A Novel Tool for Microbial Genome Editing Using the Restriction

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A novel tool for microbial genome editing using the restriction-modification system Hua Bai, Aihua Deng, Shuwen Liu, Di Cui, Qidi Qiu, Laiyou Wang, Zhao Yang, Jie Wu, Xiuling Shang, Yun Zhang, and Tingyi Wen ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.7b00254 • Publication Date (Web): 02 Oct 2017 Downloaded from http://pubs.acs.org on October 3, 2017

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ACS Synthetic Biology

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A novel tool for microbial genome editing using the restriction-modification

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system

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Hua Bai†,‡, Aihua Deng†,*, Shuwen Liu†, Di Cui†,‡, Qidi Qiu†,‡, Laiyou Wang†,‡, Zhao

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Yang†, Jie Wu†, Xiuling Shang† , Yun Zhang† and Tingyi Wen†,§,*

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†

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Microbiology, Chinese Academy of Sciences, Beijing 100101, 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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CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of

100049, China

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Graphical Table of Contents

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ACS Paragon Plus Environment


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ABSTRACT

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Scarless genetic manipulation of genomes is an essential tool for biological research.

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The restriction-modification (R-M) system is a defense system in bacteria that

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protects against invading genomes based on its ability to distinguish foreign DNA

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from self DNA. Here, we designed an R-M system-mediated genome editing (RMGE)

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technique for scarless genetic manipulation in different microorganisms. For bacteria

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with Type IV REase, an RMGE technique using the inducible DNA methyltransferase

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gene, bceSIIM (RMGE-bceSIIM), as the counter-selection cassette was developed to

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edit the genome of Escherichia coli. For bacteria without Type IV REase, an RMGE

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technique based on a restriction endonuclease (RMGE-mcrA) was established in

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Bacillus subtilis. These techniques were successfully used for gene deletion and

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replacement with nearly 100% counter-selection efficiencies, which were higher and

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more stable compared to conventional methods. Furthermore, precise point mutation

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without limiting sites was achieved in E. coli using RMGE-bceSIIM to introduce a

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single base mutation of A128C into the rpsL gene. In addition, the RMGE-mcrA

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technique was applied to delete the CAN1 gene in Saccharomyces cerevisiae DAY414

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with 100% counter-selection efficiency. The effectiveness of the RMGE technique in

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E. coli, B. subtilis, and S. cerevisiae suggests the potential universal usefulness of this

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technique for microbial genome manipulation.

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KEYWORDS: scarless genome editing, counter-selection cassette,

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restriction-modification (R-M) system, Escherichia coli, Bacillus subtilis,

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Saccharomyces cerevisiae 2


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INTRODUCTION

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Understanding the function of genes1 or developing rational design for biological

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engineering2 in bacteria heavily depends on the technology to edit the DNA sequence.

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To modify the DNA sequence, numerous genetic tools have been developed to

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manipulate bacterial chromosomes3-5. At early stages, a selectable marker was

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employed to introduce a chromosomal mutation, but the number of available markers

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limited multigene modifications. Although site-specific recombinases (FLP or Cre)

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have been used to excise marker, a scar, such as a FRT or loxP site, is left in the

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chromosome6. To overcome this problem, scarless genome editing techniques are

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particularly valuable to introduce defined mutations, especially single nucleotide

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mutations7.

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Among the recently developed methods for scarless genome editing without any

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limiting sites, the counter-selectable system is a common technique in various bacteria,

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such as Escherichia coli7, Bacillus subtilis8, 9, and Corynebacterium glutamicum10, etc.

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However, most of the existing counter-selectable systems are insufficient to introduce

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precise mutations in bacterial genomes. For example, the counter-selectable system

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mediated by rpsL11, pheS12, thyA13, tolC14, or lacY15 requires a special modification of

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the chromosome to prepare the prerequisite mutants, which is time-consuming and

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could limit its application in strains without clear genetic backgrounds. Although the

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sacB-based counter-selectable system can be directly used in the target strain, the

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counter-selection efficiency of the method is low7. In addition, the meganuclease 3



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I-SceI that recognizes an 18-bp specific sequence

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counter-selection marker for scarless genetic manipulation through introducing the

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recognition site16, 17 . Recently, a toxin-antitoxin (TA) system, a type of defense

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system in bacteria and archaea, has been used as an effective and stable genome

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manipulation techniques7, 8. However, they are inefficient to meet the demands of

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bacteria with different genetic backgrounds. Therefore, an efficient, universal, and

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scarless genome editing technique is still urgently needed for genetic manipulation of

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bacteria with different genetic backgrounds.

has been used as a

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The restriction-modification (R-M) system is a group of defense systems that

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consists of DNA methyltransferases (MTases) and restriction endonucleases (REases).

