Bacterial strains and growth experiments

Bacterial strains and plasmids used in this study are listed in appendix Table S1a and S1b. Cells were grown in Luria-Bertani (LB) medium, trypticase soy broth (TSB), modified minimal medium M9 without additional FeCl₂ (Klebensberger et al. 2006) or minimal medium M9extra (Sgobba et al. 2018). The following carbon and energy sources were used for cultivation in minimal medium: glucose (Glc), sodium acetate, glucosamine (GlcN), N-acetylglucosamine (GlcNAc) (all: Sigma Aldrich, München, Germany), N,N′-diacetylchitobiose (chitobiose, GlcNAc₂), de-N-acetylated chitobiose (GlcN₂), or chitin (France Chitine, Orange, France); concentrations of the respective carbon sources are mentioned in the results section. E. coli cells used for growth experiments were maintained on solid (1.5% (w/v) agar) medium M9 using 20 mM Glc as carbon and energy source. For E. coli strains harboring plasmids pPRII+ or carrying an integrated chloramphenicol resistance cassette, 17 μg ml⁻¹ chloramphenicol (Cm) was added. For maintenance of vector pET-22b (+) in the strain E. coli Rosetta 2 (DE3), solid LB medium (1.5% (w/v) agar) containing 100 μg ml⁻¹ ampicillin plus 34 μg ml⁻¹ chloramphenicol was used. C. glutamicum cells were maintained on solid LB medium containing 50 μg ml⁻¹ nalidixic acid. For overnight precultures of E. coli strains, 10 ml tubes with 4 ml M9 medium and 20 mM Glc were inoculated from M9 agar plates. Overnight precultures of C. glutamicum strains were cultivated in 100 ml Erlenmeyer flasks without baffles containing 10 ml of TSB. All precultures were incubated for 12–14 h. Precultures for all growth experiments were centrifuged for 5 min at 1.800 x g, and cells were resuspended in M9 medium without carbon source. Growth was monitored as optical density at 600 nm (OD₆₀₀) with the UV329 mini 1240 spectrophotometer (Shimadzu, Kyōto, Japan) or with the Camspec Visible Spectrophotometer M107 (Spectronic-Camspec Ltd., Leeds, UK). In the case of using chitin as carbon source and in co-cultivation experiments, growth was monitored as colony-forming units (CFU) as previously described by Jagmann et al. (2010) using medium M9 without carbon source for dilution. Growth experiments in single-cultures of E. coli strains were generally performed in 10 ml tubes with 4 ml liquid media inoculated from the washed pre-cultures to an OD₆₀₀ of 0.05 or, in case of isopropyl β- d-1-thiogalactopyranoside (IPTG)-induced cells, to an OD₆₀₀ of 0.4. For cultivation of lysine-auxotrophic E. coli strains in single-cultures, either 10 mM (precultures) or 2.5 mM (main cultures) l-lysine were added to the medium. Single cultures of C. glutamicum strains were performed in 100 ml Erlenmeyer flasks containing 10 ml of the respective medium with an inoculation OD₆₀₀ of 0.1. For co-cultivation experiments, 100 ml Erlenmeyer flasks containing 10 ml of medium M9extra and the respective carbon source were inoculated to an OD₆₀₀ of 0.1 or 0.4 (IPTG-induced cells) for E. coli strains and an OD₆₀₀ of 0.1 for C. glutamicum. All growth experiments were conducted at 30 °C and 200 rpm in a rotatory shaker (Ecotron, INFORS HT GmbH, Bottmingen, Switzerland) under aerobic conditions, as typically used for industrial amino acid production in C. glutamicum (Ikeda and Takeno 2013).

For growth experiments of CgLYS4 with cell-free supernatant of E. coli cultures, supernatants of EcLPP* [TkCDA] and a medium blank with 40 mM acetate which was incubated under the same conditions as the EcLPP* [TkCDA] cultures, were processed as described under preparation of cell-free supernatant of E. coli strains. As a control for this experiment, freshly prepared, non-processed M9extra medium was used for the culture of CgLYS4. All main-cultures of CgLYS4 were grown with 40 mM GlcNAc as carbon and energy source and the addition of the respective supernatants or fresh medium.

Immediately after inoculation and at several time points thereafter, samples from the cultures were taken to monitor bacterial growth and quantify metabolite concentrations. Samples for metabolite quantification were centrifuged at 18500 x g for 10 min at room temperature. The supernatants were transferred to a new 1.5 ml reaction tube and stored at −20 °C until further use.