Computational protocol: Metabolic engineering of Bacillus subtilis for growth on overflow metabolites

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Protocol publication

[…] Chromosomal DNA of Bacillus licheniformis MW3, which is a mutant of B. licheniformis DSM13 [], was used as template for the amplification of the aceBA-operon. Sequence information was derived from the published genome of B. licheniformis DSM13 [GenBank:NC_006322]. PCRs were performed with the Phusion polymerase. The utilized oligonucleotides are summarized in Table , while the constructed plasmids and used strains are listed in Table .The plasmid pACEBA was constructed by amplifying the aceBA-operon without its predicted promoter region and the hypothetical protein. The PCR product and the pBGAB [] plasmid were cut with XbaI and BamHI and ligated using T4-ligase. To integrate the native promoter and the hypothetical protein in front of the aceBA-operon, the pBGAG plasmid was cut with SmaI and NotI and an erythromycin cassette from pMTL500 [] was inserted yielding the plasmid pAMYSSE. Hereafter, this plasmid was cut with NarI and EcoRI and the PCR product resulting from the Prom1-primer pair was integrated via a modified sequence and ligation-independent cloning method []. Electroporation of the plasmids into E. coli was performed as previously described []. All utilized enzymes were purchased from New England Biolabs (Frankfurt a. M., Germany), oligonucleotides were synthesized by Life Technologies (Darmstadt, Germany).To obtain an aceBA-encoding strain, B. subtilis 60-51HGW was transformed via natural competence [] using the linearized plasmid pACEBA. The clones were selected on neomycin and the positive mutants were designated as B. subtilis ACE-P. To functionalize the operon, pProm1 was transformed in the same manner into B. subtilis ACE-P with erythromycin selection resulting in B. subtilis ACE. To examine the expression of a reporter enzyme, B. subtilis ACE and B. subtilis 6051HGW were transformed with pSacAmyE yielding a wild type (B. subtilis WTamyE) and a glyoxylate cycle - positive mutant (B. subtilis ACEamyE) that express and secrete an alpha-amylase under control of the acoA-promoter []. pSacAmyE was constructed by cutting pJK168 and pMJS2 [] with XbaI and ligating the acoA-amyE fragment from pMJS2 into the sacA landing pads of pJK168. To ensure comparable alpha-amylase measurements, the native alpha-amylase of B. subtilis was disrupted by the integration of the aceBA-operon (B. subtilis ACEamyE) and an erythromycin cassette of plasmid pAMYSSE (B. subtilis WTamyE) into the amyE-locus. All plasmid constructs and chromosomal integrations were verified by sequencing.The publicly available genome sequence of B. licheniformis was annotated in 2004 []. Since then, a large amount of novel sequences and annotations have been published, which implicates the necessity to update previous functional assignments for organisms of interest. Accordingly, we reannotated the genome sequence of B. licheniformis DSM13 in the same manner as described for B. subtilis 6051HGW []. In short, the GenBank file of B. licheniformis DSM13 [GenBank:AE017333] was imported into the GenDB 2.2 [] pipeline and the resulting database was used to analyze the ORFs with JCoast []. [...] The supernatant required for the alpha-amylase reporter enzyme assay was obtained by centrifugation of 2 ml culture at 10000 x g for 5 min at 4°C. The resulting supernatant was transferred to a new tube and stored at -20°C until further processing. The amylase activity was determined by use of the Ceralpha Assay (Megazymes, Wicklow, Ireland). To stay within the linear range, 1:200 dilutions in the suggested buffer were prepared. Measurements were done with a Tecan infinite M200 plate reader.For the cytosolic proteome analysis, 8 ml of cells were harvested by centrifugation and washed twice with TE-buffer (10 mM Tris-Cl, pH 7.5, 1 mM EDTA) and finally resuspended in 1 ml TE with 2 mM PMSF (Carl Roth, Karlsruhe, Germany). Samples were transferred to cryotubes (Sarstedt, Nümbrecht, Germany) filled with 0.25 ml glass beads (Sartorius AG, Göttingen, Germany) and