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These systems exist both in bacteria and archaea, and more than 96% of

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genome-sequenced bacteria possess R-M systems18. R-M systems have been

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classified into four types based on their subunit composition, cleavage sites, sequence

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specificity, and cofactors. Type I, II, and III REases cleave DNA without methylation

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at specific sequences, and Type IV REases cleave DNA with an exogenous

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methylation pattern19. R-M systems protect bacteria from invading exogenous DNA20,

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and therefore, this defensive machinery can be used to cleave the bacterial genome in

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a similar way to the clustered, regularly interspaced, short palindromic repeats

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(CRISPR) systems21. Unlike the CRISPR systems that can cleave genomic DNA at a

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single site directed by a single-guide RNA (sgRNA), R-M systems are able to cleave

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the genome at multiple sites22, 23. In general, exogenous REases can kill target cells by

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cleaving their genomic DNA and can be potentially used as a counter-selectable 4


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marker in a wide range of bacteria. Furthermore, for bacteria with Type IV REases,

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exogenous MTases can also kill target cells due to their exogenous methylation of

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genomic DNA, which is then cleaved by the Type IV REase of the host bacteria24.

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Accordingly, both exogenous REases and MTases can be used as counter-selectable

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markers in bacteria with Type IV REases. Therefore, the specific regulation

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mechanisms of the R-M system play pivotal roles in cell growth and death and thus

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might be powerful tools in genetic engineering for both fundamental research and

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application purposes.

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In this study, we developed an R-M system-mediated genome editing technique

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(designated as RMGE) for bacteria with or without Type IV REases. Using the model

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bacterium E. coli as an example of bacteria with Type IV REases (Table S1), a

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RMGE system based on inducible Type II MTase BceSIIM (designated as REGE-

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bceSIIM) was developed for genome editing in E. coli K-12 derivative W3110.

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Furthermore, the RMGE system based on the inducible Type IV REase McrA25

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(designated as REGE-mcrA) was constructed to manipulate the genome of the model

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Gram-positive bacterium B. subtilis W168, which was chosen as an example of

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bacteria without Type IV REases (Table S1). Additionally, RMGE-mcrA could also

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be used to manipulate the genome of Saccharomyces cerevisiae DAY414. Our results

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demonstrated that RMGE was convenient and efficient for genome editing in E. coli,

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B. subtilis and S. cerevisiae, suggesting its great potential for application in a broad

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range of microorganisms.

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RESULTS

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Design of R-M system-mediated genome editing (RMGE) for bacteria with or

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without Type IV REases. According to the genetic background of the target bacteria,

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a suitable R-M system was selected as a counter-selectable marker for RMGE. For

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example, E. coli W3110 contains Type IV REases (Figure 1a; Table S1) that can

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cleave DNA with exogenous methylation patterns. Thus, an exogenous REase or

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MTases can be used as the counter-selectable marker in genome editing of these

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bacteria. Our previous study showed that a Type II MTase BceSIIM from B. cereus

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ATCC 10987 can be expressed in E. coli26. When the bceSIIM gene is expressed in

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the E. coli, a m5C residue in the sequence GGWCC (W=A or T)26 of the genomic

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DNA is introduced by the BceSIIM and then cleaved by Type IV REases McrBC,

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which kills target cells 27. Therefore, the MTase BceSIIM was employed as a

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counter-selection cassette to construct the RMGE genome editing tool in bacteria with

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Type IV REases.

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As an effective counter-selection cassette for genetic manipulation, MTase only

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works in bacteria with Type IV REases, but not in bacteria without Type IV REases,

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such as B. subtilis W168 (Figure 1b; Table S1). The genomic DNA of W168 is

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methylated in its own pattern, so the exogenous REases that can cleave the genome

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could be used as the counter-selection cassette. For example, when the REase McrA is

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integrated into the genome and expressed in B. subtilis, the sequence YCGR (the

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recognition sequence A(/G)CGC(/T) of McrA) 28 of the genome is cleaved into pieces

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by the REase, which kills target cells. Therefore, various REases had to be screened 6


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as a counter-selection cassette to construct the RMGE genome editing tool in bacteria

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without Type IV REases.

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Construction of an RMGE system based on the inducible MTase BceSIIM

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(RMGE- bceSIIM). To assess the feasibility of the R-M system in genome editing of

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E. coli, MTase BceSIIM was selected to construct the counter-selection cassette. The

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temperature-sensitive plasmid pWYE3000, which is lost during high-temperature

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cultivation (Figure S1), was firstly constructed as a recombinant vector by overlap

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PCR. To further construct the counter-selection cassette based on MTase BceSIIM,

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the bceSIIM gene, controlled by the arabinose-inducible promoter Para, was

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PCR-amplified from WYE700 and ligated into pWYE3000 (Figure 2a). The

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pWYE3001 plasmid was generated then transferred into E. coli W3110 to test the

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regulation of cell growth and death by inducible bceSIIM. As shown in Figure 2b, no

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growth was detected in the strain EC3004 (W3110/ pWYE3001) after the addition of

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arabinose, suggesting that the inducible bceSIIM can effectively regulate the host

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cell’s growth and death. Thus, MTase BceSIIM can be used for genetic manipulation

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of E. coli, and the pWYE3001 plasmid was used as the universal genome editing

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vector for various gene modifications.

