Zhang Shuang, Luo Chao-chao, Wu Cai-xia, and Gao Xue-jun*

1Key Laboratory of Agricultural Biological Functional Genes, Northeast Agricultural University, Harbin 150030, China

2Life Science Research Center, Hebei North University, Zhangjiakou 075000, Hebei, China

Construction and Expression of Methionine-rich and Lysine-rich Fusion Gene in Bacillus natto

Zhang Shuang1, Luo Chao-chao1, Wu Cai-xia2, and Gao Xue-jun1*

1Key Laboratory of Agricultural Biological Functional Genes, Northeast Agricultural University, Harbin 150030, China

2Life Science Research Center, Hebei North University, Zhangjiakou 075000, Hebei, China

Methionine and lysine are restrictive essential amino acids of livestock, they are also the most attentive indexes in the feed production to carry out the quality control and quality evaluation. Their contents in feed directly affect livestock protein synthesis. Bacillus natto has excellent probiotic properties. In this experiment, we used the genetic engineering method, fusion PCR technique, to connect methionine-rich gene (zein) from maize endosperm protein with lysine-rich gene (Cflr) from the pepper anther, then the fusion gene was inserted into the expression vector pHT43, and the recombinant plasmid pHT43/zein-Cflr was constructed. The recombinant plasmid was transferred into Bacillus natto, and induced by IPTG for the expression of the fusion gene. We found an apparent band at 40 ku site for the recombinant strain by SDS-PAGE. The contents of methionine and lysine were individually detected with HPLC, the quantities of methionine and lysine in the recombinant strain increased by 18.37% and 24.68% than the wild one, respectively. We also verified the stability of the recombinant bacterium during passaging, and found the stability was 100%. This study provided research-basis for the application of the recombined Bacillus natto as feed additive.

methionine-rich gene, lysine-rich gene, Bacillus natto, prokaryotic expression

Introduction

Probiotics derived from microorganisms have been widely applied as an additive by food and feed industries, they provide the consumer with one or more proven health benefits (Vandenplas et al., 2014). Bacillus natto is a gram-positive spore-forming bacterium, and belongs to Bacillus subtilis. It has been included in the catalogue of the feed-grade microbes as additives of the Department of Agriculture of China (Jenny et al., 1991; Murata et al., 2013). Directfed Bacillus natto has been shown to increase the animal performance, promote growth, alter intestinal histology, and modulate immune function. Probiotics appearing in alimentary duct can increase the immunological response and improve feed conversion rate, antimicrobial effect and growth performance (Sun et al., 2011; Sun et al., 2010).

Methionine (Met) is usually the first limiting amino acid in many animal diets, especially those containing high levels of plant protein sources which have less Met content (Amira et al., 2005). Thus, adding Met in animal diets can improve the utilization of plant protein, increase the absorption rate of the vegetable protein which helps to reduce feed costs in animalfeeding, improve feed utilization and optimize growth performance (Yuan et al., 2011). The use of the probiotic fermentation production with high Met content as a feed additive has been reported (Gomes and Kumar, 2005). Also, in the feed industry, lysine (Lys) is required as a feed additive for livestock and poultry in great demand (Li et al., 2013). Lys as a feed additive is gained mostly by the fermentative production and chemical synthesis. By the chemical synthesis, large-scale production of Lys is generally achieved, but the technology is complicated, multiproduct, expensive and cannot be directly added. The fermentative production has more efficiency than chemical synthesis (Kircher and Pfefferle, 2001), and the production of the probiotics is green, nontoxic, non-polluting, safe and reliable, it also can feed directly. In this experiment, we constructed a fusion gene from Lys-rich and Met-rich gene from plants, and transformed this fusion gene into Bacillus natto, then verified whether Bacillus natto could express the target protein. This study provides a technical basis for the future application of the recombinant bacterium as a feed additive.

