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Complete genome sequence analysis of a novel granaticin producer, Streptomyces sp. A1013Y

Granaticin is a kind of antibiotics with the function of anticancer, antibacterial, anticoccidial. Streptomyces sp. A1013Y is a new strain isolated from soil with granaticin-producing ability. The genome information was analyzed for the further study of granaticin. The present work reported the complete genome of S. sp. A1013Y, which contains a 7,646,296 bp chromosome with an average GC content of 71.59%. A total of 7361 CDSs, including 66 tRNA genes, 18 rRNA genes and 23 clusters, were identified in the genome. With homologous recombination, an in-frame deletion mutant was constructed to confirm the granaticin cluster. The fermentation liquid of the mutant did not contain granaticin based on HPLC and LC-MS analysis, indicating that S. sp. A1013Y is a granaticin producer. Comparing the granaticin biosynthesis cluster of S. sp. A1013Y with Streptomycesviolaceoruber Tü22 and Streptomyces vietnamensis GIMV4.0001T, which were sequenced and produce granaticin, the similarity between the granaticin clusters was 78% and 83% respectively, but some genes still weren’t identified in the granaticin biosynthesis cluster. The phylogenetic analysis and ANI value between S. sp. A1013Y and other Streptomyces species, including S. vietnamensis GIMV4.0001T, were all below 85%, which showed that S. sp. A1013Y was probably a novel streptomyces strain that produced granaticin. The whole genome information of S. sp. A1013Y provides a valuable foundation for future granaticin analyses as well as biomedicine applications.


Introduction
Granaticin (CAS: 19879-06-2, C 22 H 10 O 10 ), a secondary metabolite, is a member of the benzoisochromanequinone (BIQ) class of aromatic polyketides whose members include actinorhodin, medermycin, and kalafungin. Some of these compounds possess anticancer, antibacterial, anticoccidial, or platelet aggregation inhibitory activities [1] . For example, granaticin has biological activity that inhibits grampositive bacteria and cytotoxic activity for tumor cells as an inhibitor of enzymes such as farnesyltransferase [2] and aminoacyl-tRNA synthetases [3] . Its derivatives, such as granaticin B, dihydrogranaticin, and dihydrogranaticin B, also have some of the same functions [4] . A new granaticin analogue and its hydrolysis products, 6deoxy-13-hydroxy-8,11-dione-dihydrogrsnsticins B and A, were isolated by Jiang et al. from Strepomyces sp. CCPC200532 and both analogue products showed similar cytotoxicity against cancer cells [5] . In recent years, granaticin B has been used in pharmaceuticals and as an excipient for treating proliferative disease and inhibiting cell growth in its new crystalline form [6] . Granaticin and its derivatives, combined with some selected carriers, were used as inhibitors to prevent and cure diseases have the characteristics of Hartnup syndrome from indoleamine 2,3-dioxygenase (IDO) or tryptophan 2,3-dioygenase (TDO). Therefore, exploring the new derivatives of granaticin has attracted great interest from researchers.
Due to the excellent bioactivity of granaticin and its derivatives, it is necessary to investigate the synthesis of granaticin. Although the mechanism of skeleton synthesis is clear, the subsequent modification process is not yet clear, and it plays an important effect on the bioactivity of the chemicals. The synthesis of granaticin, which involves a type II polyketide synthase (PKS II) including PKS, post-PKS (tailoring) modification and deoxysuger biosynthesis, was detected in the 1990s [7] ; the biosynthesis pathway was proposed and supplemented [8] , and the functions of some unknown genes have been determined gradually [9][10] . The structure of granaticin consists of a BIQ chromophore and a sugar moiety. The granaticin chromophore, a basic carbocyclic skeleton of the BIQs, is assembled by decarboxylative condensations of mathonyl residues to give a dicyclic intermediate, which is catalyzed by the minimal PKS subunits (ketosynthase (KS), chain length factor (CLF), acyl carrier protein (ACP)) and its closely associated enzyme in PKS [11] . The intermediate undergoes subsequent modifications, including stereospecific ketoreduction, enoylreduction and oxygenation, in the later "tailoring" steps. The biosynthesis of the granaticin sugar moiety is likely to follow canonical pathways for the formation of deoxysugars and aminosugars. The 2,6dideoxyhexose moiety is catalyzed from glucose and attaches to the granaticin chromophore via two carbon-carbon bonds at C-9 and C-10 with the help of glycosyltransferase (GT). The mechanism of C-glycosylation and attachment via the C-C bond in granaticin biosynthesis remains enigmatic, and further work is required to comprehend this unprecedented sugar attachment [8] .
However, some unknown genes are also presented, and the biosynthesis pathway of glycosylation has still not been characterized clearly [8] . Granaticin has attracted growing interest for various biotechnological applications in the fields of biomedicine. Therefore, it is meaningful to sequence new granaticin-producing strains for the future study of BIQ mechanisms and biomedicine applications.
In our previous study, Streptomyces sp. A1013Y, a ubiquitous gram-positive bacterium, was isolated from a soil sample from a dairy plant in Henan Province and identified. Streptomyces sp. A1013Y can produce blue pigment, which was identified as granaticin, during fermentation at 37 °C. In this article, the complete genome of S. sp. A1013Y was sequenced and assembled, and the genome information was annotated and analyzed, which included secondary metabolite biosynthetic cluster prediction and phylogenetic and ANI analyses. Homologous recombination was used to construct a nongranaticin mutant, and HPLC and LC-MS analyses were used to detect the fermentation liquid, which conformed the presence of a granaticin biosynthetic cluster. For the post-synthesis of granaticin analyses and biomedicine applications in the future, it is meaningful to sequence and analyze S. sp. A1013Y genome information.

