Whole Genome Sequencing and Analysis of Benzo(a)pyrene Degrading Bacteria Bacillus cereus ZR72-1

Whole Genome Sequencing and Analysis of Benzo(a)pyrene Degrading Bacteria Bacillus cereus ZR72-1 | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Whole Genome Sequencing and Analysis of Benzo(a)pyrene Degrading Bacteria Bacillus cereus ZR72-1 Dilibaier tuerxun, rui zhang, yanan qin, aofei jin, lirong tan, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3856829/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Benzo (a) pyrene produced by food during high-temperature process enters the body through ingestion, which causes food safety issues to the human body. In order to alleviate the harm of foodborne benzo (a) pyrene to human health, a strain that can degrade benzo (a) pyrene was screened from Kefir, a traditional fermented product in Xinjiang. Results Bacillus cereus ZR72-1 is a Gram-positive bacteria sourced from XinJiang traditional fermented product Kefir, under Benzo(a)pyrene stress conditions, there was 69.39% degradation rate of 20 mg/L Benzo(a)pyrene by strain ZR72-1 after incubation for 72 h. The whole genome of ZR72-1 sequenced using PacBio sequencing technology was reported in this study. The genome size was 5754801 bp and a GC content was 35.24%, a total of 5719 coding genes were predicted bioinformatically. Through functional database annotation, it was found that the strain has a total of 219 genes involved in the transportation and metabolism of hydrocarbons, a total of 9 metabolic pathways related to the degradation and metabolism of exogenous substances, and a total of 67 coding genes. Conclusions According to the KEGG database annotation results, a key enzyme related to Benzo(a)pyrene degradation, catechol 2,3-dioxygenase, was detected in the genome data of Bacillus cereus ZR72-1, encoding genes dmpB and xylE, respectively; There are also monooxygenases and dehydrogenases. Therefore, it can be inferred that this strain mainly degrades Benzo(a)pyrene through Benzoate metabolic. Bacillus cereus ZR72-1 Benzo(a)pyrene Biodegradation Genomics Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Background Benzo(a)pyrene is a polycyclic aromatic hydrocarbon compound composed of five Benzene rings. It is one of the three major carcinogens identified by the World Health Organization and exhibits three carcinogenic effects (carcinogenicity, teratogenicity, and mutagenicity)[ 1 ].The main sources of Benzo(a)pyrene in food are two fold. First, it can be absorbed by crops from dust and particles in the air, which eventually end up in the final products that humans rely on for sustenance, second, Benzo(a)pyrene can be formed and accumulated during heating processes such as baking, smoking, and frying of food[ 2 – 3 ].One effective strategy for the removal of Benzo(a)pyrene is microbial degradation. This strategy involves utilizing the pollutant as a carbon source to stimulate the degradation ability of microorganisms, resulting in the partial or complete removal of these pollutants[ 4 ].Microbial degradation has several advantages compared to physical and chemical methods, such as thorough degradation, lower cost, and absence of secondary pollution[ 5 ]. Barnsley et al[ 6 ]. made the discovery that Pseudomonas NCIB 9816, Pseudomonas ATCC 17483, and Pseudomonas putida PpG7 are capable of utilizing Benzo(a)pyrene in the presence of succinic acid and salicylic acid.Grosser et al.[ 7 ]used radiolabeled 14 C-Benzo(a)pyrene mineralized to 14 C-CO 2 , providing strong evidence that the genus Mycobacterium sp. is capable of utilizing Benzo(a)pyrene. Hunter et al.[ 8 ]in 2005 demonstrated that Bacillus subtilis Tgr3 (a soil bacterium) can use Benzo(a)pyrene as a carbon source and degrade it. Bacillus subtilis BMT4, isolated from soil samples enriched under Benzo(a)pyrene stress, was able to utilize Benzo(a)pyrene as a carbon and energy source and degrade approximately 85% of 50 mg·L − 1 Benzo(a)pyrene within 28 days of incubation[ 9 ],indicating the presence of microorganisms in the environment capable of degrading Benzo(a)pyrene. While microorganisms are effective at degrading Benzo(a)pyrene, their safety limits their direct application in food. Therefore, the screening of strains that are considered safe (GRAS) and have high degradation capabilities for Benzo(a)pyrene can address these issues. Halttunen et al.[ 10 ]reported several strains of lactic acid bacteria that may be beneficial for the removal of various toxic compounds.Considering the potential inhibitory activity of probiotics on polycyclic aromatic hydrocarbons (PAHs) formed in food, Abou-Arab et al.[ 11 – 12 ]studied the degradation of 16 PAH compounds (each at a concentration of 0.25 mg/L in the incubation medium) in MRS medium using different fermentation stages of Bifidobacterium bifidum, Streptococcus thermophilus, and Lb. bulgaricus. The removal abilities of PAHs were as follows: Lb. bulgaricus (91.5%), Streptococcus thermophilus (87.7%), and Lb. bulgaricus (46.6%). The potential ability of probiotics to biodegrade and eliminate toxic PAHs from the human body makes them applicable for the biodegradation of foodborne Benzo(a)pyrene. Kefir is a fermented dairy product made from the milk of cows (goats, sheep, camels, water buffalo) using kefir grains[ 13 – 14 ]. It is acidic, easily digestible, and has various potential health benefits. The main microorganisms in kefir include Lactobacillus , Lactococcus , Acetobacter , Leuconostoc , Kazachstania , Kluyveromyces , Naumovozyma , and Saccharomyces [ 15 – 16 ]. Research has shown that the microorganisms in kefir can also regulate the composition of the gut microbiota, as well as intestinal permeability, oxidative stress, and inflammation. The probiotics or fermentation products in kefir play an important role in these processes[ 17 – 19 ]. In this study, the strain Bacillus cereus ZR72-1 was selected from the Kefir microbial community for its ability to degrade Benzo(a)pyrene. The strain was subjected to whole genome sequencing using PacBio technology. By obtaining the complete genome sequence of the Benzo(a)pyrene degrading strain, the genes of the degrading strain were annotated using the KEGG database to predict the key enzymes and coding genes involved in the degradation of Benzo(a)pyrene. These research findings provide data support for further exploration of the mechanism of benzop(α)yrene degradation by Bacillus cereus ZR72-1. Methods Strains isolation and identification Kefir was sampled from Akto County ,Xinjiang Uygur Autonomous Region, China.Five grams of Kefir fermented liquor was added to Erlenmeyer flasks containing 100mL MSM supplemented with 20mgL-1 Benzo(a)pyrene.Cultures were incubated at 37°C and 120rpm for 72 h for enrichment.Thereafter,10mL of culture was sampled every 72 h and transferred to fresh MSM,Bacterial strains that degraded Benzo(a)pyrene in the Kefir were isolated and purified following the procedure described by Dilbar and Guljamal[ 20 ]. All media, pipette tips, and Erlenmeyer flasks were sterilized by autoclaving at 121℃ for20 min before use. Morphological characteristics and Gram staining were determined using an optical microscope after incubation for 24h at 37°C.Amplification was performed using 16S rRNA universal primers 27F(5'-AGAGTTTGA TCCTGGCTCAG-3') and 1492R(5'-GGTTACTTGTTACGACTT-3').PCR products were purified and sequenced by Sangon Biotech (Shanghai, China).. The resulting 16S rRNA gene sequences were submitted to GenBank,analyzed the homology of gene sequence and and submitted to NCBI for bacterial acquisition.Use GenBank login number to download sequences with high homology and construct a phylogenetic tree using MEGA11.0 software. Chemicals and reagents Minimal Salt Medium (MSM,pH 7.0)contained NH4NO3 1.00 g/L,MgSO4·7H2O 0.20 g/L, KH2PO4 0.50 g/L,K2HPO4 1.50 g/L,NaCl 0.50 g/L,(NH4)2SO4 0.50g/L.Solid medium plates were prepared by adding 18 ~ 20 gL-1 agar into the abovementioned liquid media. All media were sterilized by autoclaving at 121℃ for 30min.Benzo(a)pyrene (analytically pure, ≥ 99%), acetone, dichloromethane (all chromatographically pure, ≥ 99.9%),Methanol (chromatographically pure, ≥ 99.9%), purchased from Beijing Dingguo Changsheng Biotechnology Co., Ltd; Biodegradation of Benzo(a)pyrene by Bacillus cereus ZR72-1 One milliliters of this bacterial culture was add an equal volume of chromatography grade dichloromethane to extracted Benzo(a)pyrene, vortex mixed for 30 seconds, and thoroughly mixed,ultrasound extracted at 40 ℃ for 10 minutes, centrifuged it at 12000 r/min for 8 minutes, and removed of the lower organic phase. Add an equal volume of dichloromethane to the upper incubation solution for further extraction, merge the organic phase, and achieve a pore size of 0.45µm organic phase filtered, HPLC