*4.6. DNA Manipulation*

Genomic DNA of *P. rubra* S4059 was extracted using the Genomic DNA buffer set (QIAGEN, 19060, Hilden, Germany), as mentioned above. All primers used in this study are listed in Table S6. All purified DNA fragments were amplified using PrimeSTAR® Max Premix (TaKaRa, catalog number: R045A, Kusatsu, Japan). Blue TEMPase Hot Start Master Mix K (catalog number: 733-2584, Haasrode, Belgium) was used for homologous recombination event checking by PCR. All primers and plasmids were designed in A Plasmid Editor-ApE. The specificity of primers was checked by BLAST against the *P. rubra* S4059 genome. All primers were ordered from Integrated DNA technologies (Leuven, Belgium).

### *4.7. Construction of Suicide Plasmids for in-Frame Deletion of GH19 Chitinase in P. rubra S4059*

The suicide plasmid was constructed by the direct cloning method using pDM4 as the backbone [40,42]. The pDM4 plasmid contains an R6K replicon origin, *mob* genes and *oir*T for conjugation, and a chloramphenicol resistance gene *cat* and a *sacB* gene for counter selection [42]. An approximately 1-kb upstream and downstream region flanking of GH19 gene was amplified with primer pairs GH19-L-F/GH19-L-R, GH19-R-F/GH19-R-R (Table S6). The amplified recombining arms were fused with overlap extension PCR to form the recombining arm segments, which were cloned into pJET1.2 subcloning vector using a CloneJET PCR Cloning Kit (ThermoFisher Scientific, K1231, Vilnius, Lithuania) for sequencing. Subsequently, the homologous segment was amplified from sequencingconfirmed pJET1.2-dGH19 arms using primers GH19-pJET1.2-F and GH19-pJET1.2-R. The linear backbone was amplified from the pDM4 suicide vector using primers (GH19-pDM4- F/GH19-pDM4-R). After gel purification, the linear vector and the homologous fragment were co-electroporated into *E. coli* GB *dir-pir116* and ligated by the RecET direct cloning system [40]. The restriction cloning method was also attempted several times to construct

this plasmid. However, it was unsuccessful. All plasmids were extracted using a QIAprep Spin Miniprep Kit (QIAGEN, 27106, Hilden, Germany).

#### *4.8. Conjugation of P. rubra S4059*

The conjugation protocol was modified from Yu et al. and Wang et al. [52,53]. *E. coli* WM3064 harboring the suicide plasmid were used as the donor and *P. rubra* S4059 as the recipient. Overnight cultures of donor and recipient were prepared as pre-cultures one day before the conjugation. During conjugation, both strains were diluted 100 times and grown to OD600 ≈ 0.6. One-mL donor cells were harvested at 6000× *g* for 1 min. The pellets were resuspended and washed once using 1 mL LB + DAP. Then, 1 mL recipient was added to the *E. coli* WM3064 pellet and centrifuged at 6000× *g* for 1 min. One-mL MB + DAP was added to wash the mixture by pipetting and centrifuging at 6000× *g* for 1 min. The supernatant was removed until 20–30 μL liquid remained. The mixture of cells was resuspended and placed on a 0.2-μm pore-size membrane (MF-Millipore, GSWP02500) that was placed on an APY + DAP agar plate. The mating plates were incubated at 20 ◦C for 24 h. The cells were suspended in 1 mL MB and incubated with shaking at 750 rpm in an Eppendorf® thermomixer comfort, 25 ◦C for 1 h. After recovery, cells were spread on MA plates with 30 μg/mL chloramphinical (Table S4) and incubated at 25 ◦C for 24–48 h.

#### *4.9. Confirmation of the First Crossing over Mutants and Deletion Mutants*

Colonies from the first crossover selective plates were picked and cultured in 5 mL MB containing 30 μg/mL chloramphenicol overnight. Genomic DNA extraction from pre-cultures using NucleoSpin® Tissue kit (Macherey–Nagel, Düren, Germany, 740952.250). To determine whether the plasmid integrated into target regions, the genomic DNA was used as the template for PCR checking with primers (Cm<sup>r</sup> -F/Cmr -R, GH19-p 1/GH19-p 4, and GH19-p 2/GH19-p 3). Colonies carrying the integrated plasmid were cultured in MB with 30 μg/mL chloramphenicol at 25 ◦C overnight as pre-culture. The pre-culture was diluted 100 times and inoculated in 5 mL fresh MB without antibiotics until OD600 ≈ 0.6. The culture was 10-fold diluted and spread on the counter selection plates (half nutrients of MA) containing 10% sucrose. These counter selection plates were incubated at 20 ◦C until colonies were visible. Confirmation of the in-frame deletion mutants was carried out by PCR application and sequencing. Primers (GH19-p1/GH19-p2) were designed to amplify the mutation region, and the PCR produced was purified and sent for DNA sequencing.
