**1. Introduction**

Marine microorganisms have emerged as a promising source of novel antimicrobial compounds or hydrolytic enzymes [1,2]. In particular, the marine genus *Pseudoalteromonas* harbors a wide range of bioactive compounds with antimicrobial, antifouling, and algicidal activities [3–6]. Based on both phenotypes and genome-wide analyses, *Pseudoalteromonas* can be divided into two groups: pigmented and non-pigmented species [3]. The genomes of pigmented species contain a high number of biosynthetic gene clusters (BGCs) as compared to those of non-pigmented species [7]. Both groups carry the genetic potential to produce a wide range of glycosyl hydrolases, and the pigmented *Pseudoalteromonas* especially harbors a powerful chitin degrading machinery containing several chitinolytic enzymes [7].

Chitin is the most abundant carbon source in the marine environment [8], where it is present in three crystalline allomorphs: α-, β-, and γ-chitin. α-chitin has antiparallel chains, β-chitin has parallel chains, and γ-chitin has the mixture of both chains [9]. Chitin in nature is predominantly degraded by microorganisms [10], and chitin degradation depends on secreted extracellular chitinases (EC.3.2.1.14) and other chitinolytic enzymes/proteins, such as lytic polysaccharide monooxygenases (LPMOs) [11]. Chitinases are glycoside hydrolases (GHs) and are classified into GH18, GH19, and GH20 in the CAZy

**Citation:** Wang, X.; Isbrandt, T.; Strube, M.L.; Paulsen, S.S.; Nielsen, M.W.; Buijs, Y.; Schoof, E.M.; Larsen, T.O.; Gram, L.; Zhang, S.-D. Chitin Degradation Machinery and Secondary Metabolite Profiles in the Marine Bacterium *Pseudoalteromonas rubra* S4059. *Mar. Drugs* **2021**, *19*, 108. https://doi.org/10.3390/ md19020108

Academic Editor: Hitoshi Sashiwa

Received: 22 December 2020 Accepted: 4 February 2021 Published: 12 February 2021

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database [12,13]. The GH18 family chitinases are common in bacteria, whereas the GH19 chitinases are mostly found in plants and are believed to function as a defense mechanism against fungal pathogens [14,15]. Chitinases of both families catalyze the degradation of chitin polymers [12]. The GH 20 family β-N-acetylhexosaminidases hydrolyze amorphous chitin polymers [16], and the LPMOs are metalloenzymes that cleave glycosidic bonds in crystalline chitin and facilitate access of chitinase [17]. Paulsen et al. [7] found that pigmented and non-pigmented *Pseudoalteromonas* evolved divergent GH profiles in their genomic contents. Further, all pigmented *Pseudoalteromonas* species contain at least one GH19 chitinase, which is rarely reported in bacteria. However, only a very few nonpigmented *Pseudoalteromonase* species contain a GH19 chitinase [7]. A GH19 chitinase of the pigmented *Pseudoalteromonas tunicata* CCUG 44952T has been heterologously expressed in *Escherichia coli* and displayed antifungal activity [18]. However, whether this is the dominant role of GH19 in pigmented *Pseodoalteromonas* is yet to be investigated.

The secondary metabolome of several bacteria is influenced by carbon-source, and chitin may serve to enhance the production of secondary metabolites, as observed in strains of Vibrionaceae [19–21]. Likewise, the addition of chitin to *Streptomyces coelicolor* A3 (2) growing in autoclaved soil induced the expression of genes associated with secondary metabolites' production [22]. Due to the potent chitinolytic machinery in *Pseudoalteromonas* [7], we speculated that there could be a link between chitin degradation and secondary metabolism. Since *P. rubra* S4059 dedicates 15% of its genome to secondary metabolites [7] and as other pigmented pseudoalteromonads contain GH19 chitinases [7], we further explored the bioactivity of this prodigiosin-producing strain as a model organism. The purpose of this study was to explore the chitin degradation machinery and secondary metabolite profiles when grown on chitin and to investigate the possible function of GH19 chitinase in *P. rubra* S4059.

### **2. Results**
