Bacterial Genome Editing via a Designed ToxinâAntitoxin Cassette

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Bacterial Genome Editing via a Designed Toxin–antitoxin Cassette Jie Wu, Aihua Deng, Qinyun Sun, Hua Bai, Zhaopeng Sun, Xiuling Shang, Yun Zhang, Qian Liu, Yong Liang, Shuwen Liu, Yongsheng Che, and Tingyi Wen ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.6b00287 • Publication Date (Web): 17 Jan 2017 Downloaded from http://pubs.acs.org on January 17, 2017

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ACS Synthetic Biology

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Bacterial Genome Editing via a Designed Toxin–antitoxin Cassette

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Jie Wu,†,§,# Aihua Deng,†,# Qinyun Sun,† Hua Bai,†,§ Zhaopeng Sun,† Xiuling Shang,†

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Yun Zhang,† Qian Liu,† Yong Liang,† Shuwen Liu,† Yongsheng Che,*,‡ Tingyi

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Wen*,†,//

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†

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Microbiology, Chinese Academy of Sciences, Beijing 100101, China

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‡

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of Pharmacology & Toxicology, Beijing 100850, China

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§

University of Chinese Academy of Sciences, Beijing 100049, China

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//

Savaid medical school, University of Chinese Academy of Sciences, Beijing,

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100049, China

CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of

State Key Laboratory of Toxicology & Medical Countermeasures, Beijing Institute

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Graphical Table of Contents

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ABSTRACT

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Manipulating the bacterial genomes in an efficient manner is essential to biological 1

ACS Paragon Plus Environment


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and biotechnological research. Here, we reprogramed the bacterial TA systems as the

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toxin counter–selectable cassette regulated by an antitoxin switch (TCCRAS) for

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genetic modifications in the extensively studied and utilized Gram-positive bacteria, B.

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subtilis and Corynebacterium glutamicum. In the five characterized type II TA

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systems, the RelBE complex can specifically and efficiently regulate cell growth and

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death by the conditionally controlled antitoxin RelB switch, thereby serving as a

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novel counter-selectable cassette to establish the TCCRAS system. Using a single

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vector, such a system has been employed to perform in-frame deletion, functional

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knock-in, gene replacement, precise point mutation, large-scale insertion, and

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especially, deletion of the fragments up to 194.9 kb in B. subtilis. In addition, the

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biosynthesis of lycopene was first achieved in B. subtilis using TCCRAS to integrate

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a 5.4-kb fusion cluster (Pspac–crtI–crtE–crtB). The system was further adapted for

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gene knockdown and replacement, and large-scale deletion of the fragments up to

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179.8 kb in C. glutamicum, with the mutation efficiencies increased by 0.8−1.0 fold

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compared to conventional SacB method. TCCRAS thus holds promise as an effective

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and versatile genome-scale engineering technology for metabolic engineering and

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synthetic genomics research in a broad range of the Gram-positive bacteria.

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KEYWORDS: Toxin–antitoxin (TA) system, genome editing, large-scale deletion,

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Bacillus subtilis, Corynebacterium glutamicum

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INTRODUCTION

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Efficient tools to modify the cellular DNA sequences in a predictable fashion are

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essential to understanding the function of genes and genetic reprogramming of

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cells.1-3 Fully defined genome editing refers to deletion, insertion, replacement, and

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point mutation of the target genes, which is a vital and powerful tool for research and

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applications in the fields of biology and biotechnology.2,4 Development of genetic

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manipulation techniques, particularly those based on recombination (Red, Red/ET,

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and Cre/loxP) and nucleases (ZFNs and TALENs), have provided valuable

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approaches for the target mutations in human, animals, plants, and

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microorganisms.3,5-8

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Recently, the defense systems of bacteria and archaea, such as the

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restriction-modification (R-M), the clustered regularly interspaced short palindromic

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repeats (CRISPR), and the toxin-antitoxin (TA) systems, have attracted great interests

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and are hotspots in the development of appealing tools in genetic manipulations.9-12 In

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our previous work, a robust and efficient pipeline called

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mimicking-of-DNA-methylation-patterns (MoDMP), was designed in an endogenous

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MTases deficient Escherichia coli strain EC135 to mimic the R-M systems of the

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targeted bacteria.13 Additionally, CRISPR has showed potential as an effective

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genome editing technique in various organisms by cleaving the genomic sequences

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targeted by RNA sequences.11,12,14,15 Increasing availability of genomic sequences

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enabled elucidation of a new group of defense systems, the TA systems, which use the 3



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encoding products of the toxic and neutralizing antitoxin to regulate cell growth and

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death/dormancy under various stress conditions.9,16 In general, the toxin and antitoxin

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genes, encoding for a toxin protein and an antitoxin RNA or protein, are

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co-transcribed and co-translated from one operon. Among them, the type II system is

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most prevalent in which both toxin and antitoxin are proteins.16 The toxins inhibit cell

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growth via degradation of mRNA or inhibition of DNA replication, protein synthesis,

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cell wall biosynthesis, or ATP synthesis, while the antitoxins inhibit the activity of

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toxin in transcription, translation or catalytic progress.17 Currently, the specific

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mechanism regulating this growth-death system has been used in DNA cloning,

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protein expression, and genetic manipulation in bacteria.18,19

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The model Gram-positive bacterium B. subtilis has attracted much attention in the

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development of genetic manipulation techniques in both academic and industrial

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settings.20-22 In existing methods, the resistant genes were usually introduced into

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chromosome as the positive selectable markers for genetic modifications of

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Bacillus.23 However, it is difficult to manipulate multi-chromosomal loci due to

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limited availability of the antibiotic-resistant marker genes.22 To overcome these

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problems, markerless or scarless genetic manipulation systems including site-specific

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recombination-based methods (Cre/loxP, Xer/dif, and FLP/FLP),6,24,25

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counter-selectable systems,26-31 phage recombinase-mediated ssDNA (single-stranded

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DNA)-directed system,32,33 and the CRISPR/Cas9 system,14,15,34 have been developed

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in Bacillus. Among them, the counter-selectable systems, which use endogenous or 4


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exogenous genes encoding for the proteins that affect normal cell growth as the

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counter–selectable markers, have been widely applied in markerless or scarless

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genetic manipulations in Bacillus. Examples include the mannose-6-phosphate

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isomerase gene mamP,30 the uracil phosphoribosyl-transferase gene upp,31 the hen egg

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white lysozyme encoding gene hewl,32 the E. coli toxin gene mazF,28,29 and the

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antibiotic-resistant genes.26 However, these methods using either UPP or MamP

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required depletion of the endogenous upp or mamP gene in the target strain,30,31

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limiting their applications in the Bacillus strains without clear genetic backgrounds.

