Search

Bacillus licheniformis 2709 is a potential cell factory for the production of alkaline protease AprE, which has important value in industrial application but still lacks sufficient production capacity. To address this problem, we investigated the effects of the secretory viscous materials on the synthesis of AprE, which might seriously affect the industrial fermentation. Furthermore, an iterative chromosomal integration strategy at various chromosomal loci was implemented to achieve stable high-level expression of AprE in B. licheniformis 2709. The host was genetically modified by disrupting the native pgs cluster controlling the biosynthesis of viscous poly-glutamic acid identified in the study by GC/MS, generating a mutant with significantly higher biomass and better bioreactor performance. We further enhanced the expression of alkaline protease by integrating two additional aprE expression cassettes into the genome, generating the integration mutant BL ∆UEP-3 with three aprE expression cassettes, whose AprE enzyme activity in shake flasks reached 25,736 ± 997 U/mL, which was 136% higher than that of the original strain, while the aprE transcription level increased 4.05 times. Thus, an AprE high-yielding strain with excellent fermentation traits was engineered, which was more suitable for bulk-production. Finally, the AprE titer was further increased in a 5-L fermenter, reaching 57,763 ± 1039 U/mL. In summary, genetic modification is an enabling technology for enhancing enzyme production by eliminating the unfavorable characteristics of the host and optimizing the expression of aprE through iterative chromosomal integration. We believe that the protocol developed in this study provides a valuable reference for chromosomal overexpression of proteins or bioactive molecules in other Bacillus species. [ABSTRACT FROM AUTHOR]

Alkaline protease AprE, produced by Bacillus licheniformis 2709 is an important edible hydrolase, which has potential applications in nutrient acquisition and medicine. The expression of AprE is finely regulated by a complex transcriptional regulation system. However, there is little study on transcriptional regulation mechanism of AprE biosynthesis in Bacillus licheniformis , which limits system engineering and further enhancement of AprE. Here, the severely depressed expression of aprE in degU and degS deletion mutants illustrated that the regulator DegU and its phosphorylation played a crucial part in AprE biosynthesis. Further electrophoretic mobility shift assay (EMSA) in vitro indicated that phosphorylated DegU can directly bind to the regulatory region though the DNase I foot-printing experiments failed to observe protected region. The plasmid-mediated overexpression of degU32 (Hy) obviously improved the yield of AprE by 41.6 % compared with the control strain, which demonstrated the importance of phosphorylation state of DegU on the transcription of aprE in vivo. In this study, the putative binding sequence of aprE (5′-TAAAT......AAAAT.......AACAT...TAAAA-3′) located upstream −91 to −87 bp, −101 to −97 bp, −195 to −191 bp, −215 to −211 bp of the transcription start site (TSS) in B. licheniformis was computationally identified based on the DNA-binding sites of DegU in Bacillus subtilis. Overall, we systematically investigated the influence of the interplay between phosphorylated DegU and its cognate DNA sequence on expression of aprE , which not only contributes to the further AprE high-production in a genetically modified host in the future, but also significantly increases our understanding of the aprE transcription mechanism. [ABSTRACT FROM AUTHOR]

Bacillus licheniformis 2709 is the major alkaline protease producer, which has great potential value of industrial application, but how the high-producer can be regulated rationally is still not completely understood. It's meaningful to understand the metabolic processes during alkaline protease production in industrial fermentation medium. Here, we collected the transcription database at various enzyme-producing stages (preliminary stage, stable phase and decline phase) to specifically research the synthesized and regulatory mechanism of alkaline protease in B. licheniformis. The RNA-sequencing analysis showed differential expression of numerous genes related to several processes, among which genes correlated with regulators were concerned, especially the major differential gene abrB on enzyme (AprE) synthesis was investigated. It was further verified that AbrB is a repressor of AprE by plasmid-mediated over-expression due to the severely descending enzyme activity (11,300 U/mL to 2695 U/mL), but interestingly it is indispensable for alkaline protease production because the enzyme activity of the null abrB mutant was just about 2279 U/mL. Thus, we investigated the aprE transcription by eliminating the theoretical binding site (TGGAA) of AbrB protein predicated by computational strategy, which significantly improved the enzyme activity by 1.21-fold and gene transcription level by 1.77-fold in the mid-log phase at a cultivation time of 18 h. Taken together, it is of great significance to improve the production strategy, control the metabolic process and oriented engineering by rational molecular modification of regulatory network based on the high throughput sequencing and computational prediction. [ABSTRACT FROM AUTHOR]