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According to the standard operation procedure (Figure 3a), recombinant plasmids

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were constructed by inserting up- and down-stream homologous arms of the target

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gene into the universal vector pWYE3001 and transferred into W3110. Transformants

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were selected on LB medium with chloramphenicol (Cm) at 30 °C for 10 h. To obtain 7



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an integrated strain through a single crossover event, positive transformants harboring

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the recombinant plasmid were cultured in 5 mL of LB medium with Cm at 30 °C for

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10 h. The liquid culture was then plated on LB plates with Cm after being diluted 10-3

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times and incubated at 42 °C for 12 h. After PCR verification, the plasmid-integrated

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strain was cultured in 5 mL of LB at 30 °C for 12 h. Then, the liquid culture was

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diluted by 10-4−10-5 and grown on LB plates with the appropriate concentration of

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arabinose at 30 °C for 12 h for counter selection. The plasmid-integrated strain

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expressing exogenous MTase were killed by the Type IV REases. The colonies that

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grew on the LB plate but failed to grow on the LB plate with Cm were verified by

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PCR amplification to select positive mutant strains. DNA sequencing of the mutated

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sites and the physiological changes of the resulting mutants were also examined for

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further verification.

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Scarless single gene deletion in E. coli. To validate the RMGE- bceSIIM system, a

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diaminopimelate decarboxylase encoding gene, lysA, was chosen as the target to edit

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the chromosome of E. coli W3110. The resulting mutant EC3007 (W3110∆lysA) was

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obtained with 100% counter-selection efficiency, a similar efficiency to the traditional

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SacB system (Table S2 and Figure S2a). Physiological changes of the mutant EC3007

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were further identified in MM medium. In contrast to the normal growth of W3110,

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the mutant EC3007 could not grow on MM plates, whereas it could grow on MM

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plates supplemented with lysine (Figure 3b). The results indicated that the mutant

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could not synthetize lysine due to deletion of the lysA gene. 8


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Because the target sites might affect the counter-selection efficiency, another two

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genes, lacZ and tolC, encoding beta-galactosidase and outer membrane protein,

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respectively, were selected to further verify the RMGE- bceSIIM system. The

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lacZ-deficient strain EC3010 (W3110∆lacZ) was generated with 100%

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counter-selection efficiency, which was significantly higher compared to the 4.5 ± 4.0%

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achieved using the SacB system (Table S2 and Figure S2b). The color of the mutant

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EC3010 on LB plates with X-gal (5-Bromo-4-chloro-3-indolyl β-D-galactopyranoside)

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did not turn blue, further confirming the absence of beta-galactosidase activity

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through deletion of the lacZ gene (Figure 3c). Furthermore, the tolC-deficient strain

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EC3048 (W3110∆tolC) was obtained with 100% counter-selection efficiency, higher

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than the73.7 ± 6.2% achieved by using the SacB system (Figure S2c). Because TolC

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can confer the host cell with resistance to sodium dodecyl sulfate (SDS), the

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sensitivity of the mutant EC3048 to SDS was identified on LB medium with 0.01%

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SDS (Figure 3d).

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Precise point mutation and gene replacement in E. coli. To evaluate the feasibility

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for precise point mutations using the RMGE- bceSIIM system, a single base mutation

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(A128C) was introduced into the rpsL gene encoding the small subunit ribosomal

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protein S12, which confers streptomycin resistance. Following standard procedures,

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the mutant EC3013 (W3110 rpsLA128C) was obtained with 100% counter-selection

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efficiency (Table S2; Figure S3a). The resistance of the mutant to streptomycin was

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tested, and the nucleotide sequence of rpsL was further confirmed by DNA 9



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sequencing (Figure 4a). To assess the potential of RMGE- bceSIIM for gene replacement, the gfpmuat3a

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gene controlled by the promoter PPL29 was used to replace the promoter of the frdA

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gene encoding the fumarate reductase flavoprotein subunit. A recombinant plasmid,

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pWYE3005, with homologous arms and a fragment of PPL-gfpmuat3a was obtained.

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The mutant strain EC3016 (W3110 PfrdA::PPL-gfpmut3a) was obtained by introducing

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the pWYE3005 into W3110 with 100% counter-selection efficiency, which was

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higher than the 59.7 ± 5.1% obtained with the SacB system (Table S2; Figure S3b).

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The GFP intensity was further detected by flow cytometry analysis, which confirmed

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the expression of the gfpmut3a gene in the mutant EC3016 (Figure 4b).

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To further assess the potential of RMGE- bceSIIM for metabolic engineering, the

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cadA gene controlled by the promoter PPL was used to replace the region between the

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ygcE and ygcF genes. The mutant strain EC3053 (W3110 ygcE-ygcF::PPL-cadA) was

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generated with 98.7 ± 1.1% counter-selection efficiency, higher than the 84.6 ± 8.8%

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efficiency obtained with the SacB system (Figure S3c). Because lysine can be

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catalyzed by CadA and converted into cadaverine, accumulated cadaverine in EC3053

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was measured to be 3.35 ± 0.14 g/L, which was significantly higher than that of

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W3110 (Figure 4c and d).