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Similarly, the levansucrase (sacB), small ribosomal protein S12P (rpsL), and

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galactokinase (galK) genes were also used as the counter–selectable markers in other

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Gram-positive bacteria including Corynebacterium and Mycobacterium.35,36 Recently,

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the toxin gene-based genetic engineering strategies have been developed in B. subtilis

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using inducible mazF as the counter-selectable marker.28,29,37 However, expression of

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the toxic proteins is hardly suppressed completely, often resulting in leaky

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expression.38 To counteract the toxicity from leaky expression of the toxin,

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spontaneous generation of the resistant strains without the desired mutations was

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frequently encountered in current methods, which used the modules with only toxin

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genes as the counter–selectable markers.22,28 Although a PCR-fusion method using a

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mini-mazF-cassette was developed to decrease the frequency of the spontaneous

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resistant mutants, the long-PCR-fusion fragment-generated mutation limited its

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application in manipulating the large-scale DNA fragments by insertion or 5



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replacement.22,27 Therefore, an easy-to-use and highly designable toxin-antitoxin

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cassette that can flexibly and precisely regulate the function of toxins by the

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repressible antitoxin switch, is urgently needed for efficient genome editing of the

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Gram-positive bacteria.

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In this study, we established a new technique to genetically manipulate the

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Gram-positive bacteria by mimicking the TA behavior and editing the components of

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the type II TA systems. Specifically, a toxin counter-selectable cassette efficiently

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regulated by an antitoxin switch (designated as TCCRAS) was developed by

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designing the genetic elements to control the expression of toxin and antitoxin genes.

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Using this approach, various gene modifications have been achieved in B. subtilis and

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C. glutamicum (under the phyla of Firmicutes and Actinobacteria, respectively).

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Furthermore, the underlying mechanism for genome editing by TCCRAS was also

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explored. Our results demonstrate that the system is an efficient, versatile, affordable,

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and adaptable approach to perform sophisticated genetic modifications in a broad

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range of the Gram-positive bacteria.

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RESULTS AND DISCUSSION

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A Toxin Counter–selectable Cassette Regulated by an Antitoxin Switch

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(TCCRAS) Designed by Reprogramming of the TA Systems. To mimic the

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regulating function of the TA systems on cell growth and death in an affordable way,

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five type II TA systems (phd–doc, parDE, relBE, ccdAB, and mazEF) with diverse 6


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and ubiquitous elements were evaluated as the candidates for counter–selectable

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cassettes (Figure 1a). The TA modules were reconstructed by replacing their native

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promoters with the constitutive and inducible ones, and designated as a toxin

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counter–selectable cassette regulated by an antitoxin switch (TCCRAS). The toxins

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were constitutively expressed by PliaG, while the antitoxins were conditionally

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expressed by PxylA in B. subtilis (Figure 1b). Regulation of cell growth and death by

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TCCRAS was assessed after integrating the system into the lacA locus of B. subtilis

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W168. The negative control strain BS048 and the TA-integrated strains were routinely

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cultured in the presence of xylose (Figure 1c). However, in the absence of xylose, the

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phd-doc-integrated BS080 and the ccdAB-integrated BS085 mutants grew normally,

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while the parDE-integrated strain BS081 grew poorly. Importantly, nearly no growth

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was observed for the relBE module harboring BS084 or the mazEF module harboring

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BS086, indicating that the two modules effectively regulate cell growth and death.

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The MazF toxin has been reported as an endoribonuclease that cleaves free mRNAs at

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the ACA sequences, while the RelE toxin shows a different mechanism by cleaving

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mRNA codons in the ribosome A site and inhibiting protein synthesis.39,40 Thus, the

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RelBE complex was selected as a new counter–selectable module to establish the

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TCCRAS system.

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Scheme for Genome Modifications Using the TCCRAS System. The universal

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pWYE486 vector for genome modification in B. subtilis was constructed by inserting 7



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the TCCRAS system into the pMD19 T-simple vector (TaKaRa, Dalian, China; Figure

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2a; see Supporting Information for details). According to the standard procedures for

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gene deletion, integration, replacement, and point mutation (Figure 2b), the

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recombinant plasmid, unreplicable in B. subtilis or C. glutamicum, were constructed

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by inserting the up- and down-stream homologous arms of the target gene (or/and the

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inserted or mutated fragments) into the universal vectors (see Supporting Information

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for details). The recombinant plasmid was transformed into competent cells of the

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target strain by electro-transformation,41,42 and the transformants with the recombinant

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plasmid integrated by a single crossover event, were screened on a LB plate with

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suitable antibiotics and inducers (erythromycin and xylose for B. subtilis;

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chloramphenicol and IPTG for C. glutamicum; step 1 in Figure 2b). The antibiotics

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were used to verify the integrated antibiotic-resistant genes from the recombinant

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plasmids, and the inducers were used to express the antitoxin for cell growth. After

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PCR identification, the positive transformants were subjected to counter selection by

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sub-culturing in 2 mL LB medium with an inducer but without the antibiotic for 12 h,

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and growing on the LB plate without inducer or antibiotic after diluted by 10-5−10-7

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times (step 2 in Figure 2b). During counter-selectable process, LB medium with an

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inducer but without the antibiotic was used to culture the plasmid-integrated

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transformants, some of which eliminated the TCCRAS system, the antibiotic-resistant

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genes, and other null backbones of plasmids by the second single crossover event at

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homologous arms. Thus, only the mutants that have excised the TCCRAS cassette and 8


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other plasmid DNA can grow on LB plate without an inducer or antibiotic. In contrast,

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cells harboring the TCCRAS system failed to grow on LB plates due to the toxicity of

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sufficient toxins freed upon removal of the antitoxins in the absence of inducer. The

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resulting mutants were identified by PCR amplification and DNA sequencing of the

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mutated sites. Changes in physiological characteristics of the mutants were also

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examined to further verify the mutation. Counter–selectable and mutation efficiencies,

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the indicators for percentage of the strains excised the TCCRAS cassette and the

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desired mutations generated in all tested colonies, respectively, were finally calculated

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to verify the effectiveness of genetic manipulation.