detected.All experiments were performed three times. HPLC conditions:C18 Diamosil TM reverse phase column: chromatographic column (250 mm × 4.6 mm, particle size 5 µm) ; Mobile phase: pure methanol/water (volume ratio 100/0); UV detector: wavelength 245 nm; Injection volume: 20 µ L; Column temperature: 34 ℃; Retention time: 10 minutes.The degradation rate was calculated using the following equation: The degradation rate (%) = (1 − C/C 0 ) × 100 where C and C 0 represent the Benzo(a)pyrene concentrations in the inoculated and non-inoculated media, respectively. Whole genome sequencing Collect bacterial cultures in the logarithmic phase from MC incubation medium. Transfer an appropriate volume of bacterial solution into a 2 mL centrifuge tube and centrifuge at room temperature at 14000 r/min for 1 minute. Discard the supernatant, precipitate the bacterial cells and quickly freeze them in liquid nitrogen for at least 1–3 h (the freezing time depends on the tissue volume to ensure sufficient sample freezing), and then transfer them to -80 ℃ for long-term preservation, which is applied to the extraction of bacterial genomes.According to the instructions of the bacterial genome extraction kit, complete the extraction of bacterial genome DNA. Combine the 260/280 ratio measured by the microplate reader with the 1% agarose gel electrophoresis detection results to conduct quality detection on the extracted bacterial genome DNA. After the quality inspection is qualified, build a HiFi 8–20 Kb sequencing library based on the Pacbio platform for sequencing, and obtain the sequencing data. Prediction of Benzo(a)pyrene degradation genes Hifiasm software was used for assemb, Circulator v1.5.5 software used for cyclization and adjusting the starting site.Then Pilon v1.22 software was used for further error correction. When good accuracy genemo was generated Prodigal v2.6.3, ,KEGG databases (Kyoto Encyclopedia of Genes and Genomes, http://www.genome.jp/kegg/ ) were used for gene prediction and annotation. Results Isolation and Identifcation of Bacillus cereus ZR72-1 A strain with efective Benzo(a)pyrene-degrading ability was obtained following enrichment and purifcation from the medium with Kefir as energy sources, and was named ZR72-1.Strain ZR72-1 was a Gram-positive bacterium with a rod-like shape (Fig. S1 and Fig. 1 ) and the morphological characteristics of the colonies of strain M7-4 were as follows: ivory, rough surface, irregular edges and protruding edges, rod-like, and short (Fig. S2).Phylogenetic tree displayed the results of 16S rRNA gene sequencing analysis for strain ZR72-1 (Fig. 2 ).The 16S rRNA gene sequence of the strain had a 99% bootstrap value identical to the 16S rRNA gene of Bacillus cereus AFA01.Therefore, the isolated strain ZR72-1 was referred to as Bacillus cereus ZR72-1. The 16S rRNA gene sequence of Bacillus cereus ZR72-1 was submitted to the GenBank database (Accession No. OM570322). Benzo(a)pyrene degradation Characteristics of Bacillus cereus ZR72-1 Peak area-mass linear fitting was performed with the different concentrations of different Benzo(a)pyrene, and the respective standard curve equations were obtained, as shown in Fig. S2.In the standard curve equation, y is the HPLC peak area of the Benzo(a)pyrene (mAU*s) and x is the mass of the Benzo(a)pyrene (mg). The degradation rates of Benzo(a)pyrene when incubated with Bacillus cereus ZR72-1 are shown in Fig. 3 .It could be seen that the strains had a broad degradation spectrum for Benzo(a)pyrene,the degradation rate of Benzo(a)pyrene by strain ZR72-1 gradually increases. At 72 hours, the degradation rate of Benzo(a)pyrene reaches approximately 69.39%, indicating that the strain has reached its maximum degradation rate in the incubation medium. After this point, the degradation rate starts to decline and no longer consumes carbon sources. Therefore, we can conclude that strain ZR72-1 has the highest degradation rate at 72 hours. Whole genome analysis of Bacillus cereus ZR72-1 Genomic information helps to generate insights into the mechanisms of Benzo(a)pyrene degradation by bacteria. Hence, we carried out whole-genome sequencing using PacBio Sequencing technology platform to decipher the complete set of genes involved in the degradation of Benzo(a)pyrene .The whole genome information was deposited at NCBI( http://www.ncbi.nlm.nih.gov).Th e genome sequence length is 5754801 bp, and the GC-content is 35.21%. It contains 107 tRNAs, 42 rRNAs, and fourteen 16S-23S-5S rRNAs. It is composed of 5732 coding genes. The total length of the Coding region is 4818215 bp. The average length of the predicted genes is 840 bp, and the average length of the genes is about 851.15 bp.Te calculation method of (GC)/(G + C) was used to perform GC skew analysis and produce a schematic representation of the draft genome of Bacillus cereus ZR72-1, as detailed in Fig. 4. It has become clear from the results that the genome size was approximately 5.754 Mb, with a G + C content (Table 1). Table 1 Molecular characteristics of the genome of Bacillus cereus ZR72-1 Characteristics Bacillus cereus ZR72-1 Length (bp) GC content(%) No. of plasmid Total bases (bp) Length variation (range in bp) Average length (bp) Repeat ratio (%) Repeat region count(bp) No. of ribosomal RNA No. of transfer RNA 5,754,801 35.21 0 4,818,285 90 − 15,033 840 0.35 20323 42 107 The gene functional annotation consisted of eggNOG, GO,KEGG, were got by aligning the coding genes in the Bacillus cereus ZR72-1 genome using the BLAST database. Among the general function annotation results, eggNOG GO and KEGG database annotations were more useful, accounting for 69.6, 76.2,and 43.6% genes, respectively.While gene ontology (GO) annotated 4346 genes as 46 GO terms. As shown in Fig. S3, the supreme number of functional genes was predicted in the Metabolic Process domain.The eggNOG database was annotated to 3972 genes which were assigned to 25 functional groups.As shown in Fig. S4, among the groups, a large number of genes involved in Carbohydrate transport and metabolism were the most abundant,followed by proteins involved in Coenzyme transport and metabolism. Analysis of degradation characteristics in genome of Bacillus cereus ZR72-1 To better comprehend the metabolic pathways of Bacillus cereus ZR72-1, we analyzed the genes identifed by the KEGG database. A total of 2491 genes were annotated into 46 biological pathways in the KEGG database, Occupying 43.6% of all coding genes ,of which 127 genes were assorted as carbohydrate metabolism genes, 144 genes were assorted as amino acid metabolism genes, and 151 genes were assorted as membrane transport genes.(Fig. 5 ) Genes clearly associated with the metabolism of substances account for 80%, encompassing most of the microbial metabolic pathways and the catabolic pathways of secondary metabolites.In particular, the degradation pathways of Xenobiotics biodegradation and metabolism and aromatic compounds,such as naphthalene degradation, chlorinated cyclohexane and chlorobenzene degradation, benzoate degradation, chlorinated alkanes and chlorinated alkenes degradation, nitrotoluene degradation, styrene degradation, aminobenzoate ester degradation, and aromatic compound degradation, all fall under the category of hydrocarbons. Some of these substances contain carbon-carbon double bonds similar to those found in the molecular formula of polycyclic aromatic hydrocarbons. It is speculated that strains capable of degrading these substances may possess the ability to biodegrade polycyclic aromatic hydrocarbons, including Benzo(a)pyrene..Among these,it was analyzed that 16 genes were associated with benzoate in which, EC:1.13.11.2(Catechol 2,3-dioxygenase)were found to take a crucial role in benzoate degradation.