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Song Y.,CAS Tianjin Institute of Industrial Biotechnology | Song Y.,Chinese Academy of Sciences | Nikoloff J.M.,CAS Tianjin Institute of Industrial Biotechnology | Zhang D.,CAS Tianjin Institute of Industrial Biotechnology | And 2 more authors.
Journal of Microbiology and Biotechnology

The well-characterized gram-positive bacterium Bacillus subtilis is an outstanding industrial candidate for protein expression owing to its single membrane and high capacity of secretion, simplifying the downstream processing of secretory proteins. During the last few years, there has been continuous progress in the illustration of secretion mechanisms and application of this robust host in various fields of life science, such as enzyme production, feed additives, and food and pharmaceutical industries. Here, we review the developments of Bacillus subtilis as a highly promising expression system illuminating strong chemical- and temperatureinducible and other types of promoters, strategies for ribosome-binding-site utilization, and the novel approach of signal peptide selection. Furthermore, we outline the main steps of the Sec pathway and the relevant elements as well as their interactions. In addition, we introduce the latest discoveries of Tat-related complex structures and functions and the countless applications of this full-folded protein secretion pathway. This review also lists some of the current understandings of ATP-binding cassette transporters. According to the extensive knowledge on the genetic modification strategies and molecular biology of Bacillus subtilis, we propose some suggestions and strategies for improving the yield of intended productions. We expect this to promote striking future developments in the optimization and application of this bacterium. © 2015 by The Korean Society for Microbiology and Biotechnology. Source

Liu Y.,CAS Tianjin Institute of Industrial Biotechnology | Liu Y.,Chinese Academy of Sciences | Li F.,CAS Tianjin Institute of Industrial Biotechnology | Li F.,Chinese Academy of Sciences | And 10 more authors.
Enzyme and Microbial Technology

Amino acids are important fermentation products that are used in the food, animal feed, pharmaceutical, and cosmetics industries. Development of a better quantitation assay to measure amino acid levels will facilitate research and application in medical and industrial fields. Here we report the development of an assay that allows the rapid measurement of threonine concentration. L-threonine aldolase (LTA) catalyzes the cleavage of threonine to glycine and acetaldehyde. The resulting acetaldehyde is further converted into ethanol by alcohol dehydrogenase accompanying NADH reduction. Therefore, monitoring the changes in NADH concentration can serve as a readout for the amount of threonine present within certain range. This LTA assay can distinguish a 0.5mM difference in threonine concentration in some certain mediums. We successfully utilized this assay to measure l-threonine levels in industrial fermentation samples. Moreover, we were able to scale up this assay to 96-well format and use it in high-throughput screening of threonine-overproducing strains. We show this assay is simple, accurate, specific and suitable for determination of threonine concentration in large numbers of samples. © 2014 Elsevier Inc. Source

Dong H.,CAS Tianjin Institute of Industrial Biotechnology | Dong H.,Chinese Academy of Sciences | Liu Y.,CAS Tianjin Institute of Industrial Biotechnology | Liu Y.,Chinese Academy of Sciences | And 7 more authors.

Cytidine is an industrially useful precursor for the production of antiviral compounds and a variety of industrial compounds. Interest in the microbial production of cytidine has grown recently and high-throughput screening of cytidine over-producers is an important approach in large-scale industrial production using microorganisms. An enzymatic assay for cytidine was developed combining cytidine deaminase (CDA) and indophenol method. CDA catalyzes the cleavage of cytidine to uridine and NH3, the latter of which can be accurately determined using the indophenol method. The assay was performed in 96-well plates and had a linear detection range of cytidine of 0.058 - 10 mM. This assay was used to determine the amount of cytidine in fermentation flasks and the results were compared with that of High Perfomance Liquid Chromatography (HPLC) method. The detection range of the CDA method is not as wide as that of the HPLC, furthermore the correlation factor of CDA method is not as high as that of HPLC. However, it was suitable for the detection of large numbers of crude samples and was applied to high-throughput screening for high cytidine-producing strains using 96-well deep-hole culture plates. This assay was proved to be simple, accurate, specific and suitable for cytidine detection and high-throughput screening of cytidine-producing strains in large numbers of samples (96 well or more). © 2015 Dong et al. Source

Dong H.,CAS Tianjin Institute of Industrial Biotechnology | Dong H.,Chinese Academy of Sciences | Zhang D.,CAS Tianjin Institute of Industrial Biotechnology | Zhang D.,Chinese Academy of Sciences | Zhang D.,National Engineering Laboratory for Industrial Enzymes
Microbial Cell Factories

The complete sequencing and annotation of the genomes of industrially-important Bacillus species has enhanced our understanding of their properties, and allowed advances in genetic manipulations in other Bacillus species. Post-genomic studies require simple and highly efficient tools to enable genetic manipulation. Here, we summarize the recent progress in genetic engineering strategies for Bacillus species. We review the available genetic tools that have been developed in Bacillus species, as well as methods developed in other species that may also be applicable in Bacillus. Furthermore, we address the limitations and challenges of the existing methods, and discuss the future research prospects in developing novel and useful tools for genetic modification of Bacillus species. © 2014 Dong and Zhang; licensee BioMed Central Ltd. Source

Liu Y.H.,Key Laboratory of Industrial Fermentation Microbiology | Liu Y.H.,National Engineering Laboratory for Industrial Enzymes | Liu Y.H.,Tianjin University of Science and Technology | Hu B.,Key Laboratory of Industrial Fermentation Microbiology | And 11 more authors.
Journal of Applied Microbiology

Aims: The purpose of this research was to obtain the mutant of Bacillus licheniformis alpha amylase (BLA) with an improved acid stability and elucidate the difference in catalytic mechanism under acidic conditions between wild-type and mutant BLAs. Methods and Results: The stability of BLA under acid condition was enhanced through direct evolution using error-prone polymerase chain reaction. Two mutation sites, T353I and H400R, were obtained in BLA. To identify the mutation of amino acids in Thr353Ile/His400Arg related to its acid stability, single mutants Thr353Ile and His400Arg were obtained via site-directed mutagenesis. Among the resulting mutant enzymes, the kcat/Km values of the mutants Thr353Ile, His400Arg and Thr353Ile/His400Arg under pH 4·5 were 3·5-, 6·0- and 11·3-fold higher, respectively, than that of the wild-type. Thr353Ile/His400Arg exhibited stronger tolerance towards a lower pH without obvious difference in thermostability when compared with wild-type. Conclusions: The results combined with three-dimensional structure analysis of mutant BLAs demonstrated that Thr353Ile/His400Arg showed an improved acid stability under low pH condition as a result of the interactions of hydrogen bonding, hydrophobicity, helix propensity and electrostatic fields. Significance and Impact of Study: It provides theoretical basis and background data for the improvement of acid stability in BLA by protein engineering. © 2012 The Authors. Journal of Applied Microbiology © 2012 The Society for Applied Microbiology. Source

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