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Scarless Deletion of a Gene and a Large Operon or Chromosomal Region. To

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assess the efficiency in gene deletion by the system, the divIVA gene encoding a

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cell-division initiating protein was selected as the target. The resulting mutant BS091

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(W168∆divIVA) was conveniently obtained with 100% counter–selectable and

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mutation efficiency (Table 1; Figure S1a and b), indicating that all strains have

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excised the TCCRAS cassette and the desired mutations have been generated in all

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the tested colonies. Longer cells were observed in the divIVA-deleted strain by

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microscopy compared to the original W168, further confirming inactivation of the

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DivIVA protein (Figure 3a).

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Considering potential variation in gene deletion at different target sites, a second gene amyE encoding α-amylase was selected to further verify the system. The 9



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amylase-deficient strain BS089 (W168∆amyE) was generated with 100%

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counter–selectable efficiency, significantly increased compared to that of 78.8 ± 3.8%

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using the RelE system (Table 1; Figure S1c−e). Deletion of amyE gene (as indicated

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by the absence of clear halos in the mutants due to the lack of α-amylase activity),

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was functionally examined by starch plate assay (Figure 3b). The desired mutations

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were found in 89.6 ± 9.0% of the 240 colonies randomly selected for high throughput

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screening of α-amylase activity using a Biomek 3000 Laboratory Automation

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Workstation (Beckman Coulter, Inc., USA), compared to the mutation efficiency of

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83.1 ± 9.1% using the RelE system (Table 1; Figure S1f). Therefore, the mutation

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efficiency in deleting the 2.1-kb amyE gene (calculated as the ratio of the

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amyE-deleted mutants to the tested colonies losing the inserted plasmid by a second

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recombination event) was not significantly increased compared to those using only

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the toxins as the counter–selectable markers. Recently, the CRISPR/Cas9 system was

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used to disrupt the five genes of B. subtilis ATCC 6051a, achieving the highest

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efficiency of 53% in deleting the amyE gene,15 significantly lower than that achieved

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by the TCCRAS system.

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To evaluate the feasibility to delete a large operon or chromosomal region using

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TCCRAS, a 37.7-kb pps operon with the plipastatin peptide synthase encoding genes

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(ppsEDCBA) and a 194.9-kb dltA–rocR were separately selected as the targets, and

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successful deletion of the pps operon and dltA–rocR was confirmed by PCR (Figures

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3c; Figure S2d and f). A counter–selectable efficiency of 99.4 ± 1.1% was achieved 10


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for the resulting pps-deleted strain BS097 (W168∆ppsEDCBA), higher than that of

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69.2 ± 3.8% using the RelE system (Table 1; Figure S2a and c). While the mutation

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efficiencies using the TCCRAS and RelE systems to delete the pps operon were 61.5

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± 3.8% and 53.8 ± 3.8%, respectively (Table 1; Figure S2b and d). However, the

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mutation efficiency in pps deletion by TCCRAS was increased by approximately 9

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folds compared to that of only 6.4% using a synthetic gene circuit system.28 In

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addition, a counter–selectable efficiency of 98.8 ± 1.1% and a mutation efficiency of

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29.5 ± 2.2% were achieved in deleting a 194.9-kb fragment to generate the mutant

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strain BS214 (W168∆dltA–rocR) using TCCRAS (Table 1; Figure S2e and f). Thus,

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TCCRAS is a genome-scale engineering technology to efficiently delete DNA

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fragments in various sizes, showing superior advantage to reduce the genome sizes in

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synthetic genomics research.2,43

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Sequence-specific Integration of Various Genes. Powerful genome engineering

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technology can substantially enhance the capability to manipulate various

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organisms.1,4 To further evaluate the feasibility for gene insertion using the system,

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three fragments with sizes of 5–12 kb were successfully integrated into the original

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pps locus of strain BS097 (W168∆ppsEDCBA) following the scheme for gene

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integration (Figures 2b and 3d). Specifically, a 5.2-kb operon from E. coli W3110 for

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threonine biosynthesis, a 7.2-kb operon from C. glutamicum ATCC 13032 for

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tryptophan biosynthesis (trp), and a 12.3-kb fusion operon of trp–thrABC were 11



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integrated into the chromosome of BS097 with the counter–selectable and mutation

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efficiencies of 99.4–100% and 42.3–52.6%, respectively (Table 1; Figure S3a−f).

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To further assess the potential of TCCRAS in metabolic engineering, a 5.4-kb

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fusion cluster (Pspac–crtI–crtE–crtB) was integrated into BS097, and the resulting

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mutant BS206 (BS097::Pspac–crtI–crtE–crtB), darker in color compared to the original

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strain (Figure 3e), was generated with the counter–selectable and mutation

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efficiencies of 98.1 ± 1.9% and 50.0 ± 3.8%, respectively (Table 1; Figure S3g and h).

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Based on metabolic pathway analysis, integration of crtI, crtE, and crtB could

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synthesize lycopene using isopentenyl diphosphate (IPP) and dimethylallyl

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diphosphate (DMAPP) as the precursors (Figure 3e). A maximum level of 7.5 nM of

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lycopene was detected in the fermentation products of BS206 after cultivation in 7.5 L

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fermentor for 45 h (Figure S4), achieving the first biosynthesis of the product in B.