(Fig. 6 ) Furthermore, there were 67 genes annotated with exogenous metabolism in the Bacillus cereus ZR72-1 genome(Table S1 ), among which four were concerned with the degradation pathway of Chlorocyclohexane and Chlorobenzene degradation, ten were concerned with the degradation pathway of Benzoate, and three were concerned with the degradation pathway of Xylene degradation, Chloroalkane and Chloroalkene degradation, Naphthalene degradation, Nitrotoluene degradation, Styrene degradation, indicating that strain Bacillus cereus ZR72-1 has the potential to metabolize other types of Polycyclic aromatic hydrocarbons.Above all, there are many unknown functional genes in Bacillus cereus ZR72-1 that are worthy of further mining and analysis, and they have great potential for application in exploitation of microbial resources in the food industry. Prediction and screening of Benzo(a)pyrenedegrading related genes With the advancement of sequencing technology in recent years, it has become increasingly accurate and feasible to utilize genomic sequencing techniques to discover the complete genomic data of polycyclic aromatic hydrocarbon-degrading strains. Collecting genomic information of Benzo(a)pyrene-degrading bacteria can contribute to the exploration of their degradation mechanisms towards Benzo(a)pyrene.Even though multi-ring aromatic hydrocarbons with different molecular weights have their own distinctive degradation pathways, the enzymes involved in the degradation process are closely connected. Most multi-ring aromatic hydrocarbons utilized the same set of enzymes in the degradation of the first benzene ring or subsequent degradation pathways. According to the annotation results from the KEGG database, key enzymes related to the degradation of Benzo(a)pyrene were detected in the genome data of Bacillus cereus ZR72-1. The genes encoding these enzymes are dmpB and xylE, which code for the enzymes catechol 2,3-dioxygenase. In addition, there are monooxygenases and dehydrogenases present. Therefore, it can be inferred that this strain primarily degrades Benzo(a)pyrene through the Benzoate metabolism pathway.After comparing the amino acid sequence of the reported Benzo(a)pyrene degrading enzyme with the gene information of the ZR72-1 genome, 12 degrading enzymes with high homology were screened, as shown in Fig. 7 . Discussion In recent years, as human health issues has garnered widespread attention, there has been an increasing focus on research into preventive and control measures for toxic substances in food.The edible microbial decomposing action and mechanism of toxic substances in food have become a hot topic and trend in research[ 21 – 22 ].In 2021, Sultana et al.[ 23 ]conducted a study to isolate probiotics from fermented foods and test their ability to degrade or detoxify polycyclic aromatic hydrocarbons (PAHs). They isolated five different morphological types of probiotics from a mixture of 26 fermented food cultures co-cultured with Benzo(a)pyrene. These findings demonstrate the potential of fermented food microorganisms in biodegrading and removing toxic PAHs from the human body.In the non-targeted metabolomics analysis of microorganisms in the Kafir grain, intermediate metabolites related to the degradation of Benzo(a)pyrene have been detected[ 24 ].It is highly likely that the degrading bacteria possess key genes for metabolizing Benzo(a)pyrene and can produce key enzymes for the initial biochemical reaction in the degradation of Benzo(a)pyrene. The mechanism of bacterial degradation of Benzo(a)pyrene is as follows: Under aerobic conditions, bacteria typically use initial dioxygenases to attack the benzene ring of Benzo(a)pyrene. Due to the diversity of degradation strains, different microorganisms will choose different positions of the benzene ring for attack. Whether Benzo(a)pyrene can be completely mineralized to CO 2 and H 2 O depends on the degree of cleavage of the benzene ring[ 25 ].Different microorganisms metabolize Benzo(a)pyrene to produce different metabolites, but they eventually enter the tricarboxylic acid cycle. Pseudomonas metabolizes Benzo(a)pyrene to produce salicylic acid and catechol, which then enter the tricarboxylic acid cycle[ 26 ]. Mycobacterium, Aeromonas, Alcaligenes, Microcoecus, and Bacillus metabolize Benzo(a)pyrene to produce phthalic acid and protocatechuic acid, which then enter the tricarboxylic acid cycle[ 27 ]. There are also some strains, such as Mycobacterium sp. US6-1, that have multiple pathways for metabolizing Benzo(a)pyrene[ 28 ]. The degradation of pollutants involves a series of sequential steps mediated by different enzymes. Various oxidative enzymes, including monooxygenases and dioxygenases, play a role in the metabolism of polycyclic aromatic hydrocarbons (PAHs). Enzymes involved in decomposition and metabolism, such as oxygenases (monooxygenases and dioxygenases) and dehydrogenases, contribute to the conversion of PAHs into CO 2 and H 2 O[ 28 – 29 ].During the degradation process of aromatic compounds containing benzene rings, oxygenases are required. Microbial oxygenases mainly include dioxygenases and monooxygenases. Bacteria produce dioxygenases to cleave the benzene ring and add two oxygen atoms to the substrate, forming 1,2-dihydroxyethane. It is then further oxidized to cis-1,2-dihydroxyethanol. The cis-1,2-dihydroxyethanol can be further oxidized to intermediate metabolites such as catechol, protocatechuic acid, and gentisic acid. Then, the benzene ring is cleaved, resulting in the production of succinic acid, p-hydroxybenzoic acid, acetic acid, pyruvic acid, and acetaldehyde[ 30 ].Common bacterial dioxygenases can be classified into ring-opening dioxygenases, ring-hydroxylating dioxygenases, and terminal dioxygenases based on the different forms of ring cleavage. Tyrosine dioxygenase is a typical ring-opening dioxygenase, with tyrosine 1,2-dioxygenase being an intradiol enzyme and tyrosine 2,3-dioxygenase being an extradiol enzyme[ 31 ]..Catechol 2,3-dioxygenase is abundantly present in bacteria that degrade polycyclic aromatic hydrocarbons. Bacillus is a Gram-positive rod-shaped bacterium that can degrade polycyclic aromatic hydrocarbons (PAHs). There are several genes involved in the degradation of PAHs in Gram-positive bacteria. The first gene is nar, which is commonly found in Rhodococcus, a classic Gram-positive bacterium that can degrade PAHs and other xenobiotic compounds[ 32 ]. The second gene is phd, which is commonly found in Mycobacterium sp. SNP11[ 33 ] and Nocardioides sp. KP7[ 34 ]. The third gene is nid, which was first sequenced in Mycobacterium sp. PYR-1 and is involved in the degradation of pollutants such as phenanthrene, pyrene, anthracene, and Benzo(a)pyrene[ 35 ].The research conducted by Wei Kun et al.[ 36 ]indicates that both catechol 1,2-dioxygenase and catechol 2,3-dioxygenase are key enzymes in the degradation of Pyr by Bacillus cereus . The annotation results from the KEGG database show that catechol 2,3-dioxygenase plays a crucial role in the degradation of Benzo(a)pyrene by Bacillus cereus ZR72-1, as screened in Kefir.According to the metabolite analysis conducted by Krivobok et al. [ 37 ], it is hypothesized that Bacillus cereus ZR72-1 strain can oxidize various polycyclic aromatic hydrocarbons using multiple dioxygenases and monooxygenases. Previous studies have demonstrated that the intermediate compounds formed during PAHs removal contained carboxyl and hydroxyl groups in the presence and absence of oxygen. The compounds phenol, benzoate, salicylic acid and phthalic acid[ 38 – 39 ].In the genomic data of Bacillus cereus ZR72-1, key enzymes related to the degradation of Benzo(a)pyrene were detected. The genes encoding these enzymes are dmpB and xylE, which code for catechol 2,3-dioxygenase. Additionally, there are monooxygenases and dehydrogenases present. Therefore, it can be inferred that this strain primarily degrades Benzo(a)pyrene through the benzoate acid metabolic pathway. Kotoky rhitu et al.[ 40 ]indicates that Melia azedarach-rhizosphere mediated degradation of Benzo(a)pyrene of Serratia marcescens S2I7was identifed to contain operons for protocatechuate degradation, catechol degradation, benzoate degradation. With the advancements in sequencing technology in recent years, leveraging genomic sequencing techniques has become increasingly accurate and feasible in excavating the complete genome data of polycyclic aromatic hydrocarbon-degrading strains. Gathering genomic information of Benzo(a)pyrene-degrading bacteria can contribute to exploring the degradation mechanism of these strains towards Benzo(a)pyrene. Metabolic mechanism studies primarily involve the analysis of metabolic pathways and degradation enzyme genes, which vary significantly among different polycyclic aromatic hydrocarbons in microbial degradation processes.Up to now, there has been little research on the functional genes and enzymes involved in Benzo(a)pyrene degradation.Due to the advancement of biotechnology and the emergence of new methods, there is increasing attention on the important role of microorganisms in the degradation of polycyclic aromatic hydrocarbons (PAHs). Currently, research on the degradation pathways of low molecular weight PAHs is relatively clear, but there is limited study on the metabolism pathways of high molecular weight PAHs. The study of the microbial degradation mechanism of PAHs is essential for understanding the microbial ability to transform PAHs and the environmental impacts of their metabolites.However, it is also important to study the metabolites generated during the degradation of pollutants, as there are extensive interactions among the metabolites produced during the biodegradation of pollutants. Therefore, metabolomics is a powerful tool for elucidating the mechanisms of biodegradation/biotransformation