Complete genome sequence of Chryseobacterium sp. ZHDP1 with protease activity

This study reported a complete genome of Chryseobacterium sp. ZHDP1 isolated from the soils of a seafood market. The ZHDP1 genome with a size of 4,917,748 bp and a GC content of 35.95% possessed 4,478 coding genes, 5 rRNA genes, 26 sRNA genes, and 89 tRNA genes. The 16S rRNA gene sequence of ZHDP1 had a maximum similarity of 99.07% with that of C. gambrini 5-1St1a. The maximum values of average nucleotide identity and DNA-DNA hybridization of ZHDP1 genome were 91.39 and 47.8, respectively, which were lower than the thresholds for a new genome. Different protease genes were annotated in the genome of ZHDP1, and the protease activity was also detected in the fermentation broth of ZHDP1. Furthermore, the activity of protease in the fermentation broth was optimized through temperature, pH, and metal irons, and the results showed that 60°C and pH 7.0 were the optimum conditions and Fe 3+ could positively increase the protease activity of ZHDP1. This study provides the rst insight into the novel genomic information of Chryseobacterium sp. ZHDP1 and its protein-degrading ability, thereby broadening our knowledge of the industrial potentials in genus Chryseobacterium strains.


Introduction
Genus Chryseobacterium strains have been widely isolated from clinical specimens (Nicholson,Gulvik et al. 2020), soils (Dahal, Chaudhary et al. 2021), wastewater (Kämpfer, Dreyer et al. 2003), and plant roots (Park et al. 2006). The taxonomic information and functions of genus Chryseobacterium are also reported in former studies. Usually, genus Chryseobacterium strains emerge as causative agents in infected animal and human tissues (Lin, Lai et al. 2017). However, genus Chryseobacterium strains are recently veri ed with other properties. For example, C. antibioticum RP-3-3 isolated from Arctic soil shows antimicrobial activity against Gram-negative pathogens (Dahal, Chaudhary et al. 2021). A novel alkalistable α-amylase has been isolated from C. taeanense TKU001 which possesses the ability of starch degradation (Wang, Liang et al. 2011). Chryseobacterium sp. RBT isolated from the soils is able to degrade keratinous waste including silk, human hair, wool, and chicken feathers (Gurav, Jadhav et al 2013). Genus Chryseobacterium strains are potential sources of microbial proteases which are of great values in pharmaceutical, food, waste disposal, and detergent industries (Boominadhan, Rajakumar et al. 2009 In this work, Chryseobacterium sp. ZHDP1 with a novel genome was isolated, and the protease genes were annotated in this genome and the protease activity was also optimized. Our study supplements a potential new protein-degrading member and its genomic information in Chryseobacterium genus, which lays a foundation for the future study on the industrial application of this strain.

Materials And Methods
2.1 Isolation, puri cation, and genomic DNA extraction of ZHDP1 The sample for isolation was the soils collected from a seafood market located in Shenjianmen, Zhoushan City, China (29.95°N, 122.30°E). Approximate 10 g soils were collected into a sterile centrifuge tube by using a sterile medicine spoon. The sample was stored on ice and was immediately transferred back to the lab. The sample was kept at 4°C in the lab and the isolation must be nished in 7 days for ensuring the freshness of the sample. The sample was diluted 10 6 times with sterile physiological saline and 1 mL of diluent was spread on Casein Agar medium (10.0g/L Casein, 3.0g/L beef extract powder, 5.0g/L NaCl, 2.0g/L Na 2 HPO 4, 15.0g/L agar, 0.05g/L bromothymol blue in 1 L distilled water). The plates were cultured at room temperature for 24 h. The single colonies with clear zones were selected as proteindegrading strains and were streak on Casein Agar medium three times for puri cation. The genomic DNA of the selected strains was extracted by using TIANamp Bacteria DNA Kit (Tiangen, China). The integrity and purity of the extracted DNA were veri ed by 1% agarose gel electrophoresis and Nanodrop 2000, respectively.

Genome sequencing and annotation
The sequencing of genomic DNA was conducted by Nanopore (Oxford Nanopore technologies, UK) and Illumina (Illumina, Inc., USA) platforms. The pass reads from Nanopore and clean reads from Illumina were selected for further assembly. The assembly of the genome was performed with unicycler software