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Construction of a RMGE system based on the inducible REase McrA (RMGE-

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mcrA). To develop a general method in bacteria without Type IV REase, REases were

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used to construct a counter-selection cassette for B. subtilis W168. Type IV restriction 10


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endonucleases from E. coli W3110, including McrBC, McrA, and Mrr, were selected

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as candidates to evaluate their effectiveness25, 30. Three REase genes were separately

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inserted into the plasmid pHCMC04 under control of the xylose-inducible promoter.

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The lethal effects of the three REases on W168 were further detected on LB plates

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with Cm and xylose after the expression plasmids were introduced into the target cells.

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Figure 5a showed that the strain BS232 (W168/pWYE3008) did not grow on plates

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with Cm and xylose, indicating that the REase McrA inhibited the growth of W168.

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Thus, the inducible mcrA gene was selected as an effective counter-selection cassette

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to establish the RMGE-mcrA system. A suicide plasmid, pWYE3010, which was

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constructed by removing the replication origin reP from the plasmid pWYE3008

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(Figure 5b), was used as a universal genome editing vector for genetic manipulation

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in B. subtilis.

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Genetic manipulation in B. subtilis using the RMGE- mcrA system. For genetic

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manipulation in B. subtilis W168, the basic principle of scarless genome editing was

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illustrated in Figure 6a. The plasmid-integrated strain was obtained by

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electro-transformation of the recombinant plasmid into W168. Afterwards, the

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plasmid-integrated strain was cultured in LB medium for 12 h and then on LB plates

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with xylose at 30 °C for 12 h after first being diluted by a factor of 10-4-10-5 for

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counter selection. Cells that contained the counter-selection cassette mcrA would be

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killed, whereas colonies without mcrA were identified by PCR amplification and

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DNA sequencing of the mutated sites. 11



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To evaluate the efficiency of gene editing in B. subtilis using RMGE, the amyE

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gene encoding α-amylase was chosen as a target for deletion. The recombinant

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plasmid was constructed by inserting the up- and down-stream homologous arms of

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the amyE gene into pWYE3010. The mutant BS235 (W168∆amyE) was obtained with

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100% of counter-selection efficiency (Figure S4a). The mutant BS235 was further

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verified by PCR amplification and DNA sequencing. No clear halo was formed in the

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mutant BS235 by the starch plate assay compared to the original W168, further

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confirming the lack of α-amylase (Figure 6b).

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To assess the efficiency of RMGE in gene replacement, the gfpmuat3a gene was

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chosen to replace the upp gene in W168. The mutant strain BS237 was generated with

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100% counter-selection efficiency (Figure S4b). The GFP intensity was further

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detected by flow cytometry analysis, which showed the expression of the gfpmut3a

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gene in the mutant BS237 (Figure 6c).

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Genome editing in Saccharomyces cerevisiae using RMGE-mcrA system. To test

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whether RMGE can be applied in S. cerevisiae, McrA from E. coli W3110 as well as

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BceSIIR and BceSIIIR from B. cereus ATCC 10987 were selected as candidates to

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perform genome editing in S. cerevisiae. Three REase genes were separately cloned

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into the plasmid pYES2/CT under the control of galactose-inducible promoter to test

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the effect of REase gene expression on the cell growth. As shown in Figure 7a, the

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strain SC201 (DAY414/pWYE3023) could not grow on the SG-Ura plate, indicating

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that the expression of REase McrA resulted in the growth inhibition of DAY414. 12


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Thus, the mcrA gene was selected as an effective counter-selection cassette to

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construct the plasmid pWYE3026 in which the replication origin 2μ was removed

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from the plasmid pWYE3023 (Figure 7b).

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To evaluate the efficiency of RMGE-mcrA system in S. cerevisiae, CAN1 gene

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encoding the plasma membrane arginine permease that imports the toxic compound

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L-canavanine

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constructed by inserting the up- and down-stream homologous arms of the CAN1 gene

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into pWYE3026. The mutant SC205 (DAY414∆ CAN1) was obtained with 100% of

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counter-selection efficiency (Figure S6). The mutant SC205 was further verified by

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PCR amplification and DNA sequencing. The result showed that SC205 was able to

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grow on synthetic complete medium containing L-canavanine due to the deletion of

269

the CAN1 gene (Figure 7c).

was chosen as a target for deletion. The recombinant plasmid was

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Discussion

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Here we firstly developed an R-M system-mediated genome editing (RMGE)

273

technique for scarless genetic manipulation in E. coli W3110, B. subtilis W168 and S.

274

cerevisiae DAY414. Different types of mutations, such as deletions, replacements,

275

and point mutations, were successfully achieved. The advantages of RMGE are as

276

follows: (i) it has a high and stable counter-selection efficiency compared with

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traditional methods, such as counter-selection systems mediated by sacB, mazF9, and

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relE8; (ii) it is versatile and can be widely used in bacteria and yeasts with or without

279

Type IV REases ; and (iii) it can edit genomes at any loci without the limitation of a 13



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protospacer-adjacent motif (PAM), such as CRISPR. Therefore, the RMGE technique

281

holds promise as an efficient, versatile, and affordable strategy to perform

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sophisticated genetic modifications in a broad range of bacteria and yeasts.