Materials and Methods

Met-rich gene, Lys-rich gene, strains, plasmids and media

Maize endosperm was obtained from the farm of Northeast Agricultural University (China). Pepper anther, E. coli top10 and Bacillus natto (CCTCC 92068), plasmid pHT43 were conserved in the Key Laboratory of Agricultural Biological Functional Genes, Northeast Agricultural University. LB medium: 1% w/v peptone and NaCl, 0.5% w/v yeast extract (pH 7.0). Antibiotics were added into the medium when it was appropriate (ampicillin at 100 ug ? mL-1and chloramphenicol at 5 ug ? mL-1). Bacillus natto transformation medium: 10×Spizizen (Sp) Salts (100 mL): 2% w/v (NH4)2SO4, 14% w/v K2HPO4, 6% w/v KH2PO4, 0.2% w/v MgSO4? 7H2O, and 1% w/v sodium citrate. GM I medium: 1×Sp Salts 95.6 mL, 20% glucose 2.5 mL, 10% yeast extract 1 mL, 5% caseamino acids 0.4 mL, water to 100 mL. GM II medium: 1×Sp Salts 96.98 mL, 20% glucose 2.5 mL, 10% yeast extract 0.04 mL, 5% caseamino acids 0.08 mL, 1 mol ? L-1MgCl20.25 mL, 1 mol ? L-1CaCl20.05 mL, water to 100 mL.

Main enzymes and chemicals

PrimeSTAR? HS DNA Polymerase, DNA Marker, Protein Marker, dNTP, Taq DNA polymerase, restriction enzymes, and T4-DNA ligase were purchased from TaKaRa Biotechnology Company (Dalian, China); Trizol from Invitrogen; DNA purification kit and plasmid extraction kit from Axygen; IPTG, ampicillin, chloromycetin, 2, 4-dinitrochlorobenzene and all the chemicals used were of the analytical grade and were obtained from Shanghai Sangon Biological Engineering Technology & Services Company.

PCR and cloning

Total RNA was isolated from maize endosperm or pepper anther using Trizol reagent. The procedure of the reverse transcription was done using a PrimeScript RT-PCR Kit according to the instructions of the manufacturer (Invitrogen).

According to the published sequences of maize Met-rich protein zein mRNA (NCBI accession number, AF371266.1) and pepper anther Lys-rich protein Cflr mRNA (NCBI accession number, EU367999), two pairs of DNA oligonucleotide primers were designed and synthesized by Invitrogen Biotechnology (Shanghai, China). To acquire zein-Cflr, we used the fusion PCR method. DNA of Cflr was cloned using cDNA as the template and the primers 1 and 2 mentioned above. DNA of zein was cloned using cDNA as the template and the primers 3 and 4 mentioned above. Then acquired zein-Cflr using the two products as the template and primers 1 and 4. PCR cycles comprised an initial denaturation step at 98℃ for 3 min, followed by 35 cycles of 98℃ for 10 s, 57.8℃ for 10 s, 72℃ for 10 s, finally, 72℃ for 10 min. The amplified PCR products were analyzedby 1% agarose gel electrophoresis. The primers 1 and 4 were designed to incorporate into the restriction enzyme sites BamHⅠand AatⅡat their 5' ends (restriction sites were underlined in the primers). Primers were as the followings:

1 : 5-CGGGATCC AAAGATGGGTTGTGGGGAA T-3 (BamHⅠ)

2 : 5-CATCTTGGCTGCCATTCCTCCTGAACCA CCATCTGTTTTCAAATCTGTT-3

3 : 5-CAGGAGGAATGGCAGCCAAGATGCTTG CATT-3

4 : 5-TCGACGTCCTATCTAGAATGCAGCACCA AC-3 (AatⅡ).

Construction of expression plasmid

PCR products were double-digested by BamHⅠand AatⅡand ligated to plasmid pHT43 digested by the same restriction enzymes and the recombination plasmid pHT43/zein-Cflr was constructed. The ligation reaction was transformed into E. coli Top 10. Plasmid DNA was isolated and verified by DNA sequencing (Comate Jilin, China). The nucleotide sequence comparison was performed using the facilities of the blast server at NCBI.