Determination and optimization of the protease activity in the fermentation broth of ZHDP1
Strain ZHDP1 was cultured in the liquid medium of casein at 28°C for 36 h. The supernatant of the fermentation broth of ZHDP1 was collected by centrifugation at 3500 rpm and 4°C for 10 min and the supernatant was treated as crude protease solution for further research within 12 h. One milliliter of protease solution was mixed with 1.0 mL of 1% casein solution in 0.02 M phosphate and incubated in a water bath at 30°C for 10 min. Two milliliters of 0.4 M trichloroacetic acid were added into the mixture to terminate the reaction, then the mixture was centrifuged at 6,000 ×g for 5 min. Next, 1 mL of the solution, 5 mL of Na 2 CO 3 (0.4 M), and 1 mL of Folin reagent were mixed into a 10 mL clean tube and was incubated at 35°C for 15 min. The absorbance value of the solution was detected at 680 nm using a UVvisible spectrophotometer. The control sample was prepared by adding trichloroacetic acid solution prior to adding protease solution. In this work, the unit of enzyme activity (U) was de ned as the amount of enzyme required to decompose 1 ug tyrosine per minute under certain conditions. The protein concentration was determined by folin-ciocalteu method with casein as standard.

Description of strain ZHDP1 and its genomic information
A colony with a clear zone was obtained during the isolation process, indicating the protease activity of this strain named ZHDP1. The sequencing depths were ~230.57X and ~204.67X for the reads from Illumina and Nanopore platforms, respectively. A complete genome of ZHDP1 without any gap was assembled. The size of this genome was 4,917,748 bp and the GC content was 35.95%. A total of 4,478 coding genes was annotated in the genome, and 5 rRNA genes, 26 sRNA genes, and 89 tRNA genes were predicted. Nine gene islands and 1 prophage were found in the genome. Most genes were assigned into "Global and overview maps" (241 genes), "Carbohydrate metabolism" (252 genes), and "Amino acid metabolism" (235 genes) pathways according to the results of KEGG annotation ( Figure 1A). A total of 160 and 138 genes were annotated into "Translation, ribosomal structure and biogenesis" and "Amino acid transport and metabolism" COG groups, respectively ( Figure 1B). Both of the annotation results showed the genes related to amino acid metabolism were abundant in the genome, indicating that strain ZHDP1 had a developed metabolic system that was used to treat the hydrolysates of proteases, which endows this strain with the potential in the food industry (Neis, Dejon et al. 2015, Lin, Liu et al. 2017). The general characteristics of the genome were listed in Table 1 and Figure 1C.

Identi cation of strain ZHDP1 based on the genomic information
Based on the 16S rRNA gene sequences, strain ZHDP1 had a maximum identity of 99.07% and was phylogenetically closed to C. gambrini DSM 18014 ( Figure 2). Therefore, strain ZHDP1 was assigned into genus Chryseobacterium. The colony of strain ZHDP1 was circular, orange, and raised, which is similar with the former species (Kämpfer, Dreyer et al. 2003

Protease genes and activity optimization of strain ZHDP1
A total of 27, 22, 23 protease genes, including the genes of metalloprotease, serine protease, lon protease, thiol protease, tail-speci c protease, rhomboid protease, and alkaline protease, were annotated in the genome of this potential new species by using Uniprot, Pfam, and Refseq databases, respectively, indicating that strain ZHDP1 is a novel protein-degrading member in genus Chryseobacterium in addition to previously reported strains. Except for the analysis at genome level, the activity of protein degradation by ZHDP1 was veri ed and optimized in this study. The factors of temperature, pH, and metal ions could affect the protease activity in the fermentation broth of ZHDP1. The results showed that the optimum temperature and pH were 60°C and 7.0, respectively. Fe 3+ could mostly increase the protease activity in the broth. Finally, the maximum activity reached 25.7 U/mL under the optimum conditions (Figure 3). The protease genes annotated in the genome not only are bene cial for the related industries but also are possible participants in glycoprotein link (Parente, Casabuono et al. 2014), immune response (Lad, Yang et al. 2007), bio lm formation (Marr, Overhage et al. 2007), and pathogenesis (Miyoshi, Shinoda 2000). Therefore, the protease genes make strain ZHDP1 become a potential novel member of genus Chryseobacterium with values for industrial production, medical treatment, ecological research, and molecular biology.

Con icts of interest
Authors have no con ict of interest to declare.

Availability of data and material
The genome sequence has been deposited in Genbank database under the accession no. CP055157.

Code availability
Not applicable  Figure 1 (A) Genome circular map of ZHDP1. The meanings of the circles from outside to inside are genome sequence scales, GC contents, GC skew, depth and coverage of sequencing of Illumina and Nanopore platforms, and genes (coding and non-coding) on the positive and negative strands, respectively; (B) Annotation results of the genes in ZHDP1 genome against KEGG database; (C) Annotation results of the genes in ZHDP1 genome against COG database.

Figure 2
Phylogenetic tree constructed based on the 16S rRNA gene sequences of ZHDP1 and other genus Chryseobacterium strains by using neighborhood-joining method.