283

In this study, RMGE was efficient for deletion, replacement, and point mutation

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of 6 target loci in E. coli with nearly 100% counter-selection efficiencies, which were

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significantly increased compared to those of SacB (4.5-100%) and RelE (87-100%)

286

systems (Table S3). This method was also efficient in B. subtilis for gene deletion and

287

replacement with 100% counter-selection efficiencies, which were more stable than

288

that of TCCRAS and RelE systems (Table S4). However, mutation efficiencies of

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RMGE were varied for all 9 target loci in both E. coli, B. subtilis and S. cerevisiae,

290

and were not comparable to existing methods, such as homologous

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recombination-based counter-selection systems and nuclease-mediated CRISPR/Cas9

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systems (Table S3, 4 and 5). The high and stable counter-selection efficiency

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achieved by RMGE might be attributed to the strong ability of killing target cells

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mediated by R-M systems. In general, the R-M systems can restrict the growth of

295

phage31 and cause post-segregational killing of host strains23. Thus, the R-M systems

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can cleave DNA efficiently as shown in Figure 2b and 5a. Except for the strains

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harboring the expression plasmids, this lethal effect was also detected in the strains

298

that had integrated a single copy of the inducible R-M system, which further

299

suggested that the R-M system can cleave DNA efficiently (Figure S5).

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The RMGE technique is a versatile tool that can be used in a broad range of

301

bacteria. To our knowledge, the vast diversity and prevalence of R-M systems in the 14


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prokaryotic kingdom18 are evidence of their success in preventing exogenous DNA

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invasions23. The horizontal transfer32, 33 of many R-M systems indicates that the R-M

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system has great potential for expression in heterologous species26. Based on this

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characteristic of R-M systems, the bceSIIM and mcrA genes were successfully

306

heterologously expressed in E. coli W3110 (Figure 2b) and B. subtilis W168 (Figure

307

5a), respectively. Therefore, RMGE- bceSIIM and RMGE- mcrA systems can be

308

applied in E. coli W3110 and B. subtilis W168, respectively, and the RMGE

309

technique may be universally useful for genome manipulation in other bacterial

310

strains.

311

Genome editing mediated by R-M systems is also affordable. R-M systems used

312

for genome editing are based on the modification pattern of the target bacterial

313

genome. Different R-M systems should be chosen for genome editing in bacteria with

314

different genetic backgrounds. Information regarding R-M systems and the

315

methylation patterns of bacteria can be found in REBASE18 from genome sequencing

316

efforts, which makes it easy to construct a counter-selection cassette mediated by

317

R-M systems. Moreover, mutants achieved by RMGE only require one transformation

318

for any loci in the bacterial chromosomes.

319

In principle, the REGM technique can be established by using an exogenous

320

REase as the universal counter-selection cassette both in bacteria with and without

321

Type IV REases. In our preliminary study, BceSIIR34, a Type II REase that cleaves

322

the sequence GGWCC without methylation by BceSIIM, was used to construct the

323

universal counter-selection cassette. However, the expression plasmid harboring the 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

324

bceSIIR gene caused host cell death and was not generated due to its leaky expression

325

in E. coli. To solve this problem, the MTase bceSIIM was chosen as the

326

counter-selection marker for genome editing in E. coli. Additionally, the universal

327

counter-selection cassette could also be established by engineering a tightly regulated

328

inducible system.

329

RMGE can also be applied to genome editing of yeasts without R-M system, such

330

as S. cerevisiae. Due to the deficiency of Type IV REases in S. cerevisiae, the

331

exogenous MTases could not be used as a counter-selection marker. However, the

332

mouse-methylated DNA could be cleaved by McrBC27, indicating that REases had

333

potential to regulate the growth and death of the organisms without R-M system. As a

334

result, McrA was screened as the counter-selectable marker to successfully delete the

335

CAN1 gene in S. cerevisiae DAY414 (Figure 7a). Therefore, RMGE might be useful

336

for genome manipulation in other organisms without R-M system.

337

In summary, a novel strategy of RMGE was developed for scarless genome

338

editing in bacteria and yeasts with or without Type IV REase. Various mutations,

339

including deletions, replacements, and point mutations were achieved with nearly 100%

340

counter-selection efficiencies in E. coli, B. subtilis, and S. cerevisiae by the RMGE

341

technique using the MTase BceSIIM and REase McrA as the counter-selection

342

cassettes, respectively. Therefore, the RMGE approach is an efficient, versatile, and

343

affordable tool for genome editing in a broad range of microorganisms.

344 345

MATERIALS AND METHODS 16


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346

Bacterial strains, plasmids, and primers. The bacterial strains and plasmids used in

347

this study are listed in Table S6. EC135, which lacks all R-M systems and orphan

348

MTases, was used to construct the plasmids. All of the primers (Table S7) used in this

349

study were synthesized by Invitrogen (Shanghai, China). DNA sequencing was

350

performed by Beijing Genomics Institute (BGI, Beijing, China).