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subtilis. Thus, the extrinsic genes with a wide range in sizes (5.2 to 12.3 kb) were

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inserted into the chromosome of B. subtilis, providing an effective and versatile

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approach for metabolic engineering and synthetic biology purposes.

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Sequence-specific Gene Replacement and Precise Point Mutation. To test whether

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TCCRAS is applicable in gene replacement, the gfpmuat3a gene encoding a green

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fluorescent protein (GFP) was used to replace the upp gene in B. subtilis W168, and

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the resulting mutant BS113 (W168∆upp::P43–gfpmut3a) was produced with the

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counter–selectable and mutation efficiencies of 97.4 ± 1.1% and 57.7 ± 3.8%, 12


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respectively (Table 1; Figure S5a and b). In contrast to growth inhibition in W168

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caused by the upp gene encoding UPRTase, the BS113 mutant grew on minimal

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medium (MM) supplemented with 5-fluorouracil (5-FU; Figure 3f). Besides, flow

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cytometry analysis showed that gfpmut3a was constitutively expressed in the mutant

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(Figure 3f).

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To test the applicability of TCCRAS in precise nucleotide substitution, point

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mutation of A107C was introduced into the recU gene, which codes the general

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Holliday junction resolving enzyme in the Gram-positive bacteria.44 Following the

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described procedure (Figure 2b), the BS093 mutant (W168 recUA107C) was generated

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with the counter–selectable and mutation efficiencies of 100% and 69.2 ± 3.8%,

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respectively (Table 1; Figure S5c and d). Nucleotide sequence of the recU gene in the

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mutant was further amplified by PCR and sequenced to confirm the site of mutation

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(Figure 3g). Taken together, in addition to deletion of a specific gene, an operon, and

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a large chromosomal region, TCCRAS could also be used to replace the genes and to

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precisely introduce a nucleotide mutation in genome.

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Genome Editing in C. glutamicum Using the TCCRAS System. The regulatory

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circuits controlling the expression of relBE were designed in C. glutamicum ATCC

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13032 to test the system in other organisms, which was found to effectively control

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the growth/death of ATCC 13032 when the toxin gene relE was constitutively

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expressed by promoter P45, and the antitoxin gene relB was conditionally expressed 13



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by the IPTG-inducible promoter Ptac (Figure 4a and b). Hence, the TCCRAS cassette

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and the resistant genes were cloned into the pUC vector (TaKaRa, Dalian, China) to

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generate a universal manipulation vector pWYE587 for various genome modifications

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in C. glutamicum (Figure 4a; Table S1).

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The upp gene was first targeted for deletion to assess the system in C. glutamicum,

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and the upp-deficient strain CG709 (ATCC 13032∆upp) was generated with the

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counter–selectable and mutation efficiencies of 100% and 85.9± 5.9%, respectively,

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which were increased 1.0−1.4 folds compared to the traditional method using the

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inducible SacB as the counter-selectable marker (Table 1; Figure S6a−d). Furthermore,

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deletion of upp was functionally examined by measuring growth inhibition of the

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mutants on MM supplemented with 5-FU (Figure 4c). Besides high efficiency in

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single gene knock-out, TCCRAS was also successfully used in deleting a 179.8-kb

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chromosomal region (cg1167–cg11844) in C. glutamicum, with the counter–selectable

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and mutation efficiencies of 100% and 17.9 ± 4.4%, respectively (Table 1; Figure S6i

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and j).

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The effect of replacing the lysine efflux permease (lysE) gene in C. glutamicum

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with a lysine decarboxylase (cadA) gene from E. coli was also investigated. The

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resulting mutant CG706 (ATCC 13032∆lysE::cadA) was generated with the

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counter–selectable and mutation efficiencies of 100% and 69.2 ± 3.8%, respectively,

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0.8−1.4 folds’ increase compared to those of only 41.0 ± 1.1% and 38.5 ± 6.7% using

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the SacB method (Table 1; Figure S6e and h). Based on the results from metabolic 14


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analysis, deletion of lysE could reduce the efflux of intracellular lysine into the media,

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leading to accumulation of lysine in cells, which could be further catalyzed by the

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integrated CadA and converted to cadaverine, as detected in the CG706 mutant by

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HPLC (Figure 4d and e). Therefore, TCCRAS was also developed as a versatile

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platform to perform various genetic manipulations in C. glutamicum by mimicking

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the intricate defense behavior of RelBE.

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Regulating Mechanism of the TCCRAS System. To elucidate the regulating

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mechanism of TCCRAS, the expression of promoters PxylA and PliaG in BS216

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(W168∆lacA::PxylA-gfpmut3a-erm) and BS217 (W168∆lacA::PliaG-gfpmut3a-erm),

302

respectively, was detected using GFP as a reporter to analyse the levels of toxin or

303

antitoxin of the TCCRAS and PxylA-toxin systems (Figure S7a). In the presence of

304

xylose, PxylA produced more intense fluorescence than PliaG (Figure 4f), indicating that

305

the level of antitoxin is higher than toxin when the TCCRAS system was integrated

306

into chromosome. In the absence of xylose, PxylA driven gfp produced leaky

307

expression of fluorescence, reaching 32.9% of the maximum despite the production of

308

strong fluorescence with xylose after culturing for 6 h (Figure 4g). Additionally, leaky

309

expression of promoter PxylA can also be detected in the presence of glucose although

310

the xylose-induced expression of PxylA was repressed by glucose (Figure S7b and c).

311

Thus, the inducible promoter PxylA, used to control the toxin gene, could not be tightly

312

repressed by repressor XylR. In contrast, the toxin and antitoxin genes were 15



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

313

co-expressed in TCCRAS and the antitoxin protein was sufficient to neutralize the

314

toxic function of toxin by forming a complex in the TCCRAS-integrated strains to

315

maintain normal cell growth (Figure 4h).

