**2. Results**

#### *2.1. Genome Features of Streptomyces Strains with Differential Keratinolytic Activity*

Three strains (Table S1) from our Chilean marine actinobacterial culture collection [37–39] were selected for genome sequencing, based on a previous feather degradation screening [40]: *Streptomyces* sp. G11C, presented the highest level of keratinolytic activity, exhibiting complete feather degradation in liquid culture; *Streptomyces* sp. Vc74B-19, evidenced no keratinolytic activity, leaving the feather structure unaltered, and *Streptomyces* sp. CHD11 presented a low level of degradation, showing the partial dissolution of the fibers of the feathers [40]. To obtain insights into the genetic determinants involved in feather degradation, genomes were

sequenced using the Illumina NextSeq platform with a 150 bp × 2 configuration. Final assembly statistics, including quality metrics, for each genome, are displayed in Table 1. CHD11 and Vc74B-19 genomes, of approximately 7.5 Mbp, are larger in size than the G11C genome (nearly 6.9 Mbp), although the GC content is similar for all three strains and is comparable with percentages found in actinobacterial strains [41].


**Table 1.** Assembly statistics of analyzed *Streptomyces* genomes.

Previous taxonomic classification using the 16S rRNA gene indicated that these marine strains belong to the *Streptomyces* genus [37]. To obtain a precise taxonomic placement, we performed a phylogenomic analysis (Figure 1, Table S2) and calculated average nucleotide identity (ANI) (Figure S1, Table S3). In the case of the phylogenomic tree, the inference was made with Orthofinder [42] using 634 single-copy orthogroups. These analyses indicate that both streptomycete strains presenting either low and no keratinolytic activity, strains CHD11 and Vc74B-19, respectively, are phylogenetically close, grouping in the same sub-clade with the closest neighbor *Streptomyces emeiensis* CGMCC 4.3504<sup>T</sup> [43]. This is consistent with ANI values obtained between strains Vc74B-19 and CHD11 (88.2%) and with *S. emeiensis* type strain—87.5% for strain CHD11 and 87.4% for strain Vc74B-19. On the other hand, strain G11C, with high keratinolytic activity, groups with *Streptomyces albidoflavus* NRRL B-1271T [44], consistent with an ANIm of 96.1% that indicates strain G11C could be considered as part of the *albidoflavus* species [45]. A strain belonging to the *albidoflavus* species has been described in the literature to possess keratinolytic activity, secreting at least six extracellular proteases when cultured on a feather meal-based medium [14]. Altogether, these findings motivated us to study the minor differences between our genomes and examine a possible bioinformatic explanation for the observed differential keratinolytic activities.