Genome sequencing and assembly
The A1013Y genome was sequenced using a combination of PacBio RS and Illumina sequencing platforms with paired-end reads sequencing. The A1013Y genome contains one circular chromosome of 7,646,296 bp with an average GC content of 71.59% (Fig. 1) and no plasmid.

Genome annotation
The annotation results of the A1013Y genome sequence showed that there are 7361 CDSs, including 66 tRNA genes and 18 rRNA genes (Table 3). Twenty-three gene clusters for secondary metabolites were predicted on the chromosome. The predicted secondary metabolite biosynthetic pathways included PKSII, NRPS, and PSKIII, and the secondary metabolites contained granaticin, melanin, butyrolactone, siderophore, indole, ectoine, terpene, lassopeptide, and lantipetide. The granaticin biosynthetic gene cluster is the first cluster (Table 4). Table 4 Identified secondary metabolite regions of Streptomyces exfoliatus A1013Y.
The full length of A1013Y was 1240 bp shorter than the corresponding region of AJ011500. The overall homology rate of the granaticin cluster between these two sequences was 78%, which is relatively high but varied from gene to gene significantly. The protein-coding genes orf1 and orf31 shared the highest similarity of 93%, and orf13 had the lowest identity of 49%, but its protein sequence similarity was 79%. The full length of A1013Y was 20 bp longer than that of GU233672, and the homology rate of the granaticin cluster between these two sequences was 83%.
Orf5 had the highest similarity of 94% in the protein-coding genes; orf33 had the lowest identity of 46%, but its protein sequence similarity was 84%.

Phylogenetic and ANI analyses of S . sp. A1013Y
According to the analysis of the granaticin cluster of three Streptomyces, S. A1013Y has a relative ancestor with S. violaceoruber and S. vietnamensis. Therefore, the phylogenetic tree based on the 16S RNA sequence of granaticin producers, including S. violaceoruber and S. vietnamensis, was constructed for phylogenetic analysis of S A1013Y (Fig. 2).
The ANI results showed that the values were all under 85% when S. sp. A1013Y was compared with different Streptomyces strains, and the highest value when compared with the granaticin producer S. vietnamensis GIMV4.0001 T was 84.9268%.
According to the literature, when strains belong to different species, the ANI values are below 94% [20] . The low ANI value indicated that S. sp. A1013Y is probably a new species strain that produces granaticin (Table 5).

Identification of the granaticin biosynthetic gene cluster
The genome of the granaticin producer Streptomyces sp. A1013Y was sequenced.
According to the analysis by antiSMASH and NCBI BLAST previously, the high similarity between S. sp. A1013Y and S. violaceoruber Tü22 showed that the DNA region that contains minimal PKS genes encoding keto reductase, keto-acyl synthase, chain length factor and acyl carrier protein is the putative granaticin cluster.
The core biosynthetic genes, orf 1 (KS) and orf 2 (KS), were deleted by the method of homologous recombination (Fig. 4a). The screening and purification of the mutant were carried out and allowed the isolation of the double crossover nongranaticinproducing mutant ZSL1. ZSL1 showed growth and morphological characteristics identical to those of the wild-type strain, while the ability to produce granaticin was abolished (Fig. 4b). The genomic DNA of ZSL1 was confirmed by PCR. Due to the deletion of orf 1 and orf 2, amplicons of 1095 bp (YZ4041-F/YZ4041-R) from the mutant were detected. In contrast, a 3611 bp fragment (YZ4041-F/YZ4041-R) from the wild-type strain was observed (Fig. 4c). This result indicated that two of the minimal PKS genes were deleted by in-frame deletion. The ability of ZSL1 to produce granaticin was lost, which was further confirmed by HPLC and LC-MS analysis. No peak corresponding to granaticin was observed in ZSL1 (Fig. 4d), and a molecular weight corresponding to granaticin (C 22  was not observed (Fig. 4f).