351 352

Media and culture conditions. E. coli W3110 (F-λ- rph-1Inv(rrnD-rrnE))35 and B.

353

subtilis W168 (trp+)36 were cultured in Luria-Bertani (LB) medium. The

354

concentrations of the antibiotics used in this study were as follows: 100 µg/mL

355

ampicillin and 34 µg/mL Cm for E. coli, and 10 µg/mL Cm for B. subtilis. To induce

356

the promoters ParaB and PxylA, 0.2% arabinose and 1% xylose, respectively, were used.

357

Minimal medium (MM; 5 g/L glucose, 1 g/L Na3C6H5O7·2H2O, 2 g/L (NH4)2SO4, 0.2

358

g/L MgSO4·7H2O, 14 g/L K2HPO4·3H2O, 6 g/L KH2PO4, 100 µg/L biotin, 5 µmol/L

359

MnSO4, and 0.4 µg/mL thiamine, pH 7.1) was used to identify auxotrophic mutants.

360

Yeast was cultured in YPD medium (10 g/L yeast extract, 20 g/L peptone, and 20 g/L

361

glucose). YPG medium (10 g/L yeast extract, 20 g/L peptone, and 20 g/L galactose)

362

was used for culturing the yeast in counter-selection process. Synthetic complete

363

medium lacking uracil (SC-Ura) was used to select yeast transformants. Synthetic

364

complete galactose agar lacking uracil plates (SG-Ura) were used for testing the

365

regulation of S. cerevisiae cell growth and death.

366 367

DNA manipulation. To construct the plasmid pWYE3000, two DNA fragments of 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

368

rep101- cmR and f1 origin were amplified by polymerase chain reaction (PCR) and

369

then fused together by overlap PCR to obtain the linear plasmid pWYE3000. The

370

linear plasmid was circularized by DNA ligase in vitro and transferred into EC135 by

371

the calcium chloride technique37. The DNA fragment containing the bceSIIM gene

372

and promoter ParaB was PCR-amplified from pWYE700 and inserted into pWYE3000

373

to generate the plasmid pWYE3001, which was used for genomic modification in E.

374

coli. To obtain the plasmid pWYE3010 for genomic modification in B. subtilis, a PCR

375

fragment of the mcrA gene was inserted into the pHCMC04 plasmid to yield

376

pWYE3008. Then, the replication initiation protein-encoding gene repA was deleted

377

from pWYE3008 by PCR to yield the pWYE3010 plasmid. The recombinant

378

plasmids were constructed by inserting the up- and down-stream homologous arms of

379

the target gene (or/and the inserted or mutated fragments) into the universal genome

380

editing vectors. Transformations of E. coli, B. subtilis, and S. cerevisiae were

381

separately performed as described by Kirill et al38, Zhaopeng Sun et al39 and Gietz et

382

al40.

383 384

Counter-selection efficiencies. After the recombinant plasmid had been integrated

385

into the target strain, positive transformants were subjected to counter-selection by

386

growing them in 5 mL of LB medium for 12 h. Then, the culture was plated on the LB

387

with an inducer after dilution by 105 fold. To calculate the counter-selection

388

efficiency, 156 colonies were randomly selected from the LB plate and subcultured on

389

LB plates with or without antibiotics. Counter-selection efficiency was defined as the 18


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390

ratio of colonies that had excised the counter-selection cassettes to the total number of

391

colonies tested on the LB plate. The calculation formula was as follows:

392

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

393

Number of colonies growing on LB plate with antibiotics)/ Number of colonies

394

growing on LB plate

395 396

Flow cytometry analysis. The gfpmut3a gene encoding an enhanced green

397

fluorescent protein (GFP) was used as a reporter gene. Flow cytometry was performed

398

on a BD FACSCalibur flow cytometer (Becton, Dickinson and Company, the United

399

States). Mutants integrating the gfpmut3a gene were cultured in LB media for 12 h,

400

resuspended to an OD600 of 0.4 using PBS buffer (137 mmol/L NaCl, 2.7 mmol/L

401

KCl, 10 mmol/L Na2HPO4, 2 mmol/L KH2PO4, pH 7.2), and placed on ice prior to

402

analysis. A total of 50,000 events were collected for each sample. E. coli W3110 and

403

B. subtilis W168 were used as controls to determine the background fluorescence.

404 405

Fermentation in shake flasks. The E. coli mutant EC3053 (W3110

406

ygcE-ygcF::PPL-cadA) was cultured in 500-mL shake flasks containing 50 mL of LB

407

media. When the OD600 reached 4.5, 5% (vol/vol) of the culture was seeded into a

408

500-mL baffled shake flask containing 50 mL of fermentation media (20 g/L glucose,

409

10 g/L lysine, 10 g/L (NH4)2SO4, 2 g/L KH2PO4, 2 g/L MgSO4, 15 g/L CaCO3, and 2

410

g/L yeast extract). Throughout the experiment, 2 mL aliquots of the culture were

411

collected to analyze cell growth by a spectrophotometer as well as the cadaverine 19



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412

concentration by HPLC as described by Jie Wu et al8. The wild-type strain W3110

413

was used as the negative control. All experiments and measurements were performed

414

at least in triplicate.

