Ht multitron

The biological activity of pediocin PA-1 was determined using a growth inhibition assay [23 (link), 105 (link)]. The sensor strains L. innocua pIMK2 and L. innocua pNZ44 were grown overnight in glass tubes (filled 20% with BHI medium) on a rotary shaker (37 °C, 230 rpm, Infors HT Multitron). The samples (culture supernatant) were stepwise diluted with BHI medium in a 96-well microtiter plate. This yielded a twofold dilution series (100 µL for each dilution). In parallel, the L. innocua cultures were diluted 1:25-fold in fresh BHI medium, and the obtained suspension was mixed 1:1 with each diluted sample. The filled microtiter plate was incubated for 6 h (37 °C, 230 rpm, Infors HT Multitron). Afterwards, the cell concentration in each well was quantified at 595 nm (Labsystems iEMS Reader MF, Thermo Fisher, Waltham, MA, USA). The pediocin PA-1 activity was then estimated from the growth inhibition data [105 (link)], using software-based parameter fitting with the sigmoidal dose response tool (Origin 2021, Northampton, UK). Throughout this study, the activity is given as bacteriocin units per mL (BU mL⁻¹). Using the recently determined specific biological activity of purified, commercial pediocin PA-1 [23 (link)], allowed to infer peptide concentrations from biological activity measurement.

All succinic acid production strains were picked into 6 mL of synthetic minimal (SD) medium and grown at 30 °C, 250 rpm overnight. The next day, optical density was measured in a spectrophotometer (WPA Biowave II) and cultures were diluted to OD₆₀₀ = 0.05 in 1 mL SD media, with and without 1 µM aTc (Alfa Aesar, J66688-MB). Cultures were grown in 48-deep-well-plates (Agilent, 201238-100) at 30 °C in an Infors HT Multitron, shaking at 700 rpm. After 2 days, plates were spun down at 4000 × g, 4 °C for 10 min. Then, 300 µL of the supernatant was sampled for each well. The same day, supernatant samples were measured directly by LC-MS alongside a succinic acid standard, as follows: succinic acid was detected and measured by UPLC-MS, using an Agilent 1290 Affinity chromatograph linked to an Agilent 6550 Q-ToF mass spectrometer. Separation was achieved using an Agilent Zorbax Eclipse Plus C18 column (2.1 × 50 mm, 1.8 μm) and an acetonitrile gradient of 0% for 2 min then an increase to 98% over 0.5 min at a flow rate of 0.3 mL/min. Mass spectral data was acquired in negative ion mode from m/z 90 to 1000 at the rate of 3 spectra per second throughout the separation. In total, 0.2 µL was injected from both sample wells and standard solutions. Succinic acid concentrations were calculated from a succinic acid standard curve in Microsoft Excel.

Aspergillus nidulans ATCC 48756 (R21) and A. niger ATCC 1015 were used to confirm the existence of a stwintron in the transcript of their orthologue genes for a putative lipase characterised by the well-conserved alpha/beta hydrolase fold 3 domain (Pfam07859). Standardised medium compositions are described elsewhere [10 (link)]. Fungal biomass was generated in 500-mL Erlenmeyer flasks with 100 mL of complete medium seeded with vegetative spore inoculum, in a rotary shaker (Infors HT Multitron) at 200 rotations per min. 16 h after inoculation, mycelia were harvested by filtration, thoroughly washed with distilled water, and subsequently frozen and ground to powder under liquid nitrogen. For the extraction of genomic DNA and total RNA from the mycelial powder, Macherey–Nagel NucleoSpin kits (NucleoSpin Plant II and NucleoSpin RNA Plant) were used.