Bacterial strains, plasmids, and growth conditions
Bacterial strains and plasmids used in this study are listed in Additional file 1: Table S1. Escherichia coli DH5α was used as a host for cloning and plasmid preparation. Bacillus subtilis 1A751, which is deficient in two extracellular proteases (nprE, aprE), served as the parental strain. The plasmid pMA5 is an E. coli/B. subtilis shuttle vector and used to clone and express protein. The plasmids p7Z6 containing lox71-zeo-lox66 cassette and p148-cre containing cre expression cassette were used for the knockout of target gene. Transformants of E. coli and B. subtilis were selected on Luria–Bertani (LB) agar [1% (w/v) peptone, 0.5% (w/v) yeast extract, 1% (w/v) NaCl, and 2% (w/v) agar], supplemented with ampicillin (100 μg/ml), zeocin (20 μg/ml), or kanamycin (50 μg/ml) depending on the plasmid antibiotic marker. E. coli DH5α was incubated in LB medium supplemented with ampicillin (100 μg/ml) at 37 °C. Bacillus subtilis was cultivated in SR medium [1.5% (w/v) peptone, 2.5% (w/v) yeast extract, and 0.3% (w/v) K2HPO4, pH 7.2] containing additionally kanamycin (50 μg/ml) or zeocin (20 μg/ml) at 37 °C. All of the strains were incubated under a shaking condition at 200 rpm. Except the fed-batch fermentation, all of the experiments were repeated at least 3 times, and mean values were used for comparison.
Primers and oligonucleotides
Polymerase chain reaction (PCR) primers and oligonucleotides used in this study were synthesized by GENEWIZ (Suzhou, China) and listed in Additional file 1: Table S2.
PCRs were performed using PrimeSTAR Max DNA Polymerase (TaKaRa, Japan). DNA fragments and PCR products were excised from a 0.8% agarose gel and purified by E.Z.N.A.™ Gel Extraction Kit (200) (Omega Bio-tek, Inc., USA) according to the manufactures’ instruction. E.Z.N.A.™ Plasmid Mini Kit I (Omega Bio-tek, Inc., USA) was applied for plasmid extraction according to the manufactures’ instruction. Genomic DNA isolation was carried out by TIANamp Bacteria DNA Kit (TIANGEN BIOTECH (BEIJING) CO., LTD., China). All the DNA constructs were sequenced by GENEWIZ (Suzhou, China).
Construction of the dal deletion mutant
The deletion of the alanine racemase gene dal in B. subtilis was performed using Cre/lox system as described previously (Yan et al. 2008; Dong and Zhang 2014). The two flanking fragments upstream and downstream (~1 kb) of the dal gene were amplified using genomic DNA from B. subtilis 168 as template and UP-F/UP-R and DN-F/DN-R as primers, respectively. The lox71-zeo-lox66 cassette (~0.5 kb) was amplified from the plasmid p7Z6 using the primers lox-F and lox-R. Then, the flanking fragments and the lox71-zeo-lox66 fragment were fused together by splicing by overlap extension PCR (SOE-PCR) using the primers UP-F and DN-R. Subsequently, the fused fragment was transformed into B. subtilis 1A751. Selection of the double crossover mutant (B. subtilis 1A751D1), marker excision of by Cre-dependent recombination of the lox-sites, and selection of the dal deletion mutant (B. subtilis 1A751D2) were performed by the previous strategy (Yan et al. 2008). Finally, the entire dal gene was thus successfully deleted via double crossing over and marker excision in the chromosome of B. subtilis 1A751, which was further confirmed by PCR amplification.