316

Mechanistic study of genome editing using TCCRAS revealed several unique

317

characteristics of the system. In the PxylA-toxin system, the promoter could not be

318

guaranteed to strictly control the expression of the toxin genes (Figure 4g).

319

Consequently, frequency for spontaneous generation of the resistant strains was

320

relatively high, and the counter–selectable efficiencies of some mutations were lower

321

than 79% (Table 1). Different from conventional approaches, TCCRAS utilizes both

322

the toxin and antitoxin genes to regulate cell death and growth, and can be

323

reprogramed to conditionally express the antitoxins at a level higher than the

324

constitutively expressed toxin (Figure 4f), avoiding any spontaneous resistant strains

325

caused by promoter leakage and achieving the counter–selectable efficiencies of

326

97.4-100% in manipulating the genomes of B. subtilis and C. glutamicum (Table 1).

327

Since the antitoxins were sufficient to inhibit the toxins by forming a complex in this

328

process (Figure 4h), cells grow normally in the presence of an inducer when the

329

system and other DNA fragments were integrated into chromosome. To further

330

determine whether the toxin-antitoxin interaction happened at a transcriptional or

331

protein level, a terminator sequence was inserted between the relE and relB genes of

332

the TCCRAS cassette (designated as TCCRAS-Ter; Figure S8a). Compared to

333

TCCRAS, a similar regulating function of cell growth and death was observed in the 16


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334

TCCRAS-Ter system (Figure S8), suggesting that the control of cell growth and death

335

by the toxin-antitoxin interactions should happen at the protein level. During

336

counter-selectable process, the concentrations of antitoxins will rapidly decrease upon

337

removal of the inducers due to continuous Lon-dependent degradation, while the

338

toxins are stable in cells,39 leading to sufficient free toxins to inhibit cell growth and

339

stimulate cellular regulation mechanism for bacterial survival (Figure 4h).

340

Consequently, the host recombination machinery might be effectively activated to

341

strike out the toxin genes by recombination and contributed to recovery of cells with

342

the desired mutations in high efficiency (Table 1; Figure 4h). In addition, the

343

reprogramed MazEF system can also be effectively used for genome editing in B.

344

subtilis (Figure S9). Therefore, the TCCRAS system relies on the repressible

345

antitoxins to turn on/off the toxic functions of toxins, allowing for highly specific,

346

efficient, and predictable genome editing.

347 348 349

CONCLUSIONS In summary, TCCRAS, a novel, efficient, versatile, and affordable approach for

350

genome editing in B. subtilis and C. glutamicum has been developed by reconstructing

351

the TA systems, through which scarless genetic manipulations including in-frame

352

deletion, functional knock-in, gene replacement, precise point mutation, and

353

large-scale insertion and deletion have been achieved with confidence and high

354

efficiency. Its effectiveness in genetic modifications of the two evolutionarily distant 17



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

355

Gram-positive bacteria reveals its potential as a universal approach for a broad range

356

of Gram-positive bacteria. Thus, the system is a genome-scale engineering technology

357

for efficient deletion and insertion of the DNA fragments in various sizes, providing

358

an effective and versatile approach for metabolic engineering and synthetic genomics

359

research.

360 361

MATERIALS AND METHODS

362

Bacterial Strains, Culture Conditions, and DNA Manipulation Methods. The

363

bacterial strains and plasmids used in this study are listed in Table S1. E. coli EC135

364

lacking all known RM systems and orphan MTases was used to construct the

365

recombinant plasmids, which were replicable in E. coli, but not in B. subtilis or C.

366

glutamicum.13 B. subtilis W168 and C. glutamicum ATCC13032 were cultured at

367

30 °C in LB medium (10 g/L tryptone, 5 g/L yeast extract, 10 g/L NaCl, and 15g/L

368

agar if necessary) with appropriate antibiotics and inducers. E. coli transformation

369

was performed using the calcium chloride technique.45 Electroporation transformation

370

was used in B. subtilis41 and C. glutamicum.42

371 372

Construction of the TCCRAS System and Recombinant Plasmids. All primers

373

were listed in Table S2, and GenBank Accession numbers and the sources of all genes

374

were listed in Table S3. To generate the PliaG–toxin–antitoxin cassettes, promoter PliaG

375

was amplified from the genomic DNA of W168, and jointed with the toxin and 18


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376

antitoxin gene fragments by Splicing using Overlapping Extension (SOE) PCR. The

377

fused fragments were inserted into pAX01 to generate the recombinant plasmids

378

pWYE448-451 and pWYE481 harboring different PliaG–toxin–antitoxin–PxylA

379

cassettes (Table S1). All plasmids were verified by sequencing before integrating into

380

the lacA locus of W168. Mutants integrating the TA modules were selected on Em

381

plates (0.5 µg/mL erythromycin plus 12.5 µg/mL lincomycin) containing 1% xylose,

382

and verified by PCR amplification and DNA sequencing. The resulting mutants were

383

subjected to Em plates plus or minus xylose to evaluate cell growth and death

384

controlled by the reconstructed TA modules (Figure 1c). Detailed procedures for

385

constructing a universal manipulation vector pWYE486 (Figure 2a) and recombinant

386

plasmids for genome modification in B. subtilis were described in Supporting

387

Information.

388

For reconstruction of the TCCRAS system in C. glutamicum, the constitute

389

promoter P45 was amplified from the genomic DNA of ATCC 13032 and fused with

390

the 5’ end of the promoterless relE gene to generate the P45–relE–relB fragment. After

391

digested by SalI and HindIII, the P45–relE–relB fragment was ligated with Ptac of

392

pXMJ19 to generate the pWYE585 plasmid (pXMJ19–Ptac–relB–relE–P45). After

393

verified by PCR and DNA sequencing, pWYE585 was transformed into ATCC 13032

394

to assess the efficiency of TCCRAS on LB plate containing chloramphenicol with or

395

without IPTG. Detailed procedures for constructing a universal manipulation vector

19



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

396

pWYE587 and the recombinant plasmids for genome modification in C. glutamicum

397

were described in Supporting Information.

