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 identi�ed in the genome. With homologous recombination, an in-frame deletion mutant was constructed to con�rm 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 identi�ed 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 gram-positive 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, 6-deoxy-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 modi cation 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) modi cation 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 modi cations, including stereospeci c 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 2,6-dideoxyhexose 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] .
Granaticin has attracted growing interest for various biotechnological applications in the elds 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 identi ed.Streptomyces sp.A1013Y can produce blue pigment, which was identi ed 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 rst cluster (Table 4).

Granaticin biosynthesis gene cluster comparison with S. violaceoruber and S. vietnamensis
The complete granaticin biosynthetic cluster genome sequence was assembled to generate a 37500 bp contig, including 37 genes that were identi ed by NCBI BLAST and antiSMASH.All granaticin biosynthetic genes (orf9-orf34) were present, and the gene arrangement in A1013Y was identical to those in S. violaceoruber (AJ011500) and S. vietnamensis (GU233672) (Fig. 2).Compared with S. violaceoruber (AJ011500), the aking genes (orf7, orf8, orf35, and orf36) at the two ends of the granaticin cluster were all deleted except orf37.The orf35 deletion in A1013Y was the same as that in S. vietnamensis.
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 signi cantly.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 GIMV4.0001T 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).

Identi cation 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 puri cation of the mutant were carried out and allowed the isolation of the double crossover nongranaticin-producing 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 con rmed 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 con rmed 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 H 10 O 10, LC-MS, m/z 445 [M + H] + ) 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.

Conclusion
ANI analysis of the strain showed that Streptomyces sp.A1013Y might be a new strain able to produce granaticin and its derivatives.The complete genome was sequenced, and the genome information was analyzed.The nongranaticin-producing mutant was constructed by homologous recombination, and the granaticin cluster was con rmed by HPLC and LC-MS analysis of the fermentation liquid of the mutant.The similarity of the granaticin cluster sequence between S. sp.A1013Y, S. violaceoruber Tü22, and S. vietnamensis GIMV4.0001T and the phylogenetic and ANI analyses showed that S. sp.A1013Y is a novel granaticin-producing strain.The genome information we sequenced and analyzed provides a valuable foundation for future post-PKS modi cation analyses as well as biomedicine applications.

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 ChargeSwitch® gDNA Mini Bacteria Kit (Life Technologies) according to the manufacturer's instructions.Puri ed genomic DNA was quanti ed using a TBS-380 uorometer (Turner BioSystems Inc., Sunnyvale, CA).High quality DNA (OD260/280 = 1.8 ~ 2.0, > 10 µg) was used for further research.

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 rst using a hybrid de novo assembly solution modi ed 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 Veri cation 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-de cient 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 anking the actl-1 and actl-2 genes were ampli ed 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-actl-2-UD.After introducing pWHU2653::actl-1-actl-2-UD into S. sp.A1013Y via E. coli-Streptomyces conjugation, the apramycin-resistant colonies were selected and grown on MS agar containing 5-uorocytosine in the dark for 3 or 4 days.The 5FC-resistant colonies were then replicated on MS agar with and without apramycin to con rm plasmid loss.Colonies sensitive to apramycin were selected as the desired S. sp.A1013Y Δ actl-1 and Δ actl-2 mutant, which were veri ed by PCR using the primer pair YZ4041-F and YZ4041-R.
4.8 Detection of the production of S. sp.A1013Y and its deletion mutant.
The fermentation products of both S. sp.A1013Y and its deletion mutant were detected by using HPLC (Agilent 1260, USA) on a ZORBAX eclipse plus C18 (4.64.6 × 250 mm; 5 µm) column.The column was developed with solvent A (ddH2O with 1% (v/v) acetic acid) and acetonitrile at a ow rate of 0.5 mL/min.The percentage of acetonitrile was maintained at 50% for 0-6 min and changed from 50-75% for 50-75 min and returned to 50% for 25-30 min.The injection volume was 10 µL, and detection was performed at 580 nm (maximum absorption wavelength) using a UV variable wavelength detector.The detection wavelength was 575 nm.
LC-MS was used to analyze the granaticin production of S. sp.A1013Y and its deletion mutant.The supernatants of the two strains were ltrated with a 0.22 µm membrane and analyzed with a ZORBAX SB-AQ column (5 µm, 4.6 × 250 mm, Agilent Technologies, Santa Clara, CA, USA).The column was developed with solvent A (ddH2O with 0.1% (v/v) formic acid) and acetonitrile at a ow rate of 0.5 mL/min.The percentage of acetonitrile stayed at 50% for 0-6 min, changed from 50-75% for 50-75 min and returned to 50% for 25-30 min.Construction of S. granaticin-de cient mutant a, The in-frame deletion of act -1 and act -2 were performed using a homologous recombinant.b, S. sp.A1013Y wild-type and granaticin-de cient mutant ZSL1 grown on MS agar.c, The PCR primers used for genotype veri cation of granaticin-de cient mutant were indicated with red , and the sizes of the PCR products were also showed.d, The HPLC analysis of metabolites of S. sp.A1013Y wild-type and ZSL1 mutant.The compound 1 was granaticin.HPLC is described in "materials and methods" section.The retention times of granaticin is 11min or so.(i)

Table 1
Oligonucleotides used in this study.