Development and application of a CRISPR/Cas9 system for Bacillus licheniformis genome editing

Zhou, Cuixia; Liu, Huan; Yuan, Feiyan; Chai, Haonan; Wang, Haikuan; Liu, Fufeng; Liu, Yu; Zhang, Huitu; Lu, Fuping

doi:10.1016/j.ijbiomac.2018.10.170

Cited by 34 publications

(25 citation statements)

References 41 publications

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“…Genomic modifications including gene deletion, insertion, and replacement [30], is imperative for the development of Bacillus and other Grampositive bacteria. Although we have demonstrated that the recently developed ultramodern CRISPR/Cas9 system can be established in our host strain [23], there is a problem of complicated construction and verification of knockout vectors, as well as high cost. The markerless gene editing system with the counter-selectable upp gene based on a temperature-sensitive plasmid played an important role in genetic modification in the study.…”

Section: Discussionmentioning

confidence: 99%

“…The production of alkaline protease of the different strains in the study was determined using samples at different cultivation times in shake-flask fermentations. Because the alkaline protease activity had obviously positive correlation with the aprE expression quantity, the alkaline protease activity in culture supernatants was investigated using the detection method published by the national standardization administration commission [23].…”

Section: Analytic Methodsmentioning

confidence: 99%

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Optimized expression and enhanced production of alkaline protease by genetically modified Bacillus licheniformis 2709

Zhou

et al. 2020

Microb Cell Fact

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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.

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Section: Discussionmentioning

confidence: 99%

Section: Analytic Methodsmentioning

confidence: 99%

Optimized expression and enhanced production of alkaline protease by genetically modified Bacillus licheniformis 2709

Zhou

et al. 2020

Microb Cell Fact

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“…It was found that the mutation in the subtilisin, termed m-63, exhibited higher efficiency for catalytic activities, which was 100% much higher than that of the wild type at 10°C under N-succinyl-l-Ala-l-Ala-l-Pro-l-Phe-p-nitroanilide as a synthetic substrate for enzyme activities. It was found that the engineering for protease for cold resistance gives cold tolerance in protease, which allowed it to work even under low temperatures (Banerjee and Ray, 2017; Castilla et al, 2017; Onaizi, 2018; Zhou et al, 2019). The mutant proteases from the papain family, such as Glnl9His, Glnl9Glu, and Gin 19Ala, indicated that the Gln19Glu and Glnl9His enzymes participated in the acid-catalyzed hydrolysis in thiomidate, which was converted into amide through the provision of H+ (proton) to form the more reactive protonated thiomidate, which can work at low as well as higher levels of thermal conditions (D'Amico et al, 2002; Siddiqui and Cavicchioli, 2006; Margesin et al, 2007; De Maayer et al, 2014).…”

Section: Protease Engineeringmentioning

confidence: 99%

Microbial Proteases Applications

Razzaq

Shamsi

Ali³

et al. 2019

Front. Bioeng. Biotechnol.

391

195

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The use of chemicals around the globe in different industries has increased tremendously, affecting the health of people. The modern world intends to replace these noxious chemicals with environmental friendly products for the betterment of life on the planet. Establishing enzymatic processes in spite of chemical processes has been a prime objective of scientists. Various enzymes, specifically microbial proteases, are the most essentially used in different corporate sectors, such as textile, detergent, leather, feed, waste, and others. Proteases with respect to physiological and commercial roles hold a pivotal position. As they are performing synthetic and degradative functions, proteases are found ubiquitously, such as in plants, animals, and microbes. Among different producers of proteases, Bacillus sp. are mostly commercially exploited microbes for proteases. Proteases are successfully considered as an alternative to chemicals and an eco-friendly indicator for nature or the surroundings. The evolutionary relationship among acidic, neutral, and alkaline proteases has been analyzed based on their protein sequences, but there remains a lack of information that regulates the diversity in their specificity. Researchers are looking for microbial proteases as they can tolerate harsh conditions, ways to prevent autoproteolytic activity, stability in optimum pH, and substrate specificity. The current review focuses on the comparison among different proteases and the current problems faced during production and application at the industrial level. Deciphering these issues would enable us to promote microbial proteases economically and commercially around the world.

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“…In general, Bacillus species are efficient alkaline protease producers [5, 6], and especially Bacillus licheniformis is a promising industrial host strain for protein production, partly due to its ‘Generally Recognized as Safe (GRAS)’ status and its high enzyme secretion capacity [7, 8]. B. licheniformis is widely distributed in soils where it helps recycle nutrients by producing and secreting macromolecule-degrading hydrolases such as amylases, proteases, cellulases and phosphatases [9].…”

Section: Introductionmentioning

confidence: 99%

Optimization of alkaline protease production by rational deletion of sporulation related genes in Bacillus licheniformis

Zhou

Zhang

et al. 2019

Microb Cell Fact

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Background Our laboratory has constructed a Bacillus licheniformis strain that secretes alkaline protease (AprE) with excellent enzymatic properties. B. licheniformis is generally regarded as safe and has a high industrial exoenzyme secretion capacity, but the host retains some undomesticated characteristic that increase its competitiveness and survival, such as spore-formation, which increases the requirements and difficulties in industrial operations (e.g. sterilization and enzyme activity control). Furthermore, the influence of sporulation on alkaline protease production in B. licheniformis has not been elucidated in detail. Result A series of asporogenic variants of the parent strain were constructed by individually knocking out the master regulator genes ( spo 0A, sig F and sig E) involved in sporulation. Most of the variants formed abortively disporic cells characterized by asymmetric septa at the poles and unable to survive incubation at 75 °C for 10 min. Two of them (Δ sig F and Δ sig E) exhibited superior characteristics in protease production, especially improving the expression of the apr E gene. Under the currently used fermentation conditions, the vegetative production phase of Δ sig F can be prolonged to 72 h, and the highest protease production of Δ sig F reached 29,494 ± 1053 U/mL, which was about 19.7% higher than that of the wild-type strain. Conclusion We first constructed three key sporulation-deficient strain to investigate the effect of sporulation on alkaline protease synthesis. The sig F mutant retained important industrial properties such as facilitating the sterilization process, a prolonged stable phase of enzyme production and slower decreasing trend, which will be superior in energy conservation, simpler operations and target product controlling effect. In summary, the work provides a useful industrial host with preferable characteristics and a novel strategy to enhance the production of protease. Electronic supplementary material The online version of this article (10.1186/s12934-019-1174-1) contains supplementary material, which is available to authorized users.

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Development and application of a CRISPR/Cas9 system for Bacillus licheniformis genome editing

Cited by 34 publications

References 41 publications

Optimized expression and enhanced production of alkaline protease by genetically modified Bacillus licheniformis 2709

Optimized expression and enhanced production of alkaline protease by genetically modified Bacillus licheniformis 2709

Microbial Proteases Applications

Optimization of alkaline protease production by rational deletion of sporulation related genes in Bacillus licheniformis

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