398 399

Counter-selectable and Mutation Efficiencies. Counter–selectable efficiency was

400

defined as the ratio of colonies excised the TCCRAS cassette (colonies failed to grow

401

with antibiotic and inducer) to the total tested colonies on LB plate, and calculated

402

using the following formula:

403

Counter–selectable efficiency = (Number of colonies growing on LB plate -

404

Number of colonies growing on LB plate with antibiotic and inducer)/Number of

405

colonies growing on LB plate

406

Mutation efficiency was defined as the ratio of the desired mutants to the total

407

tested colonies losing the inserted plasmid by a second recombination event, and

408

calculated using the following formula:

409 410 411

Mutation efficiency = Number of the desired mutants/Number of the total tested colonies In this study, the counter–selectable efficiency was determined based on the

412

results obtained from the 156 randomly selected colonies from LB plates, and the

413

mutation efficiency was calculated by detecting the desired mutations in the 78

414

randomly selected positive mutants from the counter–selectable step. All

415

measurements were performed at least in triplicate and the standard deviations (SD)

416

were calculated from three independent experiments. 20


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417 418

Microscopy. To image bacterial cell morphology, approximately 1 µL mid-log phase

419

culture (OD600 = 2.0) was washed with 100 µL sterile water, resuspended in 10 µL

420

sterile water, and 1 µL resuspended cells was loaded onto a microscope slide covered

421

with 0.1% poly-L-lysine and fixed at room temperature. Images were taken with a

422

phase contrast microscope (Nikon ECLIPSE 80i, Japan).

423 424

Flow Cytometry Analysis. The gfpmut3a gene encoding a GFP with enhanced

425

intense fluorescence was used as the reporter in chromosome. Mutants integrating the

426

gfpmut3a gene were cultured in LB media with or without the inducer and glucose.

427

Cells were harvested after 2–12 h, diluted to an OD600 value of 0.4 using

428

phosphate-buffered saline buffer (pH 7.2), and placed on ice prior to analysis.33 Flow

429

cytometry analysis was performed on a BD FACS CaliburTM flow cytometer equipped

430

with an argon laser (emission at 488 nm and 15 mW) and a 525-nm band-pass filter.

431

For each sample, 20,000 events were collected at a rate of 1,000–2,000 events per

432

second. The original strain without integration of the gfpmut3a gene was used as the

433

control to determine the background fluorescence.

434 435

Statistical Analysis. Significant differences were analysed using SPSS software

436

(version 13.0; http://www-01.ibm.com/software/analytics/spss/). The data collected

437

under various conditions were compared by one-way analysis of variance (ANOVA) 21



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

438

and Duncan’s multiple range tests. All measurements were performed at least in

439

triplicate, and each value represented the mean ± standard deviation (SD). P-values

440

less than 0.05 were considered statistically significant.

441 442

ASSOCIATED CONTENT

443

Supporting Information

444

Supporting Information is available free of charge on the ACS Publications website. It

445

includes Supporting Materials and Methods, Tables S1-S3, and Figures S1−S9.

446 447

AUTHOR INFORMATION

448

Corresponding Authors

449

*E-mail: [email protected]; [email protected]

450

Author Contributions

451

#

452

the study and wrote the manuscript with assistance from Y. L., X.S., and Y. Z.. J.W.

453

and A.D. performed the experiments with assistance from Q.S., H.B., Z.S., and Q.L..

454

All authors read and approved the final manuscript.

A.D. and J.W. contributed equally to this work. T.W., A.D., J.W., and Y.C. designed

455 456

Notes

457

The authors declare no financial conflicts of interest.

458 22


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459

ACKNOWLEDGEMENTS

460

The authors would like to thank Tong Zhao for assistance with the fluorescence

461

microscopy and Dr. Kun Zhu for kindly providing the Enterobacteria phage P1. A

462

patent application related to this work has been filed (CN 201510744940.6), and the

463

authors plan to make the materials widely available to the academic community. This

464

work was supported by the National Natural Science Foundation of China (31570083

465

and 31170103), National Hi-Tech Research & Development Program of China

466

(2014AA021203), Science and Technology Service Network Initiative

467

(KFJ-EW-STS-078), and the Key Deployment Projects of Chinese Academy of

468

Sciences (KGZD-EW-606).

469 470

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Rodionov, D. A., Mironov, A. A., and Gelfand, M. S. (2001) Transcriptional

603

regulation of pentose utilisation systems in the Bacillus/Clostridium group of

604

bacteria. FEMS Microbiol. Lett. 205, 305-314.

605

39.

606 607

Cell 112, 2-4. 40.

608 609

Hayes, C. S. (2003) Toxin-antitoxin pairs in bacteria: killers or stress regulators?

Gerdes, K., Christensen, S. K., and Lobner-Olesen, A. (2005) Prokaryotic toxin-antitoxin stress response loci. Nat. Rev. Microbiol. 3, 371-382.

41.

Zhang, G. Q., Bao, P., Zhang, Y., Deng, A. H., Chen, N., and Wen, T. Y. (2011)

610

Enhancing

611

amyloliquefaciens by combining cell-wall weakening and cell-membrane

612

fluidity disturbing. Anal. Biochem. 409, 130-137.

613

42.

electro-transformation

competency

of

recalcitrant

Bacillus

Tauch, A., Kirchner, O., Loffler, B., Gotker, S., Puhler, A., and Kalinowski, J.

614

(2002) Efficient electrotransformation of Corynebacterium diphtheriae with a

615

mini-replicon derived from the Corynebacterium glutamicum plasmid pGA1.

616

Curr. Microbiol. 45, 362-367.

617

43.

618 619

Cambray, G., Mutalik, V. K., and Arkin, A. P. (2011) Toward rational design of bacterial genomes. Curr. Opin. Microbiol. 14, 624-630.

44.

