaria [DN 17/1]
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The study aimed to enhance the natural cellulase activity of Bacillus licheniformis and B. velezensis by cloning and heterologous expression of cel8A and cel48S genes from Acetivibrio thermocellus. The results demonstrate the potential for genetic complementation to significantly increase cellulase activity in bacilli.
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.
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