Construction of plasmids
The food-grade expression plasmid was constructed based on pMA5 by replacing the zeocin resistance gene zeo with the alanine racemase gene dal from B. subtilis 168 using a sequence-independent method named “simple cloning” developed by Chun You (2012). Based on the nucleotide sequence of dal, the primers dal-F/dal-R were designed to amplify the fragment dal using the B. subtilis 168 as the template. The linear vector backbone was amplified using the primers pMA5-F1 and pMA5-R1 as the primers and the plasmid pMA5 as the template. Dal-F/dal-R had the reverse complementary sequences of pMA5-F1/pMA5-R1, respectively. Then, the DNA multimer was generated based on these DNA templates by prolonged overlap extension PCR (POE-PCR). Eventually, the POE-PCR products (DNA multimer) were directly transformed into competent E. coli DH5α, yielding the recombinant plasmid pMA5-DAL. Likewise, the rdpe gene from pET-RDPE was inserted into the plasmid pMA5-DAL downstream of the promoter P
HpaII, resulting into the recombinant plasmid pMA5-DAL-RDPE.
Stability of the recombinant plasmids
The evaluation of the stability of the plasmid pMA5-DAL-RDPE was conducted using the method described by Nguyen (2005). The recombinant strains were inoculated onto Plate A (LB agar plate without supplement of d-alanine with selection pressure) and Plate B (LB agar plate with supplement of 200 μg d-alanine/ml without selection pressure). Colony numbers of the strain 1A751D2R on Plate A and Plate B were named as C
A and C
B. The value of C
B was regarded as the stability of the plasmid pMA5-DAL-RDPE at the certain generation of cultivation.
Fed-batch fermentation in 7.5 l fermentor
The food-grade RDPE production in B. subtilis 1A751D2R was evaluated in 7.5 l BIO FLO 310 fermentor (New Brunswick Scientific co Inc., USA) with a fed-batch strategy. The airflow rate was 6.0 l/min, and dissolved oxygen tension was maintained between 20 and 40% air saturation by automatic adjustment of speed of the stirrer. The temperature was kept at 37 °C and the pH was controlled at pH 7.2. Foam was controlled by the addition of a silicone-based anti-foaming agent. The fermentation medium was SR medium. The fermentation was performed with an initial working volume of 3.5 l. When the cell growth rate became constant, the substrate fed-batch mode was started by adding 8.0% soluble starch at a constant flow rate, until the final concentration of soluble starch was up to 4.0%. Cell growth was monitored by measuring dry cell weight of the fermentation broth. The activity of RDPE was determined by measuring the supernatant of broth.
The RDPE activity was analyzed by determining the amount of d-psicose obtained from d-fructose. One milliliter of reactions mixture contained d-fructose (20 g/l) in sodium phosphate buffer (50 mM, pH 8.0) and 200 μl crude enzyme. The reaction was incubated at 55 °C for 10 min,following by boiling at 100 °C for 10 min. The obtained d-psicose in the mixture was determined via high-performance HPLC system with a refractive index detector and a Sugar-PakTM column (6.5 mm × 300 mm; Waters), which was eluted with ultrapure water at 80 °C and 0.4 ml/min. One unit of DPEase activity is defined as the amount of enzyme that catalyzed the production of 1 μmol d-psicose per minute. For the determination of extracellular enzyme activity, the crude enzyme was the supernatant of fermentation broth. For the determination of intracellular enzyme activity, the cells need to be broken. Bacillus subtilis cells expressing RDPE were harvested from the culture broth by centrifugation at 6000×g for 10 min at 4 °C. The cells were then suspended in lysis buffer (25 mM Tris/HCl, 300 mM NaCl, and 40 mM imidazole, pH 8.0). The suspended cells were disrupted using a high-pressure homogenizer (APV, Denmark) at 900–1000 bar. The supernatant was obtained by centrifugation at 15,000×g for 30 min at 4 °C and filtration through a 0.45 μm filter. Then, the crude extract was applied in the enzyme assay.
Culture samples (1 ml) were harvested and the supernatant was separated from the culture medium by centrifugation (12,000g, 10 min, 4 °C). After adding 5× SDS-PAGE sample buffer, the supernatants were boiled for 10 min, and proteins were separated in SDS-PAGE using the NuPAGE 10% Bis–Tris Gel (Novex by Life Technologies, USA) in combination with MOPS SDS Running Buffer (Invitrogen Life Technologies, USA). PageRuler Prestained Protein Ladder (Invitrogen Life Technologies, USA) was used to determine the apparent molecular weight of separated proteins. Proteins were visualized with Coomassie Brilliant Blue.