415 416 417

AUTHOR INFORMATION

418

Corresponding Authors

419

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

420 421

Notes

422

The authors declare no financial conflicts of interest.

423 424

ACKNOWLEDGEMENTS

425

The authors would like to thank Tong Zhao (Institute of Microbiology, Chinese

426

Academy of Sciences) for assistance with the flow cytometry analysis. This work was

427

supported by the National Natural Science Foundation of China (31570083 and

428

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

429

(2014AA021203), Science and Technology Service Network Initiative

430

(KFJ-STS-QYZD-047 and KFJ-EW-STS-078), and the Key Deployment Projects of

431

Chinese Academy of Sciences (KGZD-EW-606).

432 433

ASSOCIATED CONTENT

434

Supporting Information 20


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435

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

436

Table S1. The MTases and REases of target strains in this study.

437

Table S2. Counter-selection efficiencies of the genome-scale engineering using the

438

RMGE system.

439

Table S3. Counter-selection efficiencies and mutation efficiencies of the RMGE

440

system in E. coli.

441


442

system in B. subtilis.

443


444

system in S. cerevisiae.

445

Table S6. Bacterial strains and plasmids used in this study

446

Table S7. Primers used in this study.

447

Figure S1. Temperature sensitive assay of the plasmid pWYE3000.

448

Figure S2. Counter-selection efficiencies for the gene-deficient strains in E. coli.

449

Figure S3. Counter-selection efficiencies for point mutation and gene replacement in

450

E. coli.

451

Figure S4. Counter-selection efficiencies for genome editing in B. subtilis by RMGE.

452

Figure S5. The lethal effect detected in the strains that had integrated a single copy of

453

the inducible R-M system in the chromosome.

454

Figure S6. Counter-selection efficiencies for deletion of the CAN1 gene in S.

455

cerevisiae by RMGE

456 457 458 459 460

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restriction-modification systems from Bacillus cereus strains ATCC 10987 and ATCC 14579, J Bacteriol 194, 49-60. 35

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Gietz, R. D., and Woods, R. A. (2002) Transformation of yeast by lithium acetate/single-stranded carrier DNA/polyethylene glycol method, In Guide to Yeast Genetics and Molecular and Cell Biology, Pt B (Guthrie, C., and Fink, G. R., Eds.), pp 87-96, Elsevier Academic Press Inc, San Diego.

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570

Figures legends

571

Figure 1. Schematic illustration of RMGE for genetic manipulation. (a) A schematic

572

illustration of genome editing by RMGE in bacteria with Type IV REase. (b) A

573

schematic illustration of genome editing by RMGE in bacteria without Type IV

574

REase. R represents A or G, Y represents C or T and W represents A or T.

575 576

Figure 2. Construction and functional characterization of the plasmid pWYE3001. (a)

577

Construction of the plasmid pWYE3001. The plasmid pWYE3000 was constructed by

578

removing the sacB gene and a useless DNA fragment (marked in the gray) from the

579

plasmid pKOV. The fragment of the inducible bceSIIM gene was inserted into

580

pWYE3000 to generate the plasmid pWYE3001. (b) The regulation effect of the

581

bceSIIM gene on cell growth/death in E. coli. The strain EC3003 (W3110/pWYE3000)

582

was used as the control.

583 584

Figure 3. Genome editing in E. coli using RMGE- bceSIIM. (a) Schematic illustration

585

of the standard operating procedure for genetic manipulation in E. coli. UHA and

586

DHA represent the up- and down-stream homologous arms, respectively. The

587

asterisks indicate the deletion, replacement, or point mutation. (b) MM plate assay for

588

the deletion of the lysA gene. Due to lacking the lysA gene, the mutant EC3007 cannot

589

grow on the MM plate unless 0.5 g/L lysine has been added. (c) The deletion of the

590

lacZ gene was detected on LB plates with 0.1 g/L X-gal. (d) The deletion of the tolC

591

gene was detected by testing the sensitivity of the mutant EC3048 to SDS. 25



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

592 593

Figure 4. Point mutation and gene replacement using RMGE- bceSIIM. (a)

594

Verification of the mutant strain EC3013 (W3110 rpsLA128C) by testing for

595

streptomycin resistance and DNA sequencing. (b) Flow cytometry analysis of the

596

expression of gfpmut3a in EC3016 (W3110frdA::PPL- gfpmut3a). (c) Detection of

597

cadaverine produced by EC3053 (W3110ygcE-ygcF::PPL-cadA) and W3110 by

598

HPLC. (d) Cell growth and cadaverine production of W3110 and EC3053.

599 600

Figure 5. Design and construction of the plasmid pWYE3010 in B. subtilis. (a)

601

Screening for an effective REase to inhibit cell growth in B. subtilis. The strain W168

602

containing pHCMC04 was used as a control. (b) Construction of the plasmid

603

pWYE3010. The plasmid pWYE3010 cannot replicate in B. subtilis because the rep

604

gene fragment was removed.

