Engineering B. subtilis ATCC 6051a to raise its natural competence by expressing recombinant comK
Compared with traditional genetic manipulation through circular plasmid mediated homologous recombination, double crossover by homologous fragments exchange through linear DNA eases the handling of genome engineering in prokaryotic cells in terms of efficiency and labor intensity. However, comparing with circular plasmid transformation, homologous integration through linear DNA requires much higher transformation efficiency. Therefore, it was an intimidating task to perform one step gene replacement in B. subtilis ATCC 6051a, a genetically indocile, however industrial favored strains. The alignment analysis using the genomic sequences of B. subtilis ATCC 6051a and B. subtilis 168 revealed a frameshift mutation from AA to A that leads to functional disability of ComP in B. subtilis ATCC 6051a, subsequently reducing the cellular level of ComK [6], one of the key activators modulating natural competence in Bacillus. Overexpression of ComK was proposed and confirmed to improve the transformation efficiency even earlier [9-11, 24, 25]. Thus, we proposed to perform homologous expression of ComK in 6051a by cloning a copy of the native comk gene under a xylose inducible promoter, PxylA from B. megaterium [9], then inserting the cassette into 6051a’s chromosome via double-crossover recombination. However, that could not be directly accomplished duo to host cells’ low transformation efficiency. Therefore, we constructed pMK4-comk, a self-replicating plasmid containing the expression cassette, and transformed the shuttle vector into B. 6051a (as shown in Fig. 1a, more details are shown in Additional file 1: Fig S1). With the lifted transformation efficiency gained through the plasmid mediated expression of ComK, linear comk cassette together with kanamycin resistance marker (kan) was integrated into the genome and replaced native nprE in 6051a (Fig. 1b). Finally, a plasmid expressing Cre integrase was transformed into the bacillus mutant (Fig. S1), creating a markerless strain, which was named as B. subtilis 164S.
Enhanced transformation efficiency upon induction of ComK
The transformation efficiency of B. subtilis 164S was determined by transforming 1.0 μg of pMK4 DNA (circular DNA of 5585 bp in length) to properly prepared competence cells. As the result of induced expression of ComK by culturing cells in medium containing D-xylose, the transformation efficiency reached 3×104 transformants per μg DNA, which increased more than one thousand times over 6051a (data not shown).
To determine the recombination efficiency through double crossover, linear DNAs with varying sizes of homologous fragments flanking four different open reading frames (encoding spoII AC, aprE, amyE and srfAC,respectively, Table 1) were prepared by fusion PCR [26, 27]. The numbers of transformants obtained after transformation into 164S or 6051a using each linear DNA were counted. As seen in Table 1, 164S transformants’ colony numbers in all experiments fell into a range of 70~160, in contrast, no transformant was obtained from 6051a using the same linear DNA (data not shown).
Previous studies postulated that the minimal length of DNA fragments required for homologous recombination was around 400~500 bp [27, 28], it has never been confirmed in industrial bacillus strain such as 6051a. To determine the relationship between the length of the homologous flanking region and the double crossover efficiency, we prepared linear DNAs for in-frame deletion of spoII AC with different sizes of homologous arms (200 to 1000 bp, respectively, Fig. 2a), used them for gene replacement in 164S. As presented in Fig. 2b, linear DNA mediated transformations in 164S were DNA size dependent, longer DNA arms evoked much higher recombination efficiency in 164S, and the minimal length to initiate an efficient DNA exchange event was about 400 bp, that is in agreement with the previous report [27].
Table 1 The efficiency of linear DNA mediated transformation in B. subtilis 164S
Integration
loci
|
Homology length (bp)
|
Transformation efficiencya
|
Upstream
|
Downstream
|
spoII AC
|
1010
|
1012
|
1.6×102
|
aprE
|
619
|
600
|
0.9×102
|
amyE
|
815
|
807
|
1.3×102
|
srfAC
|
928
|
1008
|
0.7×102
|
a Transformation experiments were repeated three times, the transformation efficiency was calculated as the average number of ErmRcolonies formed on plates per μg of linear DNAs.
Seamless cloning method for one step construction of Bacillus expression vector
Seamless cloning is advantageous in preparing faithful DNA constructs. The method takes advantage of restriction-independent cloning strategies, such as overlap PCR, DNA recombination or DNA ligation using type IIs restriction enzymes. Available commercial kits for seamless cloning include Golden Gate cloning technology, Gibson assembly, or In-fusion Cloning. Additionally, an enzyme-free cloning method known as “Simple Cloning” was developed and proved to have high transformation efficiency in B. subtilis SCK6 [24]. According to the report, ~107 transformants per μg of DNA multimer can be achieved. Here we slightly modified the method and developed it as one step seamless method for the construction of desired plasmids. As demonstrated in Fig. 3a, the backbone plasmid was linearized by restriction enzyme, it joined with the insert through overlap PCR using primers without restriction site sequences that originally existed on the plasmid DNA; during PCR program, the unmatched sequences would be automatically trimmed by 3'- 5' exonuclease of high-fidelity DNA polymerase; after transformation into B. subtilis cell, linear DNA multimers generated by PCR trigger efficient in vivo splicing that leads to formation of circular DNA [24].