of Benzo(a)pyrene by studying the subsequent analysis of degrading bacterial strains. Conclusion In this study, a Benzo(a)pyrene strain was isolated from Xinjiang Kefir and identified as Bacillus cereus ZR72-1,there was 69.39% degradation rate of 20 mg/L Benzo(a)pyrene by strain ZR72-1 after incubation for 72 h. The genome of the test strain Bacillus cereus ZR72-1 consists of a total length of 5,745,611 base pairs, with a GC content of 35.24%. It is predicted to have a total of 5,719 coding genes. Through functional database annotation, it was found that there are 219 genes involved in the transportation and metabolism of hydrocarbons in this strain, as well as 9 metabolic pathways related to the degradation and metabolism of exogenous substances. There are a total of 67 genes encoding these functions. In the genome data of Bacillus cereus ZR72-1, key enzymes related to the degradation of Benzo(a)pyrene, namely catechol 2,3-dioxygenase, were detected. The corresponding genes encoding these enzymes are dmpB and xylE. Additionally, monooxygenases and dehydrogenases were also found, suggesting that this strain mainly degrades Benzo(a)pyrene through the salicylic acid metabolic pathway. Declarations Acknowledgements We thank Aofei Jin and Lirong Tan for help with the screening and identifcation of strain TL106. We also thank Yanan Qin and Zhixian Duo for technical assistance. All authors participated in the writing of the manuscript and agreed with the final format. Authors ’ contributions Dilibaier Tuerxun and Aofei Jin conceived and designed the experiments. Lirong Tan, Zhuonan Yang,Zhixian Duo contributed to the samples collection and performed the experiments. Dilibaier Tuerxun performed the bioinformatics analysis and wrote the frst draft of manuscript. Rui Zhang and Yanan Qin revised the fnal manuscript. All the authors contributed to the article and approved the submitted version. Funding This work was supported by Natural Science Foundation of Xinjiang Uygur Autonomous Region(2022D01D42),, the National Natural Science Foundation of China (31560440),Natural Science Foundation of Xinjiang (grant numbers 2022D01C404). Availability of data and materials All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials. Ethics approval and consent to participate The study were approved and instructed by the Xinjiang Key Laboratory of Special Species Conservation and Regulatory Biology and Normal University, Xinjiang, China Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests. References Ma L, Xing J, Li A, et al. Changes of microbial community structure in different fermentation stages of Kefir[J]. Modern Food Science and Technology, 2019, 35(8): 27-34+26.) Wang X ,ZHou ZH H,ZHao X L,et al.The harm and its detection techniques of benzo(a)pyrene[J].Cereals and Oils,2011(03):48-49. Bakircioglu D, Topraksever N, Yurtsever S, et al. 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Modern perspectives on the health benefits of kefir in next generation sequencing era: Improvement of the host gut microbiota. Critical reviews in food science and nutrition,59(11), 1782–1793. https://doi.org/10.1080/10408398.2018.1428168 Baars, T., van Esch, B., van Ooijen, L., Zhang, Z., Dekker, P., Boeren, S., Diks, M., Garssen, J., Hettinga, K., & Kort, R. (2023). Raw milk kefir: microbiota, bioactive peptides, and immune modulation.Food & function,14(3), 1648–1661. https://doi.org/10.1039/d2fo03248a Dilbar T,Guljamal A,ZHeng B,et al.Selection of benzo (a) pyrene degrading strains in Kefir and optimization of degradation conditions.[J/OL].Food and Fermentation Industries,1-11[2023-12-25] https://doi.org/10.13995/j.cnki.11-1802/ts.036763. Nachvak S M, Soleimani D, Gholizadeh S, et al. Kebab, A Delicious Food, but Contaminated with Harmful Compounds: A Literature Review[J]. Journal of Isfahan Medical School, 2021, 39(626): 376-383. Shoukat, S., Liu, Y., Rehman, A., & Zhang, B. (2019). Screening of Bifidobacterium strains with assignment of functional groups to bind with benzo[a]pyrene under food stress factors.Journal of chromatography. B, Analytical technologies in the biomedical and life sciences,1114-1115, 100–109. https://doi.org/10.1016/j.jchromb.2019.03.024 Sultana O, Lee S, Seo H, et al. Biodegradation and Removal of PAHs byBacillus velezensisIsolated from Fermented Food[J]. Journal of microbiology biotechnology, 2021, 31 (7):999-1010. Aisa G, Xing J, Li A, et al. Microbial para benzo in Kefir grains(a)Non targeted metabonomic analysis of pyrene [J]. Biotechnology Bulletin , 2022,38 (6): 18-29. ZHang X N. Optimization of conditions for the degradation of benzopyrene by Bacillus M1 and its mechanism research.[D]. Hebei Agricultural University，2017. Ma J. Screening of Polycyclic Aromatic Hydrocarbon Degrading Bacteria, Investigation of Degradation Mechanisms and Performance Studies[D]. Dalian University of Technology, 2013. Bouchez, M., Blanchet, D., & Vandecasteele, J. P. (1995). Degradation of polycyclic aromatic hydrocarbons by pure strains and by defined strain associations: inhibition phenomena and cometabolism. Applied microbiology and biotechnology,43(1), 156–164. https://doi.org/10.1007/BF00170638 Cui H. Screening, identification, and study of the degradation characteristics of a strain of polycyclic aromatic hydrocarbon-degrading bacteria.[D]. Jilin Agricultural University, 2013. Lyu, Y., Zheng, W., Zheng, T., & Tian, Y. (2014). Biodegradation of polycyclic aromatic hydrocarbons by Novosphingobium pentaromativorans US6-1.PloS one,9(7), e101438. https://doi.org/10.1371/journal.pone.0101438 ZHang J and ZHeng T Progress in controlling and bioremediation of polycyclic aromatic hydrocarbon pollution in the environment.[J]. Fujian Environment, 2002, 19 (2):26-29. Cao x, Tian W, Hu j, et al. Research progress on genes and enzymes involved in the degradation of PAHs.[J]. Journal of Ecology, 2007, 26 (6):917-924. Kulakov, L. A., Chen, S., Allen, C. C., & Larkin, M. J. (2005). Web-type evolution of rhodococcus gene clusters associated with utilization of naphthalene.Applied and environmental microbiology,71(4), 1754–1764. https://doi.org/10.1128/AEM.71.4.1754-1764.2005 Pagnout, C., Frache, G., Poupin, P., Maunit, B., Muller, J. F., & Férard, J. F. (2007). Isolation and characterization of a gene cluster involved in PAH degradation in Mycobacterium sp. strain SNP11: expression in Mycobacterium smegmatis mc(2)155. Research in microbiology, 158(2), 175–186. https://doi.org/10.1016/j.resmic.2006.11.002 ZHang D, Li ZH G，Bao X G ，et al. Research progress on the Bacterial Degradation of Naphthalene and Phenanthrene, [J]. Journal of Genetics and Genomics, 26 (06):726-734. Kumari, S., Regar, R. K., Bajaj, A., Ch, R., Satyanarayana, G. N. V., Mudiam, M. K. R., & Manickam, N. (2017). Simultaneous Biodegradation of Polyaromatic Hydrocarbons by a Stenotrophomonas sp: Characterization of nid Genes and Effect of Surfactants on Degradation.Indian journal of microbiology,57(1), 60–67. https://doi.org/10.1007/s12088-016-0612-6 Wei K, CHen SH M, YiH, et al Research on the degradation characteristics of polycyclic aromatic hydrocarbonpyrene and the associated degradation enzymes in Bacillus subtilis.[J]. Journalof Environmental Sciences, 2016, 36 (2):7. Krivobok, S., Kuony, S., Meyer, C., Louwagie, M., Willison, J. C., & Jouanneau, Y. (2003). Identification of pyrene-induced proteins in Mycobacterium sp. strain 6PY1: evidence for two ring-hydroxylating oxygenates.Journal of bacteriology,185(13), 3828–3841. https://doi.org/10.1128/JB.185.13.3828-3841.2003 Zhou, X., Lei, D., Tang, J., Wu, M., Ye, H., & Zhang, Q. (2022). Whole genome sequencing and analysis of fenvalerate degrading bacteria Citrobacter freundii CD-9.AMB Express,12(1), 51. https://doi.org/10.1186/s13568-022-01392-z Qin, W., Fan, F., Zhu, Y., Wang, Y., Liu, X., Ding, A., & Dou, J. (2017). Comparative proteomic analysis and characterization of benzo(a)pyrene removal by Microbacterium sp. strain M.CSW3 under denitrifying conditions.Bioprocess and biosystems engineering,40(12), 1825–1838. https://doi.org/10.1007/s00449-017-1836-5 Kotoky, R., & Pandey, P. (2020). Rhizosphere assisted biodegradation of benzo(a)pyrene by cadmium resistant plant-probiotic Serratia marcescens S2I7, and its genomic traits.Scientific reports,10(1), 5279. https://doi.org/10.1038/s41598-020-62285-4 Additional Declarations No competing interests reported. 