Transfection into Bacillus natto

Plasmids pHT43/zein-Cflr were isolated from E. coli Top 10 by plasmid extraction kit. We developed a new protocol to prepare Bacillus natto competent cells based on the methods reported by simple, fast and high-efficiency transformation system for directed evolution of cellulase in Bacillus subtilis (Zhang and Zhang, 2011).

Detection of zein and Cflr gene expressions by SDS-PAGE

Bacillus natto containing pHT43/zein-Cflr was streaked on a nutrient agar plate supplemented with chloramphenicol (5 μg ? mL-1), followed by incubation at 37℃ overnight. A single colony was inoculated into 5 mL of LB at 37℃ with shaking at 200 r ? min-1for 12 h. The inoculated medium was then transferred to 30 mL of LB at a dilution factor of 5%, followed by incubation at 37℃ with shaking at 200 r ? min-1. When OD600reached 0.6-0.8, protein production was induced by IPTG (1 mmol ? L-1) at 37℃ for 2, 12 and 24 h. The samples were loaded onto the gradient polyacrylamide gel made of 5% stacking gel and 12% separating gel as described by Molecular Cloning. Fixative was used to fix the protein gel for 30 min after electrophoresis, then the gels were stained in Coomassie blue and destained in a 40% methanol and 10% acetic acid solution to watch the band of the product.

Met and Lys contents detected by HPLC in Bacillus natto

For HPLC assay, the standard samples of Met and Lys were hydrolyzed according to the acid hydrolysis method of GB/T 18246-2000, and the mixture of amino acids was obtained. The amino acid mixture reacted with 2, 4-dinitrochlorobenzene to make Met into dinitrochlorobenzene-Met. The product was filtrated through a 0.45 μm filter membrane. The chromatographic system used was equipped with a 20 uL loop and UV detector. To detect Met, separation was performed in a 150×4.6 mm reversed-phase C18 analytical column. The mobile phase was made of 30% methyl cyanides in the sodium acetate buffer (pH 5.2). The flow rate was 0.8 mL ? min-1and the analytical wavelength was 350 nm. Determination of Lys content was the same as Met. The amino acid mixture was reacted with 2, 4-dinitrochlorobenzene to make Lys into dinitrochlorobenzene-Lys. The mobile phase was made of 25% methanol in the sodium acetate buffer.

Plasmid stability in recombinant Bacillus natto

The transformation of the plasmid vector containing exogenous genes into Bacillus natto always brings a series of physiological burdens affecting plasmid stability. Plasmid stability was measured by calculating the ratio between the number of the colony forming units on the plate with the antibiotic and on the plate without the antibiotic. A recombinant strain of Bacillus natto containing a pHT43/zein-Cflr was grown for 30generations under selective conditions in LB plate with or without the chloramphenicol (5 μg ? mL-1) continuous culture. The rate of the recombinant plasmid stability was counted.

Curves of growth of Bacillus natto and recombinant

OD values at a wavelength of 630 nm of Bacillus natto and Bacillus natto with pHT43/zein-Cflr liquid culture medium were measured by using a spectrophotometer from 2 h to 22 h after these bacteria were incubated.

Results

Gene cloning and transfection of recombinant plasmids into Bacillus natto

Zein (476 bp) fragment was cloned from maize endosperm. Cflr (720 bp) was cloned from pepper anther. zein-Cflr (1 141 bp) was obtained by fusion PCR (Fig. 1A). The recombinant plasmid pHT43/ zein-Cflr (9 198 bp) was successfully constructed by restriction enzyme analysis and nucleotide sequencing. The recombinant clone was confirmed by BamHⅠ and AatⅡ restriction enzyme analyses and nucleotide sequencing of plasmid extracted (Fig. 1B).

Fig. 1　APCR products of zein-Cflr gene

Fig. 2　SDS-PAGE detection of zein-Cflr in Bacillus natto

Detection of zein and Cflr gene expressions by SDS-PAGE

The expression of the recombinant protein was induced by IPTG in supernatant. A single 40 ku protein band was invisible in SDS-PAGE for the induced culture (Fig. 2).