605 606

Figure 6. Genome editing in B. subtilis using RMGE- mcrA. (a) Schematic

607

illustration of the standard operating procedure for genetic manipulation in B. subtilis.

608

UHA and DHA represent the up- and down-stream homologous arms, respectively.

609

The asterisks indicate a deletion or replacement. (b) Detection of the amyE gene

610

deletion using starch plates. The strains were cultured on LB plates containing 1%

611

starch for 12 h, and then the plate was stained with iodine. W168 was used as a

612

control. (c) Flow cytometry analysis of the expression of GFP in BS237

613

(W168upp::P43-gfpmut3a). 26


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614

Figure 7. Genome editing in S. cerevisiae using RMGE- mcrA. (a) Screening for an

615

effective REase to regulate cell growth and death in S. cerevisiae. The strain DAY414

616

containing pYES2.0/CT was used as a control. (b) Construction of the plasmid

617

pWYE3026. The plasmid pWYE3026 was generated by removing the 2µ gene

618

fragment from pWYE3023. (c) Detection of the CAN1 gene using synthetic complete

619

medium plates with 60 µg/mL L-canavanine. DAY414 was used as a control.

620

27



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Figure 1. Schematic illustration of RMGE for genetic manipulation. (a) A schematic illustration of genome editing by RMGE in bacteria with Type IV REase. (b) A schematic illustration of genome editing by RMGE in bacteria without Type IV REase. R represents A or G, Y represents C or T and W represents A or T. 107x64mm (300 x 300 DPI)


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Figure 2. Construction and functional characterization of the plasmid pWYE3001. (a) Construction of the plasmid pWYE3001. The plasmid pWYE3000 was constructed by removing the sacB gene and a useless DNA fragment (marked in the gray) from the plasmid pKOV. The fragment of the inducible bceSIIM gene was inserted into pWYE3000 to generate the plasmid pWYE3001. (b) The regulation effect of the bceSIIM gene on cell growth/death in E. coli. The strain EC3003 (W3110/pWYE3000) was used as the control. 240x325mm (300 x 300 DPI)



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Figure 3. Genome editing in E. coli using RMGE- bceSIIM. (a) Schematic illustration of the standard operating procedure for genetic manipulation in E. coli. UHA and DHA represent the up- and down-stream homologous arms, respectively. The asterisks indicate the deletion, replacement, or point mutation. (b) MM plate assay for the deletion of the lysA gene. Due to lacking the lysA gene, the mutant EC3007 cannot grow on the MM plate unless 0.5 g/L lysine has been added. (c) The deletion of the lacZ gene was detected on LB plates with 0.1 g/L X-gal. (d) The deletion of the tolC gene was detected by testing the sensitivity of the mutant EC3048 to SDS. 139x109mm (300 x 300 DPI)


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Figure 4. Point mutation and gene replacement using RMGE- bceSIIM. (a) Verification of the mutant strain EC3013 (W3110 rpsLA128C) by testing for streptomycin resistance and DNA sequencing. (b) Flow cytometry analysis of the expression of gfpmut3a in EC3016 (W3110frdA::PPL- gfpmut3a). (c) Detection of cadaverine produced by EC3053 (W3110ygcE-ygcF::PPL-cadA) and W3110 by HPLC. (d) Cell growth and cadaverine production of W3110 and EC3053. 168x159mm (300 x 300 DPI)



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Figure 5. Design and construction of the plasmid pWYE3010 in B. subtilis. (a) Screening for an effective REase to inhibit cell growth in B. subtilis. The strain W168 containing pHCMC04 was used as a control. (b) Construction of the plasmid pWYE3010. The plasmid pWYE3010 cannot replicate in B. subtilis because the rep gene fragment was removed. 141x112mm (300 x 300 DPI)


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Figure 6. Genome editing in B. subtilis using RMGE- mcrA. (a) Schematic illustration of the standard operating procedure for genetic manipulation in B. subtilis. UHA and DHA represent the up- and downstream homologous arms, respectively. The asterisks indicate a deletion or replacement. (b) Detection of the amyE gene deletion using starch plates. The strains were cultured on LB plates containing 1% starch for 12 h, and then the plate was stained with iodine. W168 was used as a control. (c) Flow cytometry analysis of the expression of GFP in BS237 (W168upp::P43-gfpmut3a). 147x121mm (300 x 300 DPI)



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Figure 7. Genome editing in S. cerevisiae using RMGE- mcrA. (a) Screening for an effective REase to regulate cell growth and death in S. cerevisiae. The strain DAY414 containing pYES2.0/CT was used as a control. (b) Construction of the plasmid pWYE3026. The plasmid pWYE3026 was generated by removing the 2µ gene fragment from pWYE3023. (c) Detection of the CAN1 gene using synthetic complete medium plates with 60 µg/mL L-canavanine. DAY414 was used as a control. 98x54mm (300 x 300 DPI)


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A Novel Tool for Microbial Genome Editing Using the Restriction

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