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University","correspondingAuthor":false,"prefix":"","firstName":"rui","middleName":"","lastName":"zhang","suffix":""},{"id":271304559,"identity":"7136dcc6-b43a-4345-8a53-2df29e37925e","order_by":2,"name":"yanan qin","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAq0lEQVRIiWNgGAWjYDACZgbGBww8IFYC8VqYDUjUwsDAJgGhidXCd5z3WDWPzGEGfvYcA4afO4jQInmYL+3mDJ7DDJI9bwwYe88QocXgMI/ZjQ9ALQY3cgyYGduI1FKQANRiT5IWBrAtEsRqkTzMYyw5gyedR+LMs4KDvcRo4Tt/xvAzb4+1HH978sYHP4nRwnAAiBl7IJF5gBgNUGU/iFM7CkbBKBgFIxQAAIDAL+yxHIeaAAAAAElFTkSuQmCC","orcid":"","institution":"Xinjiang University","correspondingAuthor":true,"prefix":"","firstName":"yanan","middleName":"","lastName":"qin","suffix":""},{"id":271304560,"identity":"1aed215a-4414-4a7d-9f15-46defe6a3762","order_by":3,"name":"aofei jin","email":"","orcid":"","institution":"Xinjiang Normal 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11:59:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3856829/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3856829/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":50811332,"identity":"c57e5f58-aeca-476a-afed-21744c0f63e8","added_by":"auto","created_at":"2024-02-07 18:06:40","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":43630,"visible":true,"origin":"","legend":"\u003cp\u003eScanning electron micrograph of Strain\u003cem\u003e \u003c/em\u003eZR72-1\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-3856829/v1/5dfb89cdd8ed6c6407400397.jpeg"},{"id":50811330,"identity":"8804f0d8-810b-445e-8711-1293b3bca1b6","added_by":"auto","created_at":"2024-02-07 18:06:40","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":138280,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic tree based on the 16S rRNA sequence of strain ZR72-1\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-3856829/v1/b72d75aacf2d027209407f1a.png"},{"id":50811329,"identity":"57dc8f3f-0d51-47ba-9224-c20819c1db5d","added_by":"auto","created_at":"2024-02-07 18:06:40","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":47014,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eBacillus cereus \u003c/em\u003eZR72-1degradation of Benzo(a)pyrene\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-3856829/v1/b00f2ad5c9601395a4bd7f83.png"},{"id":50811333,"identity":"65da30a0-deb3-4b7a-bde3-5c3b95faff08","added_by":"auto","created_at":"2024-02-07 18:06:41","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":598362,"visible":true,"origin":"","legend":"\u003cp\u003eWhole genome \u003cem\u003eBacillus cereus \u003c/em\u003eZR72-1 The first circle is contigs and corresponding length information, the second circle is GC content information, the third circle is the corresponding base sequencing depth, the fourth circle and the fifth circle are CDS, rRNA, tRNA distribution information, and the sixth circle and the seventh circle are the corresponding COG function classification. (From outmost to innermost)\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-3856829/v1/db9b068efd7a47d8e3ed5499.png"},{"id":50811334,"identity":"9fec22b4-6f8e-4a05-8edd-5e738153c1b2","added_by":"auto","created_at":"2024-02-07 18:06:41","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":66894,"visible":true,"origin":"","legend":"\u003cp\u003eThe map of KEGG pathway classifcation histogram\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-3856829/v1/b08a194e71944f382c30c07c.png"},{"id":50811549,"identity":"0218833b-6674-47f4-ba9c-c3a021de2fa3","added_by":"auto","created_at":"2024-02-07 18:14:41","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":196912,"visible":true,"origin":"","legend":"\u003cp\u003eDegradation pathway of Benzoate.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-3856829/v1/4941426374e3f6f752e9de57.png"},{"id":50811337,"identity":"92dd8e22-2035-4a0e-a0be-45fdc2e90fdf","added_by":"auto","created_at":"2024-02-07 18:06:41","extension":"jpeg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":65084,"visible":true,"origin":"","legend":"\u003cp\u003eThe evolutionary relationship between twelve predicted proteins in the \u003cem\u003eBacillus cereus\u003c/em\u003e ZR72-1 genome and the reported proteins that degrade Benzo(a)pyrene\u003c/p\u003e","description":"","filename":"floatimage7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-3856829/v1/5da287ebbd2fc9463223757e.jpeg"},{"id":51740417,"identity":"c5ab454d-8e62-413e-b6cb-6a9cb4d8dee0","added_by":"auto","created_at":"2024-02-28 08:35:31","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1490486,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3856829/v1/2be0262d-a646-41cf-8aef-6d7f88f2129f.pdf"},{"id":50811335,"identity":"4afa48e6-611a-4620-8eae-4d480cac7ec2","added_by":"auto","created_at":"2024-02-07 18:06:41","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":437021,"visible":true,"origin":"","legend":"","description":"","filename":"Additionalfile1.docx","url":"https://assets-eu.researchsquare.com/files/rs-3856829/v1/862774577ea6c4bdee27cebf.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Whole Genome Sequencing and Analysis of Benzo(a)pyrene Degrading Bacteria Bacillus cereus ZR72-1","fulltext":[{"header":"Background","content":"\u003cp\u003eBenzo(a)pyrene is a polycyclic aromatic hydrocarbon compound composed of five Benzene rings. It is one of the three major carcinogens identified by the World Health Organization and exhibits three carcinogenic effects (carcinogenicity, teratogenicity, and mutagenicity)[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e].The main sources of Benzo(a)pyrene in food are two fold. First, it can be absorbed by crops from dust and particles in the air, which eventually end up in the final products that humans rely on for sustenance, second, Benzo(a)pyrene can be formed and accumulated during heating processes such as baking, smoking, and frying of food[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].One effective strategy for the removal of Benzo(a)pyrene is microbial degradation. This strategy involves utilizing the pollutant as a carbon source to stimulate the degradation ability of microorganisms, resulting in the partial or complete removal of these pollutants[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].Microbial degradation has several advantages compared to physical and chemical methods, such as thorough degradation, lower cost, and absence of secondary pollution[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eBarnsley et al[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. made the discovery that Pseudomonas NCIB 9816, Pseudomonas ATCC 17483, and Pseudomonas putida PpG7 are capable of utilizing Benzo(a)pyrene in the presence of succinic acid and salicylic acid.Grosser et al.[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]used radiolabeled \u003csup\u003e14\u003c/sup\u003eC-Benzo(a)pyrene mineralized to \u003csup\u003e14\u003c/sup\u003eC-CO\u003csub\u003e2\u003c/sub\u003e, providing strong evidence that the genus Mycobacterium sp. is capable of utilizing Benzo(a)pyrene. Hunter et al.[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]in 2005 demonstrated that Bacillus subtilis Tgr3 (a soil bacterium) can use Benzo(a)pyrene as a carbon source and degrade it. Bacillus subtilis BMT4, isolated from soil samples enriched under Benzo(a)pyrene stress, was able to utilize Benzo(a)pyrene as a carbon and energy source and degrade approximately 85% of 50 mg\u0026middot;L\u0026thinsp;\u0026minus;\u0026thinsp;1 Benzo(a)pyrene within 28 days of incubation[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e],indicating the presence of microorganisms in the environment capable of degrading Benzo(a)pyrene.\u003c/p\u003e \u003cp\u003eWhile microorganisms are effective at degrading Benzo(a)pyrene, their safety limits their direct application in food. Therefore, the screening of strains that are considered safe (GRAS) and have high degradation capabilities for Benzo(a)pyrene can address these issues. Halttunen et al.[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]reported several strains of lactic acid bacteria that may be beneficial for the removal of various toxic compounds.Considering the potential inhibitory activity of probiotics on polycyclic aromatic hydrocarbons (PAHs) formed in food, Abou-Arab et al.[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]studied the degradation of 16 PAH compounds (each at a concentration of 0.25 mg/L in the incubation medium) in MRS medium using different fermentation stages of Bifidobacterium bifidum, Streptococcus thermophilus, and Lb. bulgaricus. The removal abilities of PAHs were as follows: Lb. bulgaricus (91.5%), Streptococcus thermophilus (87.7%), and Lb. bulgaricus (46.6%). The potential ability of probiotics to biodegrade and eliminate toxic PAHs from the human body makes them applicable for the biodegradation of foodborne Benzo(a)pyrene.