Nucleotide sequence accession numbers
Genome information for the chromosome of S A1013Y and the 16S rDNA sequence are available under the accession number MK801227 in the GenBank database.

Fermentation conditions
The fermentation conditions were optimized to produce blue pigments. S. sp.
A1013Y was active at 37 °C for 24 h on activated medium (10 g lactose, 5 g tryptone, 10 g yeast extract powder, 4 g NaCl, and 20 g agar per liter, pH 7.0) and then inoculated from activated plate medium into the seed culture liquid medium (same ingredients as the activated medium except agar) at 37 °C for 40-42 h on a rotary shaker (180 rpm) for subsequent fermentation and granaticin production. The fermentation medium (same ingredients as the activated medium except agar) was inoculated with 5% inoculum size of seed culture broth for 90-92 h at 37 °C on a rotary shaker (180 rpm) [21] .

DNA extraction
A total of 5 ml collected broth from YEME liquid medium was centrifuged at 4000 g for 10 min. Genomic DNA was isolated from the cell pellets using the

Genome sequencing and assembly
The whole genome of Streptomyces sp. A1013Y was sequenced using a combination of PacBio RS and Illumina sequencing platforms. The Illumina data were used to evaluate the complexity of the genome. The complete genome sequence was assembled using both the PacBio reads and Illumina reads. The assembly was produced first using a hybrid de novo assembly solution modified by Koren, S., et al [22] , in which a de-Bruijn-based assembly algorithm and a CLR read correction algorithm were integrated into the "PacBioToCA with Celera Assembler" pipeline [23] .
The whole genome was sequenced and analyzed on the free online platform of the Majorbio i-Sanger Cloud Platform (www.i-sanger.com).

Phylogenetic and ANI analyses
For the phylogenetic and ANI analyses, all 16S rRNA sequences and the complete sequences used in this study were obtained from the FTP site of NCBI at https://www.ncbi.nlm.nih.gov/nuccore.
The 16S rRNA sequences of S. sp. A1013Y and concatenated sequences were aligned with ClustalW, and a neighbor-joining tree was constructed in MEGA version 10 with 1000 bootstrap runs.
The average nucleotide identity was calculated using the ANI calculator available at https://www.ezbiocloud.net/tools/ani [24] . 4.7 Verification of the expression of the granaticin cluster gene 4.7.1 DNA manipulation General DNA manipulations were performed as described [24] . PCRs were performed with PrimeSTAR HS DNA polymerase (Takara, Shiga, Japan) or Taq DNA polymerase (TransGene, Beijing, China) according to the manufacturer's instructions. All PCR primers used in this study are listed in Table 1, and all the strains and plasmids used in this study are listed in Table 2. Homologous recombination (HR) was performed as described. Transformation of Streptomyces-E. coli conjugations were carried out according to standard protocols [25] .

Construction of a granaticin-deficient mutant
In-frame deletion mutants of S. sp. A1013Y Δ actl-1 and Δ actl-2 were constructed using a homologous recombination method. Two 2.0-kb fragments flanking the actl-1 and actl-2 genes were amplified from the genomic DNA of S. sp. A1013Y with primer pairs Gra4041L-F/Gra4041L-R and Gra4041R-F/Gra4041R-R, respectively. The two fragments were cloned into the HindIII site of pWHU2653 using a one-step cloning strategy, generating the Δ actl-1 and Δ actl-2 plasmid pWHU2653::actl-1-

Consent for publication
Not applicable

Availability of data and materials
All data generated or analysed during this study are include in this published article.
The completed genome sequence and 16sRNA sequence of Streptomyces sp. A1013Y and the granaticin biosynthesis cluster genome sequences of Streptomyces violaceoruber Tü22 and Streptomyces vietnamensis GIMV4.0001 T are available in the NCBI database.

Competing interests
The authors declare that they have no competing interests

Funding
This study was financially supported by the Program for the National Natural The comparison of the biosynthesis gene clusters of granaticin from three strains. Minimal PK Figure 3 The phylogenetic tree of the genus Streptomyces based on 16S rDNA gene sequences. The g