Production of the antimicrobial peptide pediocin PA-1 with Corynebacterium glutamicum on spent sulfite liquor

Description

Cultivation data of the recombinant production of the antimicrobial peptide (AMP) pediocin PA-1 with Corynebacterium glutamicum on spent sulfit liquor using various process modes. For all experiments, C. glutamicum CR099::U pXMJ19 ped-ACD(Cg) was used, which, in contrast to the wild-type, contains genes for improved mannose uptake and the ability to metabolize xylose integrated into the genome (denoted by ::U), as well as an codon optimized product biosythesis genes on a plasmid. Low molecular weight permeate of ultra-filtered spent sulfite liquor (UF-SSL) obtained from Norway spruce (Picea abies) pulping (Borregaard AS, Sarpsborg, Norway) was used as substrate for all experiments. The UF-SSL contained 4 metabolizable carbon sources (6.4 g/L acetate, 42 g/L glucose, 135 g/L mannose and 57 g/L xylose) and was stored at 4°C. The valorization of UF-SSL was performed without additional prior detoxification steps such as bioling or overliming using a minimal medium based on UF-SSL. 3 different process modes were compared for AMP production: an induced batch, an uninduced batch + induced fed batch and an uninduced batch + induced extended batch approach. The dataset consists of on-line data recorded during cultivation (pH, T, dO2, O2,offgas, CO2,offgas, weight of reactor and feeds, gassing, ...), off-line data of samples taken every 3h (BM; PA-1; HPLC: glc, man, gal, ara, xyl, urea; Enzyme Assay: ace, lac, glu, NH4, PO4) and meta data (feed composition, quantity, density...). UF-SSL was diluted with water to 25% (~10 g/L initial glucose concentration), sterile filtered (0.2µm) and supplemented (0.2 mg/L biotin, 12 mg/L chloramphenicol, 5 ml/L polyproylenglycol 2000) and used as minimal medium. Due to strong precipitation when PO4 was added to UF-SSL (containing 14 g/L Ca), a separate nitrogen and phosphate (NP) feed was used instead of mixing with the medium. For the NP feed concentrations of urea and KH2PO4 were adapted to adjust the specific carbon to nitrogen (CN) and phosphate (CP) ratios upon full addition of the feed for each respective process phase. The induced batch was performed at growth optimized conditions (pH 7.0, CN 1:10, CP 1:30, dO2 30%). Fed batch processes were operated at optimized grwth conditions during the uninduced batch and under enhanced production conditions (pH 6.0, CN 1:10, CP 1:45, dO2 15%). Extended batch processes were operated similarly to the fed batch processes but using a constant CP 1:45 to increase process simplicity. The antimicrobial activity of Pediocin PA-1 was measured using a growth inhibition assay with Listeria innocua pNZ44 as the indicator strain and evaluated by fitting a dose-response curve using a nonlinear iterative least squares algorithm. Activity was defined as 50% growth inhibition compared to the blank (inflection point of the dosage-response curve) and reported in biological units (BU/ml). An approximate conversion factor of 2050BU/ml per mg/L can be used for comparison with other products.

Steps to reproduce

Pre-culture was started from glycerol stocks stored at -80°C by plating cells on 2TY agar plates (16 g/L tryptone, 10 g/L yeast extract, 5 g/L NaCl, 10 g/L agar, heat sterilized, 12 mg/L chloramphenicol) and incubated at 30°C for 72h. Subsequently, 2 seeding steps were performed on liquid 2TY complex medium (without agar) for 24h (1 colony, 12.5 ml medium) and 18h (12.5 ml seed 1, 225 ml medium, 25 ml 100% UF-SSL with 100 g/L 3-(N-morpholino)propanesulfonic acid (MOPS), pH 7, sterile filtered) at 30°C with 230 rpm shaking speed. The addition of UF-SSL in the Seed 2 step is to allow for short term adaptation of the cells to the substrate (sugar utilization genes, inhibitor detoxification genes). Prior to innoculation, Seed 2 was harvested and resuspended in 0.9 g/L NaCl (saline) solution for an inoculum of 1 g/L in 75 ml transferred via styringe to avoid transferring complex components from the preculture into the batch. Innoculum volume and medium dilution (initial cell to inhibitor ratio), freshly incubated agar plates plus precise timing of preculture plus rapid handling of cells when not in medium (cell viability) were identified as critical parameters for reproducible growth in the batch. 3L working volume glass bioreactors (Labfors 5, Infors, Germany) equipped with optical dO2 probes (Visferm DO, Hamilton, Switzerland), potentiometric pH probes (Easyferm PHI, Hamilton, Switzerland) and off-gas analyzers (BlueInOne Ferm, BlueSense, Germany) were used for cultivation. Samples were collected every 3 h and stored at 4°C until analysis using a custom sampling device. pH was maintained at its setpoint +/- 0.02 by adding 2.5 M KOH and 2.5 M H2SO4, temperature at 30°C, and dO2 at its setpoint by increasing the stirrer speed at a constant air flow of 0.3 vvm. Prior to analysis, samples were separated into supernatant and pellet by centrifugation (3420 RCF, 4°C, 5 min). Supernatants were analyzed by HPLC (UltiMate U3000, ThermoFisher, USA) with an RI detector (RI-100, Shodex, USA) using a Pb column (Nucleogel Sugar Pb 300mm, Macherey-Nagel, Germany) with an isocratic flow of 0.4 ml/min of ultrapure water at 79°C and automated enzymatic photometric assays (CEDEX Bio HT Analyzer, Roche, Switzerland). Pellets were analyzed for biomass concentration by resuspension in saline twice, followed by serial dilution on 96-well plates (PP black, Microplate, 96-well, F-Bottom;) , followed by fluorescence measurement (EX: 280/15 nm; EM: 340/20 nm) using a plate reader (Spark, Tecan, Switzerland). The indicator strain was stored at -80°C and incubated (37°C, overnight) in BHI medium [37.5g/L BHI (Oxoid Brain-Hart-Infusion), heat sterilized, 12mg/L chloramphenicol]. A serial dilution of the supernatant in BHI medium was performed on a 96-well plate (sterile Microplate, 96-well, F-Bottom), BHI medium with grown indicator strain added 1:1 and subsequently incubated (37°C, 6h) and optical density measured at 600nm using a plate reader (Spark, Tecan, Switzerland).

Institutions

Categories

Funding

Licence