\u003c/p\u003e \u003cp\u003eKefir is a fermented dairy product made from the milk of cows (goats, sheep, camels, water buffalo) using kefir grains[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. It is acidic, easily digestible, and has various potential health benefits. The main microorganisms in kefir include \u003cem\u003eLactobacillus\u003c/em\u003e, \u003cem\u003eLactococcus\u003c/em\u003e, \u003cem\u003eAcetobacter\u003c/em\u003e, \u003cem\u003eLeuconostoc\u003c/em\u003e, \u003cem\u003eKazachstania\u003c/em\u003e, \u003cem\u003eKluyveromyces\u003c/em\u003e, \u003cem\u003eNaumovozyma\u003c/em\u003e, and \u003cem\u003eSaccharomyces\u003c/em\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Research has shown that the microorganisms in kefir can also regulate the composition of the gut microbiota, as well as intestinal permeability, oxidative stress, and inflammation. The probiotics or fermentation products in kefir play an important role in these processes[\u003cspan additionalcitationids=\"CR18\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn this study, the strain \u003cem\u003eBacillus cereus\u003c/em\u003e ZR72-1 was selected from the Kefir microbial community for its ability to degrade Benzo(a)pyrene. The strain was subjected to whole genome sequencing using PacBio technology. By obtaining the complete genome sequence of the Benzo(a)pyrene degrading strain, the genes of the degrading strain were annotated using the KEGG database to predict the key enzymes and coding genes involved in the degradation of Benzo(a)pyrene. These research findings provide data support for further exploration of the mechanism of benzop(α)yrene degradation by \u003cem\u003eBacillus cereus\u003c/em\u003e ZR72-1.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStrains isolation and identification\u003c/h2\u003e \u003cp\u003eKefir was sampled from Akto County ,Xinjiang Uygur Autonomous Region, China.Five grams of Kefir fermented liquor was added to Erlenmeyer flasks containing 100mL MSM supplemented with 20mgL-1 Benzo(a)pyrene.Cultures were incubated at 37\u0026deg;C and 120rpm for 72 h for enrichment.Thereafter,10mL of culture was sampled every 72 h and transferred to fresh MSM,Bacterial strains that degraded Benzo(a)pyrene in the Kefir were isolated and purified following the procedure described by Dilbar and Guljamal[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. All media, pipette tips, and Erlenmeyer flasks were sterilized by autoclaving at 121℃ for20 min before use.\u003c/p\u003e \u003cp\u003eMorphological characteristics and Gram staining were determined using an optical microscope after incubation for 24h at 37\u0026deg;C.Amplification was performed using 16S rRNA universal primers 27F(5'-AGAGTTTGA TCCTGGCTCAG-3') and 1492R(5'-GGTTACTTGTTACGACTT-3').PCR products were purified and sequenced by Sangon Biotech (Shanghai, China).. The resulting 16S rRNA gene sequences were submitted to GenBank,analyzed the homology of gene sequence and and submitted to NCBI for bacterial acquisition.Use GenBank login number to download sequences with high homology and construct a phylogenetic tree using MEGA11.0 software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eChemicals and reagents\u003c/h2\u003e \u003cp\u003eMinimal Salt Medium (MSM,pH 7.0)contained NH4NO3 1.00 g/L,MgSO4\u0026middot;7H2O 0.20 g/L, KH2PO4 0.50 g/L,K2HPO4 1.50 g/L,NaCl 0.50 g/L,(NH4)2SO4 0.50g/L.Solid medium plates were prepared by adding 18\u0026thinsp;~\u0026thinsp;20 gL-1 agar into the abovementioned liquid media. All media were sterilized by autoclaving at 121℃ for 30min.Benzo(a)pyrene (analytically pure, \u0026ge; 99%), acetone, dichloromethane (all chromatographically pure, \u0026ge; 99.9%),Methanol (chromatographically pure, \u0026ge; 99.9%), purchased from Beijing Dingguo Changsheng Biotechnology Co., Ltd;\u003c/p\u003e \u003cp\u003e \u003cb\u003eBiodegradation of Benzo(a)pyrene by\u003c/b\u003e \u003cb\u003eBacillus cereus\u003c/b\u003e \u003cb\u003eZR72-1\u003c/b\u003e\u003c/p\u003e \u003cp\u003eOne milliliters of this bacterial culture was add an equal volume of chromatography grade dichloromethane to extracted Benzo(a)pyrene, vortex mixed for 30 seconds, and thoroughly mixed,ultrasound extracted at 40 ℃ for 10 minutes, centrifuged it at 12000 r/min for 8 minutes, and removed of the lower organic phase. Add an equal volume of dichloromethane to the upper incubation solution for further extraction, merge the organic phase, and achieve a pore size of 0.45\u0026micro;m organic phase filtered, HPLC detected.All experiments were performed three times.\u003c/p\u003e \u003cp\u003eHPLC conditions:C18 Diamosil TM reverse phase column: chromatographic column (250 mm \u0026times; 4.6 mm, particle size 5 \u0026micro;m) ; Mobile phase: pure methanol/water (volume ratio 100/0); UV detector: wavelength 245 nm; Injection volume: 20 \u0026micro; L; Column temperature: 34 ℃; Retention time: 10 minutes.The degradation rate was calculated using the following equation:\u003c/p\u003e \u003cp\u003eThe degradation rate (%) = (1\u0026thinsp;\u0026minus;\u0026thinsp;C/C\u003csub\u003e0\u003c/sub\u003e) \u0026times; 100\u003c/p\u003e \u003cp\u003ewhere C and C\u003csub\u003e0\u003c/sub\u003e represent the Benzo(a)pyrene concentrations in the inoculated and non-inoculated media, respectively.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eWhole genome sequencing\u003c/h2\u003e \u003cp\u003eCollect bacterial cultures in the logarithmic phase from MC incubation medium. Transfer an appropriate volume of bacterial solution into a 2 mL centrifuge tube and centrifuge at room temperature at 14000 r/min for 1 minute. Discard the supernatant, precipitate the bacterial cells and quickly freeze them in liquid nitrogen for at least 1\u0026ndash;3 h (the freezing time depends on the tissue volume to ensure sufficient sample freezing), and then transfer them to -80 ℃ for long-term preservation, which is applied to the extraction of bacterial genomes.According to the instructions of the bacterial genome extraction kit, complete the extraction of bacterial genome DNA. Combine the 260/280 ratio measured by the microplate reader with the 1% agarose gel electrophoresis detection results to conduct quality detection on the extracted bacterial genome DNA. After the quality inspection is qualified, build a HiFi 8\u0026ndash;20 Kb sequencing library based on the Pacbio platform for sequencing, and obtain the sequencing data.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003ePrediction of Benzo(a)pyrene degradation genes\u003c/h2\u003e \u003cp\u003eHifiasm software was used for assemb, Circulator v1.5.5 software used for cyclization and adjusting the starting site.Then Pilon v1.22 software was used for further error correction. When good accuracy genemo was generated Prodigal v2.6.3, ,KEGG databases (Kyoto Encyclopedia of Genes and Genomes, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.genome.jp/kegg/\u003c/span\u003e\u003cspan address=\"http://www.genome.jp/kegg/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) were used for gene prediction and annotation.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eIsolation and Identifcation of\u003c/strong\u003e \u003cstrong\u003eBacillus cereus\u003c/strong\u003e \u003cstrong\u003eZR72-1\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA strain with efective Benzo(a)pyrene-degrading ability was obtained following enrichment and purifcation from the medium with Kefir as energy sources, and was named ZR72-1.Strain ZR72-1 was a Gram-positive bacterium with a rod-like shape (Fig. \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e) and the morphological characteristics of the colonies of strain M7-4 were as follows: ivory, rough surface, irregular edges and protruding edges, rod-like, and short (Fig. S2).Phylogenetic tree displayed the results of 16S rRNA gene sequencing analysis for strain ZR72-1 (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e).The 16S rRNA gene sequence of the strain had a 99% bootstrap value identical to the 16S rRNA gene of \u003cem\u003eBacillus cereus\u003c/em\u003e AFA01.Therefore, the isolated strain ZR72-1 was referred to as \u003cem\u003eBacillus cereus\u003c/em\u003e ZR72-1. The 16S rRNA gene sequence of \u003cem\u003eBacillus cereus\u003c/em\u003e ZR72-1 was submitted to the GenBank database (Accession No. OM570322).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBenzo(a)pyrene degradation Characteristics of\u003c/strong\u003e \u003cstrong\u003eBacillus cereus\u003c/strong\u003e \u003cstrong\u003eZR72-1\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePeak area-mass linear fitting was performed with the different concentrations of different Benzo(a)pyrene, and the respective standard curve equations were obtained, as shown in Fig. S2.In the standard curve equation, y is the HPLC peak area of the Benzo(a)pyrene (mAU*s) and x is the mass of the Benzo(a)pyrene (mg).