McGregor, N., Ayora, S., Sedelnikova, S., Carrasco, B., Alonso, J. C., Thaw, P.,

620

and Rafferty, J. (2005) The structure of Bacillus subtilis RecU Holliday junction

621

resolvase and its role in substrate selection and sequence-specific cleavage.

622

Structure 13, 1341-1351. 28


Page 29 of 37

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623

45.

Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular cloning: a

624

laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring

625

Harbor, New York. 53-84.

626 627

29



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

628

Figure legends

629

Figure 1. Design and functional characterization of the TCCRAS system. (a) Five

630

type II TA systems were selected to design the systems. Constitutive and inducible

631

promoters were separately used to control the expression of toxins and antitoxins. (b)

632

Plasmid and the TCCRAS-integrated strain were used to characterize different TA

633

systems. Plasmid pAXTA was generated by inserting different TCCRAS systems into

634

pAX01, and integrated to the lacA locus of B. subtilis W168. (c) Screening for an

635

effective TCCRAS system to regulate cell growth and death in B. subtilis. Strain

636

W168 integrating pAX01 was used as the control.

637 638

Figure 2. Construction of a universal vector pWYE486 in B. subtilis and the

639

schematic illustration of the TCCRAS-based genetic manipulation. (a) The

640

homologous regions of amyE were amplified using P23/P24 and P25/P26, and fused

641

through SOE-PCR by P23/P26 (Table S3). The fused product was treated with Taq

642

DNA polymerase to generate 3'-dA overhangs and then inserted into pMD19 T-simple

643

vector to create pWYE467, which was digested by ClaI, dephosphorylated, and

644

ligated with the reconstructed relBE fragments from pWYE448 to generate pWYE469.

645

Plasmid pWYE469 was then digested by KpnI to remove the homologous regions of

646

amyE and self-ligated to generate pWYE486. (b) Schematic illustration of the

647

TCCRAS-based approach for deletion, integration, replacement, and point mutation

648

of the target genes or sites. UHA and DHA indicate the upstream and downstream 30


Page 30 of 37

Page 31 of 37

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649

homologous arms, respectively. DG, RG, and IG represent the deleted, replaced, and

650

integrated/inserted genes, respectively. Asterisk represents the mutated site.

651 652

Figure 3. Gene deletion, integration, replacement, and point mutation in B. subtilis

653

using the TCCRAS system. (a) Microscopic images for changes in cell morphology of

654

the divIVA knockout strains. Scale bar, 10 µm. (b) Verification of amyE deletion using

655

a starch plate assay. (c) and (d) PCR detection for deletion of the pps operon, and

656

integration of the 5.2-, 7.2-, and 12.3-kb operons, respectively. Positions of the

657

molecular size markers (left), and sizes of the amplified fragments (right) are

658

indicated. IG represents the integrated genes. UHA and DHA represent the upstream

659

and downstream homologous arms, respectively. (e) Biosynthetic pathway of

660

lycopene by introducing crtE, crtB, and crtI genes (green), and HPLC analysis of

661

lycopene in B. subtilis. The inserted tubes indicates darker red for mutant BS206

662

(BS097::Pspac–crtI–crtE–crtB) compared to the original BS097 (W168∆ppsEDCBA).

663

(f) Functional verification for loss of the uracil phosphoribosyl transferase activity

664

and expression of GFP in the BS113 mutant (W168∆upp::P43–gfpmut3a). (g)

665

Functional verification of the recombination deletion and repair activity and

666

confirmation of the recU mutation by DNA sequencing in the BS093 mutant (W168

667

recUA107C).

668 669

Figure 4. Genome editing in C. glutamicum and the underlying mechanism of 31



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

670

TCCRAS system. (a) Construction of the TCCRAS system used in C. glutamicum.

671

(b) Screening for a TCCRAS system that effectively regulate cell growth and death

672

in C. glutamicum. (c) Functional verification for loss of the uracil phosphoribosyl

673

transferase activity in the upp deleted CG709 (ATCC 13032∆upp). (d) HPLC

674

detection of cadaverine produced by the CG706 mutant (ATCC 13032∆lysE::cadA).

675

(e) Cell growth and cadaverine production of the WT ATCC 13032 and the CG706

676

mutant. (f) GFP expression controlled by promoter PliaG or PxylA in the presence of

677

xylose. GFP expression driven by PliaG in BS217 (W168∆lacA::PliaG-gfpmut3a-erm)

678

represents constitutive expression of the toxin, while the GFP expression driven by

679

PxylA in BS216 (W168∆lacA::PxylA-gfpmut3a-erm) represents inducible expression of

680

the antitoxin in TCCRAS. (g) GFP expression driven by promoter PxylA in the

681

absence of xylose or by adding xylose after culturing for 6 h. Protein expression of

682

gfp driven by PxylA in the absence of xylose was determined in BS216, which

683

represents the level of toxin when the toxin gene was integrated into chromosome in

684

the RelE system. According to the traditional counter-selectable process, upon

685

addition of an inducer, GFP expression by adding xylose at 6 h indicates possible

686

expression level of the toxins. (h) The proposed model for manipulating process by

687

the TCCRAS system. TG and HA represent target gene and homologous arm,

688

respectively. IP and CP indicate inducible and constitutive promoters, respectively.