Construction of pMK4-T7-abfA1 demonstrated the feasibility of the novel cloning strategy. First, pMK4-T7 was digested with BamHI to give linear double strand DNAs that were used in PCR reaction for preparation DNA multimers, the PCR system also included PCR prepared abfA1 fragment containing homologous ends to the vector DNA, however without BamHI residue site that originally existed immediate downstream of T7 promoter on pMK4-T7; PCR generated multimers were subsequently transformed into 164S, that promptly produced transformants harboring circularized pMK4-T7-abfA1. As shown in Fig. 3b, the DNA sequencing confirmed BamHI site was eliminated on generated pMK4-T7-abfA1. The transformation efficiency can be over 1.0×106 transformants per μg of DNA multimer, which was lower than the report [24] but sufficient for most strain engineering jobs.
Construction of inducible T7 expression system in B. subtilis
T7 RNA polymerase over-expressing strains, such as BL21 (DE3) or JM109 (DE3), were required for T7 promoter guided robust expression in E. coli, for the same reason, T7 RNA polymerase has to be expressed in B. subtilis if T7 promoter is recruited for transcription of target genes [20-22]. However, most bacterial systems utilize IPTG as the expression switch, however, IPTG is costly and unhealthy if not removed from the products, thereby limiting the system’s application in the food or pharmaceutical field [29]. In this study, a D-xylose inducible T7 expression system was constructed. As demonstrated in Fig. 4a, we first assembled an artificial cassette encoding T7 RNA polymerase under the inducible promoter PxylA, delivery of the linear DNA into 164S knocked out aprE in 164S and replaced it with the DNA cassette expressing T7 RNA polymerase. A copy of xylR was included in the cassette to stop the “leaky expression” of the polymerase when D-xylose is not available. 164T7P was tested in the expression of GFP (cloned in plasmid pMK4-T7-gfp, as seen in Additional file 2: Fig S2). Since P43 was recognized as a strong and constitutive promoter in B. subtilis [12, 30], a 164T7P control strain harboring the plasmid pMK4-P43-gfp (Additional file 2: Fig S2) was made for comparison between PxylA and P43. To examine GFP expression levels, the average fluorescence intensity in cells grown upon the presence of different concentrations of D-xylose was determined in a microplate reader. As shown in Fig. 4b, the level of heterologous expressed GFP in 164T7P correlated with the concentration of D-xylose supplemented in mediums (Fig. 4b and Fig. 4c). It showed that in medium with 1.0% D-xylose, the fluorescence intensity of GFP expressed by T7 promoter reached 2.83×107 au, which is more than 13 times of that achieved by P43 promoter (2.08×106 au) (Fig. 4c, Additional file 3: Fig. S3), besides, in the absence of xylose (Fig. 4b, Fig. 4c), recombinant B. subtilis cells exhibited very low level of background expression of GFP, emitting fluorescence equivalent of only 1% that induced level, whereas, lacO/IPTG inducible control system in B. subtilis displayed a rather high level of leaky expression [23].
Heterologous expression and characterization of AbfA1
L-arabinosyl residues are widely distributed in hemicelluloses as one of the main side chains. Therefore, α-L-arabinofuranosidase (EC 3.2.1.55) is indispensable for the full degradation of polysaccharide xylan into xylose [31-33]. Through preliminary search and screening (data not shown), an ORF (abfA1) from the B. licheniformis ATCC 14580 encoding putative abfA1 was found and cloned into the plasmid pMK4-T7 using Simple and Seamless method, in vivo generated plasmid pMK4-T7-abfA1 rendered B. subtilis 164T7P ability of producing arabinofuranosidase (AbfA1) using mRNA transcribed by inducible T7 expression system. For full induction, 10 g L-1 of D-xylose was supplemented into the culture growing at 37ºC. After the cells were spun down, 10 μL of supernatant was analyzed by SDS-PAGE. As seen in Fig. 5a, a high level of α-L-arabinofuranosidase was secreted out of host cells, with an estimated mass of 56.9 kDa. The total protein concentration in the supernatant was measured to be 0.8 g L-1 in Bradford assay, and the recombinant AbfA1 represented more than 90% of the total protein in the supernatant(Fig. 5a). The activity and the kinetic parameters of AbfA1 were determined using pNPA (4-nitrophenyl α-L-arabinofuranoside) as the substrate. The maximum activity during fermentation was measured to be 90.6±2.0 U mL-1 at 52 h (Fig. 5b), with a Km value of 1.4±0.1 mM and the kcat value of 139.4 s−1. The enzyme remained to be active in the pH range of 3.5-9.0 with an optimal pH at 6.5 (Fig. 5c). Its optimal temperature was determined to be 45 ºC while working between 25-55 ºC (Fig. 5d). This is by far the first report on the heterologous secretion expression of α-L-arabinofuranosidase in B. subtilis. It is worth mentioning that the protein sequence of AbfA1 showed neither Sec- nor Tat- signal peptide when analyzed by SignalP-5.0 Server [34]. And, even the score of the prediction of non-classical protein secretion on AbfA1 is rather low (<0.5) [35], we still speculate that it is secreted through a non-classical pathway when expressed in B. subtilis [36].