\u003c/p\u003e\n\u003cp\u003eThe degradation rates of Benzo(a)pyrene when incubated with \u003cem\u003eBacillus cereus\u003c/em\u003e ZR72-1 are shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e.It could be seen that the strains had a broad degradation spectrum for Benzo(a)pyrene,the degradation rate of Benzo(a)pyrene by strain ZR72-1 gradually increases. At 72 hours, the degradation rate of Benzo(a)pyrene reaches approximately 69.39%, indicating that the strain has reached its maximum degradation rate in the incubation medium. After this point, the degradation rate starts to decline and no longer consumes carbon sources. Therefore, we can conclude that strain ZR72-1 has the highest degradation rate at 72 hours.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWhole genome analysis of\u003c/strong\u003e \u003cstrong\u003eBacillus cereus\u003c/strong\u003e \u003cstrong\u003eZR72-1\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGenomic information helps to generate insights into the mechanisms of Benzo(a)pyrene degradation by bacteria. Hence, we carried out whole-genome sequencing using PacBio Sequencing technology platform to decipher the complete set of genes involved in the degradation of Benzo(a)pyrene .The whole genome information was deposited at NCBI(\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.ncbi.nlm.nih.gov).Th\u003c/span\u003e\u003c/span\u003ee genome sequence length is 5754801 bp, and the GC-content is 35.21%. It contains 107 tRNAs, 42 rRNAs, and fourteen 16S-23S-5S rRNAs. It is composed of 5732 coding genes. The total length of the Coding region is 4818215 bp. The average length of the predicted genes is 840 bp, and the average length of the genes is about 851.15 bp.Te calculation method of (GC)/(G\u0026thinsp;+\u0026thinsp;C) was used to perform GC skew analysis and produce a schematic representation of the draft genome of \u003cem\u003eBacillus cereus\u003c/em\u003e ZR72-1, as detailed in Fig.\u0026nbsp;4. It has become clear from the results that the genome size was approximately 5.754 Mb, with a G\u0026thinsp;+\u0026thinsp;C content (Table\u0026nbsp;1).\u003c/p\u003e\n\u003cp\u003eTable 1 Molecular characteristics of the genome of \u003cem\u003eBacillus cereus\u003c/em\u003e\u0026nbsp; ZR72-1\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003ctable id=\"Taba\" border=\"1\"\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eCharacteristics\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003eBacillus cereus\u003c/em\u003e ZR72-1\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eLength (bp)\u003c/p\u003e\n\u003cp\u003eGC content(%)\u003c/p\u003e\n\u003cp\u003eNo. of plasmid\u003c/p\u003e\n\u003cp\u003eTotal bases (bp)\u003c/p\u003e\n\u003cp\u003eLength variation (range in bp)\u003c/p\u003e\n\u003cp\u003eAverage length (bp)\u003c/p\u003e\n\u003cp\u003eRepeat ratio (%)\u003c/p\u003e\n\u003cp\u003eRepeat region count(bp)\u003c/p\u003e\n\u003cp\u003eNo. of ribosomal RNA\u003c/p\u003e\n\u003cp\u003eNo. of transfer RNA\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e5,754,801\u003c/p\u003e\n\u003cp\u003e35.21\u003c/p\u003e\n\u003cp\u003e0\u003c/p\u003e\n\u003cp\u003e4,818,285\u003c/p\u003e\n\u003cp\u003e90\u0026thinsp;\u0026minus;\u0026thinsp;15,033\u003c/p\u003e\n\u003cp\u003e840\u003c/p\u003e\n\u003cp\u003e0.35\u003c/p\u003e\n\u003cp\u003e20323\u003c/p\u003e\n\u003cp\u003e42\u003c/p\u003e\n\u003cp\u003e107\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eThe gene functional annotation consisted of eggNOG, GO,KEGG, were got by aligning the coding genes in the Bacillus cereus ZR72-1 genome using the BLAST database. Among the general function annotation results, eggNOG GO and KEGG database annotations were more useful, accounting for 69.6, 76.2,and 43.6% genes, respectively.While gene ontology (GO) annotated 4346 genes as 46 GO terms. As shown in Fig. S3, the supreme number of functional genes was predicted in the Metabolic Process domain.The eggNOG database was annotated to 3972 genes which were assigned to 25 functional groups.As shown in Fig. S4, among the groups, a large number of genes involved in Carbohydrate transport and metabolism were the most abundant,followed by proteins involved in Coenzyme transport and metabolism.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnalysis of degradation characteristics in genome of\u003c/strong\u003e \u003cstrong\u003eBacillus cereus\u003c/strong\u003e \u003cstrong\u003eZR72-1\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo better comprehend the metabolic pathways of \u003cem\u003eBacillus cereus\u003c/em\u003e ZR72-1, we analyzed the genes identifed by the KEGG database. A total of 2491 genes were annotated into 46 biological pathways in the KEGG database, Occupying 43.6% of all coding genes ,of which 127 genes were assorted as carbohydrate metabolism genes, 144 genes were assorted as amino acid metabolism genes, and 151 genes were assorted as membrane transport genes.(Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e)\u003c/p\u003e\n\u003cp\u003eGenes clearly associated with the metabolism of substances account for 80%, encompassing most of the microbial metabolic pathways and the catabolic pathways of secondary metabolites.In particular, the degradation pathways of Xenobiotics biodegradation and metabolism and aromatic compounds,such as naphthalene degradation, chlorinated cyclohexane and chlorobenzene degradation, benzoate degradation, chlorinated alkanes and chlorinated alkenes degradation, nitrotoluene degradation, styrene degradation, aminobenzoate ester degradation, and aromatic compound degradation, all fall under the category of hydrocarbons. Some of these substances contain carbon-carbon double bonds similar to those found in the molecular formula of polycyclic aromatic hydrocarbons. It is speculated that strains capable of degrading these substances may possess the ability to biodegrade polycyclic aromatic hydrocarbons, including Benzo(a)pyrene..Among these,it was analyzed that 16 genes were associated with benzoate in which, EC:1.13.11.2(Catechol 2,3-dioxygenase)were found to take a crucial role in benzoate degradation.(Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e)\u003c/p\u003e\n\u003cp\u003eFurthermore, there were 67 genes annotated with exogenous metabolism in the \u003cem\u003eBacillus cereus\u003c/em\u003e ZR72-1 genome(Table \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e), among which four were concerned with the degradation pathway of Chlorocyclohexane and Chlorobenzene degradation, ten were concerned with the degradation pathway of Benzoate, and three were concerned with the degradation pathway of Xylene degradation, Chloroalkane and Chloroalkene degradation, Naphthalene degradation, Nitrotoluene degradation, Styrene degradation, indicating that strain \u003cem\u003eBacillus cereus\u003c/em\u003e ZR72-1 has the potential to metabolize other types of Polycyclic aromatic hydrocarbons.Above all, there are many unknown functional genes in \u003cem\u003eBacillus cereus\u003c/em\u003e ZR72-1 that are worthy of further mining and analysis, and they have great potential for application in exploitation of microbial resources in the food industry.\u003c/p\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n\u003ch2\u003ePrediction and screening of Benzo(a)pyrenedegrading related genes\u003c/h2\u003e\n\u003cp\u003eWith the advancement of sequencing technology in recent years, it has become increasingly accurate and feasible to utilize genomic sequencing techniques to discover the complete genomic data of polycyclic aromatic hydrocarbon-degrading strains. Collecting genomic information of Benzo(a)pyrene-degrading bacteria can contribute to the exploration of their degradation mechanisms towards Benzo(a)pyrene.Even though multi-ring aromatic hydrocarbons with different molecular weights have their own distinctive degradation pathways, the enzymes involved in the degradation process are closely connected. Most multi-ring aromatic hydrocarbons utilized the same set of enzymes in the degradation of the first benzene ring or subsequent degradation pathways.\u003c/p\u003e\n\u003cp\u003eAccording to the annotation results from the KEGG database, key enzymes related to the degradation of Benzo(a)pyrene were detected in the genome data of \u003cem\u003eBacillus cereus\u003c/em\u003e ZR72-1. The genes encoding these enzymes are dmpB and xylE, which code for the enzymes catechol 2,3-dioxygenase. In addition, there are monooxygenases and dehydrogenases present. Therefore, it can be inferred that this strain primarily degrades Benzo(a)pyrene through the Benzoate metabolism pathway.After comparing the amino acid sequence of the reported Benzo(a)pyrene degrading enzyme with the gene information of the ZR72-1 genome, 12 degrading enzymes with high homology were screened, as shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn recent years, as human health issues has garnered widespread attention, there has been an increasing focus on research into preventive and control measures for toxic substances in food.The edible microbial decomposing action and mechanism of toxic substances in food have become a hot topic and trend in research[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].In 2021, Sultana et al.