689

32


Page 32 of 37

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690

Table 1. Counter-selectable and Mutation Efficiencies of the Genome-scale

691

Engineering Using the TCCRAS System Genome modifica tion

Strain and genotype

Length (kb) of target sequence

Counter-select able efficiency (%)

Mutation efficiency (%)

BS089 (W168∆amyE)

2.1

100.0 ± 0.0

89.6 ± 9.0

BS089 (W168∆amyE)a

2.1

78.8 ± 3.8a

83.1 ± 9.1a

BS091 (W168∆divIVA)

0.6

100.0 ± 0.0

100.0 ± 0.0

BS097 (W168∆ppsEDCBA)

37.7

99.4 ± 1.1

61.5 ± 3.8

BS097 (W168∆ppsEDCBA)a

37.7

69.2 ± 3.8a

53.8 ± 3.8a

BS214 (W168∆dltA-rocR)

194.9

98.1 ± 1.9

29.5 ± 2.2

CG709 (ATCC 13032∆upp)

0.6

100.0 ± 0.0

85.9 ± 5.9

CG709 (ATCC 13032∆upp)b

0.6

42.3 ± 5.1b

43.6 ± 4.4b

CG711(ATCC 13032∆cg11675-cg11844)

179.8

100.0 ± 0.0

17.9 ± 4.4

BS206(W168∆pps::Pspac-crtI-crtE-crtB)

5.4

98.1 ± 1.9

50.0 ± 3.8

BS105 (W168∆pps::thrABC)

5.2

99.4 ± 1.1

52.6 ± 5.8

BS109 (W168∆pps::trp)

7.2

100.0 ± 0.0

42.3 ± 3.8

BS115(W168∆pps::trp-thrABC)

12.3

100.0 ± 0.0

52.6 ± 2.2

BS113 (W168∆upp::P43-gfpmut3a)

0.8/1.1c

97.4 ± 1.1

57.7 ± 3.8

CG706 ( ATCC 13032∆lySE::cadA)

0.7/2.1c

100.0 ± 0.0

69.2 ± 3.8

CG706 ( ATCC 13032∆lySE::cadA)b

0.7/2.1c

41.0 ± 1.1b

38.5 ± 6.7b

1 bp

100.0 ± 0.0

69.2 ± 3.8

Deletion

Insertion

Replacement

Point mutation BS093 (W168recUA107C)

692 693 694 695

a

b

Genome modification using PxylA-relE as the counter-selectable marker; Genome modification using the sacB gene encoding levansucrase as the counter-selectable marker; c Size of the deleted/inserted gene.

33



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 1. Design and functional characterization of the TCCRAS system. (a) Five type II TA systems were selected to design the systems. Constitutive and inducible promoters were separately used to control the expression of toxin and antitoxin. (b) Plasmid and the TCCRAS-integrated strain were used to characterize different TA systems. Plasmid pAXTA was generated by inserting different TCCRAS systems into pAX01, and integrated to the lacA locus of B. subtilis W168. (c) Screening for an effective TCCRAS system to regulate cell growth and death in B. subtilis. Strain W168 integrating pAX01 was used as the control.


Page 34 of 37

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Figure 2. Construction of a universal vector pWYE486 in B. subtilis and the schematic illustration of the TCCRAS-based genetic manipulation. (a) The homologous regions of amyE were amplified using P23/P24 and P25/P26, and fused through SOE-PCR by P23/P26 (Table S3). The fused product was treated with Taq DNA polymerase to generate 3'-dA overhangs and then inserted into pMD19 T-simple vector to create pWYE467, which was digested by ClaI, dephosphorylated, and ligated with the reconstructed relBE fragments from pWYE448 to generate pWYE469. Plasmid pWYE469 was then digested by KpnI to remove the homologous regions of amyE and self-ligated to generate pWYE486. (b) Schematic illustration of the TCCRAS-based approach for deletion, integration, replacement, and point mutation of the target genes or sites. UHA and DHA indicate the upstream and downstream homologous arms, respectively. DG, RG, and IG represent the deleted, replaced, and integrated/inserted genes, respectively. Asterisk represents the mutated site.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 3. Gene deletion, integrations, replacement, and point mutation in B. subtilis using the TCCRAS system. (a) Microscopic images for changes in cell morphology of the divIVA knockout strains. Scale bar, 10 µm. (b) Verification of amyE deletion using a starch plate assay. (c) and (d) PCR detection for deletion of the pps operon, and integration of the 5.2-, 7.2-, and 12.3-kb operons, respectively. Positions of the molecular size markers (left), and sizes of the amplified fragments (right) are indicated. IG represents the integrated genes. UHA and DHA represent the upstream and downstream homologous arms, respectively. (e) Biosynthetic pathway of lycopene by introducing crtE, crtB, and crtI genes (green), and HPLC analysis of lycopene in B. subtilis. The inserted tubes indicates darker red for mutant BS206 (BS097::Pspac–crtI–crtE– crtB) compared to the original BS097 (W168∆ppsEDCBA). (f) Functional verification for loss of the uracil phosphoribosyl transferase activity and expression of GFP in the BS113 mutant (W168∆upp::P43– gfpmut3a). (g) Functional verification of the recombination deletion and repair activity and confirmation of the recU mutation by DNA sequencing in the BS093 mutant (W168 recUA107C).


Page 36 of 37

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Figure 4. Genome editing in C. glutamicum and the underlying mechanism of TCCRAS system. (a) Construction of the TCCRAS system used in C. glutamicum. (b) Screening for a TCCRAS system that effectively regulate cell growth and death in C. glutamicum. (c) Functional verification for loss of the uracil phosphoribosyl transferase activity in the upp deleted CG709 (ATCC 13032∆upp). (d) HPLC detection of cadaverine produced by the CG706 mutant (ATCC 13032∆lysE::cadA). (e) Cell growth and cadaverine production of the WT ATCC 13032 and the CG706 mutant. (f) GFP expression controlled by promoter PliaG or PxylA in the presence of xylose. GFP expression driven by PliaG in BS217 (W168∆lacA::PliaG-gfpmut3aerm) represents constitutive expression of toxin, while the GFP expression driven by PxylA in BS216 (W168∆lacA::PxylA-gfpmut3a-erm) represents inducible expression of antitoxin in the TCCRAS system. (g) GFP expression driven by promoter PxylA in the absence of xylose or by adding xylose after culturing for 6 h. Protein expression of gfp driven by PxylA in the absence of xylose was determined in BS216, which represents the level of toxin when the toxin gene was integrated into chromosome in the RelE system. According to the traditional counter-selectable process, upon addition of inducer, GFP expression by adding xylose at 6 h indicates possible expression level of the toxins. (h) The proposed model for manipulating process by the TCCRAS system. TG and HA represent target gene and homologous arm, respectively. IP and CP indicate inducible and constitutive promoters, respectively.


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