*Article* **Chitin Degradation Machinery and Secondary Metabolite Profiles in the Marine Bacterium** *Pseudoalteromonas rubra* **S4059**

**Xiyan Wang, Thomas Isbrandt, Mikael Lenz Strube, Sara Skøtt Paulsen, Maike Wennekers Nielsen, Yannick Buijs, Erwin M. Schoof, Thomas Ostenfeld Larsen, Lone Gram and Sheng-Da Zhang \***

> Department of Bioengineering, Technical University of Denmark, DK-2800 Kgs Lyngby, Denmark; xwan@dtu.dk (X.W.); tispe@bio.dtu.dk (T.I.); milst@dtu.dk (M.L.S.); ssp@sbtinstruments.com (S.S.P.); mweni@dtu.dk (M.W.N.); yabu@dtu.dk (Y.B.); erws@dtu.dk (E.M.S.); tol@bio.dtu.dk (T.O.L.); gram@bio.dtu.dk (L.G.)

**\*** Correspondence: shez@dtu.dk; Tel.: +45-5011-7765

**Abstract:** Genome mining of pigmented *Pseudoalteromonas* has revealed a large potential for the production of bioactive compounds and hydrolytic enzymes. The purpose of the present study was to explore this bioactivity potential in a potent antibiotic and enzyme producer, *Pseudoalteromonas rubra* strain S4059. Proteomic analyses (data are available via ProteomeXchange with identifier PXD023249) indicated that a highly efficient chitin degradation machinery was present in the red-pigmented *P. rubra* S4059 when grown on chitin. Four GH18 chitinases and two GH20 hexosaminidases were significantly upregulated under these conditions. GH19 chitinases, which are not common in bacteria, are consistently found in pigmented *Pseudoalteromonas,* and in S4059, GH19 was only detected when the bacterium was grown on chitin. To explore the possible role of GH19 in pigmented *Pseudoalteromonas*, we developed a protocol for genetic manipulation of S4059 and deleted the GH19 chitinase, and compared phenotypes of the mutant and wild type. However, none of the chitin degrading ability, secondary metabolite profile, or biofilm-forming capacity was affected by GH19 deletion. In conclusion, we developed a genetic manipulation protocol that can be used to unravel the bioactive potential of pigmented pseudoalteromonads. An efficient chitinolytic enzyme cocktail was identified in S4059, suggesting that this strain could be a candidate with industrial potential.

**Keywords:** chitin; chitinase; chitin degradation machinery; *Pseudoalteromonas*; secondary metabolites