the cells were disrupted by bead-beating for 30 seconds at 6.5 m/s. After cooling for 5 min on ice, this procedure was repeated twice. Cell debris was removed by 30 min centrifugation at 17000 x g. The supernatant was stored at -20°C. 5 μg of protein mixture were separated by SDS-PAGE [] and stained with silver-blue Coomassie [].The bands were excised using a sterile scalpel and transferred into 96 well micro titer plates (Greiner bio one, Frickenhausen, Germany). The tryptic digest with subsequent spotting on a MALDI-target was carried out automatically with the Ettan Spot Handling Workstation (Amersham Biosciences, Uppsala, Sweden). The gel pieces were washed twice with 100 μl of a solution of 50% CH3OH and 0% 50 mM NH4HCO3 for 30 min and once with 100 μl 75% CH3CN for 10 min. After drying at 37°C for 17 min 10 μl trypsin solution containing 4 μg/ml trypsin (Promega, Madison, WI, USA) was added and incubated at 37°C for 120 min. For extraction gel pieces were covered with 60 μl 0.1% TFA in 50% CH3CN and incubated for 30 min at 40°C. The peptide-containing supernatant was transferred into a new microtiter plate and the extraction was repeated with 40 μl of the same solution. The supernatants were completely dried at 40°C for 220 min. The dry residue was resuspended in 0.9 μl α-cyano-4-hydroxy-cinnamic acid matrix (3.3 mg/ml in 50%/49.5%/0.5% (v/v/v) CH3CN/H2O/TFA) and 0.7 μl of this solution was deposited on the MALDI target plate. The samples were allowed to dry on the target 10 to 15 min before measurement by MALDI-TOF mass spectrometry.The MALDI-TOF measurement was carried out on the AB SCIEX TOF/TOF™ 5800 Analyzer (ABSciex /MDS Analytical Technologies, Darmstadt, Germany). This instrument is designed for high throughput measurement, being automatically able to measure the samples, calibrate the spectra and analyze the data using the TOF/TOF™ Series Explorer™ SoftwareV4.1.0. The spectra were recorded in a mass range from 900 to 3700 Da with a focus mass of 1700 Da. For one main spectrum 25 sub-spectra with 100 shots per sub-spectrum were accumulated using a random search pattern. If the autolytical fragment of trypsin with the mono-isotopic (M + H) + m/z at 2211.104 reached a signal to noise ratio (S/N) of at least 40, an internal calibration was automatically performed as one-point-calibration using this peak. The standard mass deviation was less than 0.15 Da. If the automatic mode failed (in less than 1%), the calibration was carried out manually. The five most intense peaks from the TOF-spectra were selected for MS/MS analysis. For one main spectrum 20 sub-spectra with 125 shots per sub-spectrum were accumulated using a random search pattern. The internal calibration was automatically performed as one-point-calibration with the mono-isotopic arginine (M + H) + m/z at 175,119 or Lysine (M + H) + m/z at 147,107 reached a signal to noise ratio (S/N) of at least 5. The peak lists were created by using GPS Explorer™ Software Version 3.6 (build 332) with the following settings for TOF-MS: mass range, 900–3700 Da; peak density, 20 peaks per 200 Da; minimum S/N ratio of 15 and maximal 65 peaks per spot. The TOF-TOF-MS settings were a mass range from 60 to Precursor - 20 Da; a peak density of 50 peaks per 200 Da and maximal 65 peaks per precursor. The peak list was created for an S/N ratio of 10. All peak lists were analysed using Mascot search engine version 2.4.0 (Matrix Science Ltd, London, UK) with a specific user sequence database and specific B. subtilis and B. licheniformis databases. [...] Graphs were created using R [] and, where applicable, with the ggplot2 package []. Graphs for metabolites were created with VANTED V2.1.3 []. Genome analysis was done with Geneious version 5.6.2 created by Biomatters (Auckland, NZ) available from http://www.geneious.com. Free Gibbs-Energy (-∆G) of mRNA structures was determined with UNAfold []. […]

Pipeline specifications

Software tools Mascot Server, Ggplot2, VANTED, Geneious, UNAFold
Applications Miscellaneous, MS-based untargeted proteomics
Organisms Bacillus subtilis, Bacillus licheniformis
Chemicals Acetoin, Carbon, Glucose