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Current Development in Genetic Engineering Strategies of Bacillus Species Huina Dong1,2 and Dawei Zhang1,2,3*

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Dong and Zhang Microbial Cell Factories 2014, 13:63 http://www.microbialcellfactories.com/content/13/1/63 REVIEW Open Access Current development in genetic engineering strategies of Bacillus species Huina Dong1,2 and Dawei Zhang1,2,3* Abstract 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. Keywords: Bacillus species, Genetic engineering strategies, Counter-selection marker, Operator-repressor system, Toxin gene, Site-specific recombination Introduction Bacillus species (Bacillus licheniformis,GenBankac- Bacillus subtilis and some related Bacillus species are cession number CP000560 and Bacillus amyloliquefa- non-pathogenic, free of exotoxins and endotoxins, and ciens, GenBank accession number AE017333) revealed have a recognized history of safe use in foods. These ahighdegreeofhomologytoB. subtilis [8-10]. These species are also useful for fermentation and large-scale complete genome sequences not only enhance our un- cultivation. Despite these natural advantages, protocols derstanding of these strains, but also allow advances in using Bacillus species lag far behind those for Escherichia genetic manipulations in other Bacillus species [6]. coli and Saccharomyces cerevisiae, which are the most With rapid developments in post-genomic studies, widely used cellular factories for producing industrially- simple and efficient genetic tools are required to con- important enzymes and biochemicals [1,2]. Therefore, veniently enable multiple modifications of the genomes efficient genetic manipulation and highly developed of Bacillus species. systems-level strategies for developing Bacillus species Classical chromosomal modification is based on the in- as microbial cell factories are needed. Recently, Bacillus sertion of a selectable marker, usually a drug resistance species have received increased attention for their use in gene, into the chromosome of a bacterial strain [11]. Using genetic engineering and production of heterologous pro- this strategy, a second selective maker gene is required to teins [3], valuable enzymes, vitamins, platform chemicals, introduce another chromosomal modification, so the and antibiotics [4-6]. number of available selection genes limits the feasibility The complete genome sequences of several B. subtilis of multiple chromosomal modifications. Moreover, the strains have been determined and annotated, stimulating selectable gene should be removed by single-crossover novel methods for interpreting metabolic pathways and recombination if the strain is to be used for further gen- supplying an overview of protein machinery [7]. The com- etic manipulation. In addition, the chance of obtaining a pletion of the genome sequences of industrially-important positive strain is relatively low, and the selection process is laborious. To overcome these problems, methods that * Correspondence: [email protected] can eliminate marker cassettes in the primary transfor- 1 Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, mants are desperately needed. Tianjin 300308, China 2Key Laboratory of Systems Microbial Biotechnology, Chinese Academy of Recently, useful tools for genetic modification of Bacillus Sciences, Tianjin 300308, China species have emerged from the fields of systems and Full list of author information is available at the end of the article © 2014 Dong and Zhang; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Dong and Zhang Microbial Cell Factories 2014, 13:63 Page 2 of 11 http://www.microbialcellfactories.com/content/13/1/63 synthetic biology. Thus, it is necessary to summarize re- [13-15]. Similarly, Uotsu-Tomita et al. [16] developed a cent progress, current obstacles, and future goals to in- double cI-Pr method for positive selection of B. subtilis spire more research interest and advance studies in this recombinants, and Tsuge et al. [17] obtained markerless field. First, we summarize the progress in genetic modifi- deletion of sfp, degQ, and ppsABCDE using the cI-Pr sys- cation strategies of Bacillus species, including several tem. This method can be repeatedly used by changing kinds of marker-free genetic modification methods and the location of the cI gene [18]. site-specific recombination strategies. Next, we discuss some strategies developed in other species that could also araR as a counter-selection marker be used in Bacillus species. Lastly, we compare current araR encodes a negative regulator of the ara operon that genetic engineering strategies, analyze their challenges can be induced by L-arabinose in B. subtilis [19]. Liu et al. and limitations, and discuss future research goals for de- [20] developed a method in which the chromosomal araR veloping novel and useful tools for genetic modification of locus was replaced by Para-neo, which confers neomycin re- Bacillus species. sistance. By adding L-arabinose to the growth medium, the expression of Para-neo canbeinducedbasedonthepres- Operator-repressor system-based genetic engineering ence of araR in the chromosome. First, the selection strategies marker cassette cat-araR is inserted upstream of the target The development of various inducible promoter systems gene via recombination, and then selected for with chlor- has played an important role in the analysis of gene amphenicol. Next, single-crossover recombination between expression and function. Promoters responsive to an as- two downstream regions removes the cat-araR cassette, to- sortment of inducing agents, including heavy metals, gether with the target gene. The remaining Para-neo in the hormones, and heat shock, as well as several viral, cellu- genome can be used again for further genetic modification. lar, and bacterial regulatory factors, have been success- The authors obtained 3.8-kb and 41.8-kb deletion strains fully used to manipulate gene expression. Some systems using this system that left no selective marker gene in the have been applied to genetically engineer Bacillus spe- targeted loci. cies, and the selection and counter-selection marker cassettes are shown in Figure 1. Pyrimidine metabolism-based genetic engineering strategies Common approaches for counter-selection exploit genes cI as a counter-selection marker involved in purine or pyrimidine metabolism and are The CI repressor gene (cI857 or cI) from E. coli bacterio- based on the fact that purine or pyrimidine analogs can phage lambda encodes the CI repressor, which can bind be converted to toxic compounds. Plating cells on media to the Pr promoter of lambda phage to suppress its pro- containing the analog leads to a strong selection for moter activity. Itaya [12] developed a counter-selection clones that have lost the chromosomally-integrated copy method in which a neomycin-resistance gene is regu- of the gene encoding the converting enzyme. Therefore, lated by Pr. This system, controlled by the presence/ab- parental strains used for genome modification must lack sence of the CI repressor, allows precise selection for the respective gene for purine or pyrimidine nucleotide marker-free genetic modification of the chromosome biosynthesis. Recently, upp and pyrF, encoding uracil Figure 1 Operator-repressor system-based genetic engineering strategy. (A) In the presence of the R gene, the constitutively expressed R protein specifically binds to the Pr promoter and represses promoter activity. (B) When the R gene is excised from the genome, the Pr promoter is activated and mutants can be selected by antibiotic resistance. (C) Diagrams of selection and counter-selection marker cassettes. R: Repressor, R S R S AR: Antibiotic resistance, Pr: Promoter. [AR1 ], [AR1 ], [AR2 ], and [AR2 ] are AR1-resistant, AR1-sensitive, AR2-resistant, and AR2-sensitive bacterial phenotypes, respectively. Dong and Zhang Microbial Cell Factories 2014, 13:63 Page 3 of 11 http://www.microbialcellfactories.com/content/13/1/63 phosphoribosyltransferase (UPRTase) and orotidine 5′- knock out genes. Compared with the parental B. subtilis phosphate decarboxylase (OMPdecase), respectively, have 168 genome, the genome of strain MGB874 is depleted by been used as counter-selection markers in Bacillus species. 874 kb (20.7%), including 865 genes. The relevant pyrimidine metabolism pathway and mecha- Tanaka et al. [27] developed an improved counter- nisms of action of pyrimidine analogs are shown in selection system that combines the upp and cI-Pr sys- Figure 2. tems. In
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