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Enhanced Activity by Genetic Complementarity: Heterologous Secretion of Clostridial Cellulases by Bacillus licheniformis and Bacillus velezensis.

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.

SUBMITTER: Arsov A

PROVIDER: S-EPMC8468253 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Transcriptome analysis was used to investigate the global stress response of the Gram-positive bacterium Bacillus subtilis caused by overproduction of the well-secreted AmyQ α-amylase from Bacillus amyloliquefaciens. Analyses of the control and overproducing strains were carried out at the end of exponential growth and in stationary phase, when protein secretion from B. subtilis is optimal. Among the genes that showed increased expression were htrA and htrB, which are part of the CssRS regulon that responds to high-level protein secretion and heat stress. The analysis of the transcriptome profiles of a cssS mutant compared to the wild-type, under identical secretion stress conditions, revealed several genes with altered transcription in a CssRS-dependent manner, for example citM, ylxF, yloA, ykoJ and several genes of the flgB operon. However, a high affinity CssR-binding was only observed for htrA and htrB, and possibly for citM. In addition, the DNA macroarray approach reveal that several genes of the sporulation pathway are downregulated by AmyQ overexpression, and a group of motility-specific (σD-dependent) transcripts were clearly upregulated. Subsequent flow cytometric analyses demonstrate that upon overproduction of AmyQ as well as a non-secretable variant of the α-amylase, the process of sporulation is severely inhibited. The same experiments were implemented to investigate the expression levels of the hag promoter, a well-established reporter for σD-dependent gene expression. This approach confirmed the observations based on our DNA macroarray analyses and led us to conclude that expression levels of several genes involved in motility are maintained at high levels under all conditions of α-amylase overproduction. Secretion stress was applied by overproducing the well-secreted AmyQ α-amylase (pKTH10 vector) from B. amyloliquefaciens. Besides examining secretion stress in wild-type cells, we compared transcriptome profiles of a cssS mutant strain under conditions of high-level AmyQ production. Samples for transcriptome analyses were collected at the late exponential growth stage (one hour before the transition point) and 3 hours upon entry in the stationary growth phase. Three independent cultures of each strain were used and cells were sampled for macroarray experiments. Duplicate spots were averaged in Array-Pro software (Media Cybernetics, Inc.) and the signal was normalized after background subtraction by calculation of the percentage of total signal per gene using Microsoft Excel.

Project description:BACKGROUND:Bacillus licheniformis 2709 is extensively applied as a host for the high-level production of heterologous proteins, but Bacillus cells often possess unfavorable wild-type properties, such as production of viscous materials and foam during fermentation, which seriously influenced the application in industrial fermentation. How to develop it from a soil bacterium to a super-secreting cell factory harboring less undomesticated properties always plays vital role in industrial production. Besides, the optimal expression pattern of the inducible enzymes like alkaline protease has not been optimized by comparing the transcriptional efficiency of different plasmids and genomic integration sites in B. licheniformis. RESULT:Bacillus licheniformis 2709 was genetically modified by disrupting the native lchAC genes related to foaming and the eps cluster encoding the extracellular mucopolysaccharide via a markerless genome-editing method. We further optimized the expression of the alkaline protease gene (aprE) by screening the most efficient expression system among different modular plasmids and genomic loci. The results indicated that genomic expression of aprE was superior to plasmid expression and finally the transcriptional level of aprE greatly increased 1.67-fold through host optimization and chromosomal integration in the vicinity of the origin of replication, while the enzyme activity significantly improved 62.19% compared with the wild-type alkaline protease-producing strain B. licheniformis. CONCLUSION:We successfully engineered an AprE high-yielding strain free of undesirable properties and its fermentation traits could be applied to bulk-production by host genetic modification and expression optimization. In summary, host optimization is an enabling technology for improving enzyme production by eliminating the harmful traits of the host and optimizing expression patterns. We believe that these strategies can be applied to improve heterologous protein expression in other Bacillus species.

Dataset Information

Enhanced Activity by Genetic Complementarity: Heterologous Secretion of Clostridial Cellulases by Bacillus licheniformis and Bacillus velezensis.

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