Met and Lys contents detected by HPLC

The standard curve of Met was experimentally calculated from the peak capacity by the average (Fig. 3A). Fig. 3B showed a HPLC profile of metionine standard sample, the column hold-up time was 3.319 min. Fig. 3A showed the standard curve of Lys. HPLC results of Lys standard liquid of 80 μg ? mL-1is shown in Fig. 4B, and it showed the retention time was 9.709 min. The contents of Met and Lys were individually detected with HPLC, and the quantities of Met and Lys in the recombinant strain were increased by 18.37% and 24.68% than those of the wild one, respectively.

Fig. 1 B　Identification results of recombinant plasmid with endonuclease reaction

Fig. 3　Contents of Met detected by HPLC in fermentation broth of bacterium

Fig. 4　Contents of Lys detected by HPLC in fermentation broth of bacterium

Detection of plasmid stability in Bacillus natto

Bacillus natto and the recombinant strain of Bacillus natto containing a pHT43/zein-Cflr was grown for 30 generations under selective conditions in LB plate with or without the chloramphenicol (5 μg ? mL-1) continuous culture. The experimental data showed that all of them had a good stability, and all the rates of the plasmid stability were 100% (Fig. 5).

Growth curves of Bacillus natto and recombinant

Fig. 6 showed the growth curves of Bacillus natto and the recombinant, there was almost no difference between this two strains. The quantity of the recombinant was gently higher than that of Bacillus natto in the exponential phase. These experimental data showed that pHT43/zein-Cflr had almost no influence of physiological burdens on Bacillus natto.

Fig. 5　Plasmid stability test for recombinant plasmid

Fig. 6　Growth curve

Discussion

10-ku-zein gene was a maize storage protein in which about 25% of total amino acids contain sulphur. The protein that was rich in Met was an essential amino acid in livestock fodder (Kirihara et al., 1988). The gene was already transferred into tobacco and Sweetpotato (Randall et al., 2000). Another reported Lys-rich protein genes mainly included Cflr, tsb and wblrp, and Lys content in the protein expressed by Cflr was the highest. It had been successfully introduced into wheat, maize and rice plants and proved to increase the contents of Lys (Sun et al., 2010; Gao et al., 2001). Fusion PCR method was used to acquire zein-Cflr gene in this study.

Bacillus natto as a heterologous expression strain, which was not familiar to us. Hence, whether Metrich and Lys-rich protein genes could be expressed in Bacillus natto and raising its Met and Lys contents was not known. Bacillus natto belonged to Bacillus subtilis, and PHT43 was a vector of Bacillus subtilis. So it was good for heterologous gene transfer into Bacillus natto. This was the first time to transfer Lys-rich and Metrich protein genes from plants into Bacillus natto.

The sample of induced culture was detected by SDS-PAGE and the recombinant protein band had been found. The quantities of Met and Lys in the recombinant strain increased by 18.37% and 24.68% than those of the wild one, respectively. So this study provided research-basis for the application of the recombined Bacillus natto as a feed additive.

Conclusions

In this study, zein and Cflr genes were cloned from maize endosperm and pepper anther, respectively. Therecombinant plasmid pHT43/zein-Cflr was constructed by fusion PCR and had a good plasmid stability, then it was expressed significantly in Bacillus natto, and the contents of Met and Lys increased by 18.37% and 24.68% than those of the wild one, respectively.

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Q819 Document code: A Article ID: 1006-8104(2015)-02-0022-07

10 March 2015

Supported by the Funding of High Technology Project of Ministry of Science and Technology of China (863 Project, 2013AA102504-03)

Zhang Shuang (1991-), female, Master, engaged in the research of genetically modified microorganisms. E-mail: zhuimengzs@163.com

. Gao Xue-jun, professor, supervisor of Ph. D student, engaged in the research of animal biotechnology. E-mail: gaoxj5390@ sina.com