*2.4. Influence of Chitin on the Metabolome of S4059*

To investigate the potential link between chitin degradation and secondary metabolite production, the S4059 wild type was grown on mannose, crystalline chitin, colloidal chitin, or N-acetyl glucosamine (NAG) containing marine minimal medium (MMM) in stationary phase. Chemical analysis using high-performance liquid chromatography coupled to diode array detection and high-resolution mass spectrometry (HPLC-DAD-HRMS) showed that mannose and crystalline chitin resulted in largely the same chemical profiles, although two unknown tentative prodigiosin analogs were produced in higher amounts on mannose (Figure S1). NAG containing media resulted in an overall higher production of secondary metabolites compared to mannose, crystalline, and colloidal chitin, and NAG and colloidal chitin both led to increased production of two unknown non-prodigiosin derived (based on UV-Vis absorption) compounds (Figure S1). Prodigiosin, hexyl prodigiosin, and heptyl prodigiosin (Figure S1B–D) were produced in varying amounts on all media. The identity of prodigiosin was confirmed using an authentic standard in combination with our inhouse MS/MS library, and the two analogs were identified based on similarities with MS/MS and UV-Vis absorption spectra. Additionally, using our in-house MS/MS library, combined with a compound list generated from all *Pseudoalteromonas*-derived secondary metabolites found in the Reaxys database, no other known secondary metabolites were identified in S4059.

### *2.5. The Deletion of GH19 Chitinase Does Not Affect Growth or Chitin Degradation*

To explore the function of GH19 chitinase in pigmented *P. rubra* S4059 and investigate a possible link between chitin degradation and secondary metabolites production, an in-frame deletion of GH19 chitinase gene (the GH19 A0A5S3UX38 with a signal peptide) was generated in S4059 by homologous recombination (Figure 2A,B). Wild type S4059 and GH19 deletion mutant (Δ*GH19*) were grown in MMM supplemented with crystalline

chitin, colloidal chitin, NAG, or mannose. The mutant grew similarly to the wild type in all substrates with the same growth rate and maximum cell density (Figure 2C,D and Figure S2). The maximum cell density reached 109 colony-forming unit (CFU)/mL in all substrates supplemented with casamino acid while only reaching 10<sup>8</sup> CFU/mL when the strains grew in chitin containing MMM without casamino acid. The generation time of wild type and the mutant was 43.80 ± 8.46 min in all substrates with casamino acid, while without casamino acid, the value was 91.27 ± 2.23 min in crystalline chitin and 74.87 ± 0.30 min in colloidal chitin contained MMM.

**Figure 2.** The in-frame deletion of the GH19 chitinase gene in *Pseudoalteromonas rubra* S4059 was verified by PCR with primers P1 and P2 that target the left and right homology arm of the GH19 chitinase gene (**A**). The PCR products were analyzed by electrophoresis (**B**). M: DNA ladder; NC: negative control with water as the PCR template; WT: PCR products with gDNA of wild type strain S4059 as a template; Δ*GH19*: PCR products with gDNA of Δ*GH19* as a template. Growth kinetics of *Pseudoalteromonas rubra* S4059 wild type (red lines) and Δ*GH19* (black lines) when grown in a marine minimal medium containing (**C**) colloidal chitin or (**D**) crystalline chitin (shrimp chitin) without casamino acids at 25 ◦C for 24 h, shaking at 200 rpm. Data were analyzed on three biological replicates, and the error bars represent the standard deviation.

The chitin degradation capacity of the *P. rubra* S4059 wild type and the GH19 mutant was also tested on chitin (crystalline and colloidal chitin) plates. As expected from the growth experiment, the mutant had the same chitinolytic ability (the size of clearing zone) as the wild type (Figure S2E–F). According to a previous study, the heterologously expressed GH19 chitinase in *E. coli* showed antifungal activity against *Aspergillus niger*. Therefore, the antifungal activity was also explored by co-cultivating both the strains with the fungus *Aspergillus niger* on marine agar (MA) plates, showing that the GH19 mutant displayed the same inhibitory effects as the wild type (data not shown).