The expression of enzymes in Bacillus licheniformis , such as the valuable extracellular alkaline protease AprE, is highly regulated by a complex transcriptional regulation mechanism. Here, we found that the transcript abundance of aprE varies >343-fold in response to the supply of nutrients or to environmental challenges. To identify the underlying regulatory mechanism, the core promoter of aprE and several important upstream regulatory regions outside the promoter were firstly confirmed by 5′-RACE and mutagenesis experiments. The specific proteins that bind to the identified sequences were subsequently captured by DNA pull-down experiments, which yielded the transcriptional factors (TFs) Spo0A, CggR, FruR, YhcZ, as well as fragments of functionally unassigned proteins. Further electrophoretic mobility shift assay (EMSA) and DNase I foot-printing experiments indicated that Spo0A can directly bind to the region from −92 to −118 nucleotides upstream of the transcription start site, and the deletion of this specific region drastically decreased the production of AprE. Taken together, these results indicated that the expression of aprE was mainly regulated by the interplay between Spo0A and its cognate DNA sequence, which was successfully applied to overproduce AprE in a genetically modified host harboring three aprE expression cassettes. The DNA binding proteins may serve to increase the efficiency of transcription by creating an additional binding site for RNA polymerase. The discovery of this mechanism significantly increases our understanding of the aprE transcription mechanism, which is of great importance for AprE overproduction. • We first discovered the direct regulatory of Spo0A on aprE in Bacillus. • The regulatory region of aprE in B. licheniformis was first precisely mapped. • The mechanism was successfully used to hyper-express of aprE in industrial host. • A stable alkaline protease high-yielding strain was engineered. [ABSTRACT FROM AUTHOR]

Bacillus licheniformis 2709 is the main industrial producer of alkaline protease (AprE), but its biosynthesis is strictly controlled by a highly sophisticated transcriptional network. In this study, the UP elements of aprE located 74–98, 98–119 and 140–340 bp upstream of the transcriptional start site (TSS) were identified, which presented obvious effects on the transcription of aprE. To further analyze the transcriptional mechanism, the specific proteins binding to the approximately 500-bp DNA sequences were subsequently captured by reverse-chromatin immunoprecipitation (reverse-ChIP) and DNA pull-down (DPD) assays, which captured the transcriptional factors CggR, FruR, and YhcZ. The study demonstrated that CggR, FruR and YhcZ had no significant effect on cell growth and aprE expression. Then, aprE expression was significantly enhanced by deleting a potential negative regulatory factor binding site in the genome. The AprE enzyme activity in shake flasks of the genomic mutant BL ∆1 was 47% higher than in the original strain, while the aprE transcription level increased 3.16 times. The protocol established in this study provides a valuable reference for the high-level production of proteins in other Bacillus species. At the same time, it will help reveal the molecular mechanism of the transcriptional regulatory network of aprE and provide important theoretical guidance for further enhancing the yield of AprE. • The regulatory region of aprE in the industrial strain B. licheniformis 2709 was investigated firstly. • The aprE expression level was remarkably improved through removing some sequence in the regulatory region. • Functions of the captured transcriptional factors were analyzed. • Help reveal the molecular mechanism of the transcriptional regulatory network of aprE. [ABSTRACT FROM AUTHOR]

Abstract A highly efficient genome editing system for Bacillus licheniformis was developed based on single-plasmid CRISPR/Cas9. For highly efficient genome editing the shuttle vector pWH1520 was selected to construct the knockout plasmids. A construct harboring a pS promoter driving cas 9 endonuclease expression, a strong pLY-2 promoter driving the transcription of a single guide RNA was demonstrated as being the most effective. To verify the feasibility of the method the uprT gene coding uracil phosphoribosyltransferase was selected as the reporter gene. The efficiency of introducing nucleotide point mutations and single gene deletion reached an editing efficiency of up to 99.2% and 97.3%, respectively. After a upp -deficient strain was engineered, the system and strain were applied to introduce genomic deletions of another two genes, amy L and chi A (encoding amylase and chitinase, respectively) with about 90% deletion efficiency. As two native extracellular proteins with relatively high secretion in the host, amylase and chitinase can hamper the secretion and expression of alkaline protease. It was demonstrated that the mutant with deletions of the two genes effectively improved the alkaline protease yield by 24.8%. The results illustrated that the establishment of a CRISPR/Cas9 system for Bacillus licheniformis is of significance, and confirmed the system's high efficiency. The system provides support for effective molecular modification and metabolic regulation of Bacillus licheniformis , and offers promise for applications in genetic modification of other industrially relevant Bacillus species. [ABSTRACT FROM AUTHOR]