[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]conducted a study to isolate probiotics from fermented foods and test their ability to degrade or detoxify polycyclic aromatic hydrocarbons (PAHs). They isolated five different morphological types of probiotics from a mixture of 26 fermented food cultures co-cultured with Benzo(a)pyrene. These findings demonstrate the potential of fermented food microorganisms in biodegrading and removing toxic PAHs from the human body.In the non-targeted metabolomics analysis of microorganisms in the Kafir grain, intermediate metabolites related to the degradation of Benzo(a)pyrene have been detected[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].It is highly likely that the degrading bacteria possess key genes for metabolizing Benzo(a)pyrene and can produce key enzymes for the initial biochemical reaction in the degradation of Benzo(a)pyrene.\u003c/p\u003e \u003cp\u003eThe mechanism of bacterial degradation of Benzo(a)pyrene is as follows: Under aerobic conditions, bacteria typically use initial dioxygenases to attack the benzene ring of Benzo(a)pyrene. Due to the diversity of degradation strains, different microorganisms will choose different positions of the benzene ring for attack. Whether Benzo(a)pyrene can be completely mineralized to CO\u003csub\u003e2\u003c/sub\u003e and H\u003csub\u003e2\u003c/sub\u003eO depends on the degree of cleavage of the benzene ring[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].Different microorganisms metabolize Benzo(a)pyrene to produce different metabolites, but they eventually enter the tricarboxylic acid cycle. Pseudomonas metabolizes Benzo(a)pyrene to produce salicylic acid and catechol, which then enter the tricarboxylic acid cycle[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Mycobacterium, Aeromonas, Alcaligenes, Microcoecus, and Bacillus metabolize Benzo(a)pyrene to produce phthalic acid and protocatechuic acid, which then enter the tricarboxylic acid cycle[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. There are also some strains, such as Mycobacterium sp. US6-1, that have multiple pathways for metabolizing Benzo(a)pyrene[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe degradation of pollutants involves a series of sequential steps mediated by different enzymes. Various oxidative enzymes, including monooxygenases and dioxygenases, play a role in the metabolism of polycyclic aromatic hydrocarbons (PAHs). Enzymes involved in decomposition and metabolism, such as oxygenases (monooxygenases and dioxygenases) and dehydrogenases, contribute to the conversion of PAHs into CO\u003csub\u003e2\u003c/sub\u003e and H\u003csub\u003e2\u003c/sub\u003eO[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].During the degradation process of aromatic compounds containing benzene rings, oxygenases are required. Microbial oxygenases mainly include dioxygenases and monooxygenases. Bacteria produce dioxygenases to cleave the benzene ring and add two oxygen atoms to the substrate, forming 1,2-dihydroxyethane. It is then further oxidized to cis-1,2-dihydroxyethanol. The cis-1,2-dihydroxyethanol can be further oxidized to intermediate metabolites such as catechol, protocatechuic acid, and gentisic acid. Then, the benzene ring is cleaved, resulting in the production of succinic acid, p-hydroxybenzoic acid, acetic acid, pyruvic acid, and acetaldehyde[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].Common bacterial dioxygenases can be classified into ring-opening dioxygenases, ring-hydroxylating dioxygenases, and terminal dioxygenases based on the different forms of ring cleavage. Tyrosine dioxygenase is a typical ring-opening dioxygenase, with tyrosine 1,2-dioxygenase being an intradiol enzyme and tyrosine 2,3-dioxygenase being an extradiol enzyme[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]..Catechol 2,3-dioxygenase is abundantly present in bacteria that degrade polycyclic aromatic hydrocarbons.\u003c/p\u003e \u003cp\u003eBacillus is a Gram-positive rod-shaped bacterium that can degrade polycyclic aromatic hydrocarbons (PAHs). There are several genes involved in the degradation of PAHs in Gram-positive bacteria. The first gene is nar, which is commonly found in Rhodococcus, a classic Gram-positive bacterium that can degrade PAHs and other xenobiotic compounds[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. The second gene is phd, which is commonly found in Mycobacterium sp. SNP11[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e] and Nocardioides sp. KP7[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. The third gene is nid, which was first sequenced in Mycobacterium sp. PYR-1 and is involved in the degradation of pollutants such as phenanthrene, pyrene, anthracene, and Benzo(a)pyrene[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e].The research conducted by Wei Kun et al.[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]indicates that both catechol 1,2-dioxygenase and catechol 2,3-dioxygenase are key enzymes in the degradation of Pyr by \u003cem\u003eBacillus cereus\u003c/em\u003e. The annotation results from the KEGG database show that catechol 2,3-dioxygenase plays a crucial role in the degradation of Benzo(a)pyrene by \u003cem\u003eBacillus cereus\u003c/em\u003e ZR72-1, as screened in Kefir.According to the metabolite analysis conducted by Krivobok et al. [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e], it is hypothesized that \u003cem\u003eBacillus cereus\u003c/em\u003e ZR72-1 strain can oxidize various polycyclic aromatic hydrocarbons using multiple dioxygenases and monooxygenases.\u003c/p\u003e \u003cp\u003ePrevious studies have demonstrated that the intermediate compounds formed during PAHs removal contained carboxyl and hydroxyl groups in the presence and absence of oxygen. The compounds phenol, benzoate, salicylic acid and phthalic acid[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e].In the genomic data of \u003cem\u003eBacillus cereus\u003c/em\u003e ZR72-1, key enzymes related to the degradation of Benzo(a)pyrene were detected. The genes encoding these enzymes are dmpB and xylE, which code for catechol 2,3-dioxygenase. Additionally, there are monooxygenases and dehydrogenases present. Therefore, it can be inferred that this strain primarily degrades Benzo(a)pyrene through the benzoate acid metabolic pathway. Kotoky rhitu et al.[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]indicates that Melia azedarach-rhizosphere mediated degradation of Benzo(a)pyrene of \u003cem\u003eSerratia marcescens\u003c/em\u003e S2I7was identifed to contain operons for protocatechuate degradation, catechol degradation, benzoate degradation.\u003c/p\u003e \u003cp\u003eWith the advancements in sequencing technology in recent years, leveraging genomic sequencing techniques has become increasingly accurate and feasible in excavating the complete genome data of polycyclic aromatic hydrocarbon-degrading strains. Gathering genomic information of Benzo(a)pyrene-degrading bacteria can contribute to exploring the degradation mechanism of these strains towards Benzo(a)pyrene. Metabolic mechanism studies primarily involve the analysis of metabolic pathways and degradation enzyme genes, which vary significantly among different polycyclic aromatic hydrocarbons in microbial degradation processes.Up to now, there has been little research on the functional genes and enzymes involved in Benzo(a)pyrene degradation.Due to the advancement of biotechnology and the emergence of new methods, there is increasing attention on the important role of microorganisms in the degradation of polycyclic aromatic hydrocarbons (PAHs). Currently, research on the degradation pathways of low molecular weight PAHs is relatively clear, but there is limited study on the metabolism pathways of high molecular weight PAHs. The study of the microbial degradation mechanism of PAHs is essential for understanding the microbial ability to transform PAHs and the environmental impacts of their metabolites.However, it is also important to study the metabolites generated during the degradation of pollutants, as there are extensive interactions among the metabolites produced during the biodegradation of pollutants. Therefore, metabolomics is a powerful tool for elucidating the mechanisms of biodegradation/biotransformation of Benzo(a)pyrene by studying the subsequent analysis of degrading bacterial strains.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn this study, a Benzo(a)pyrene strain was isolated from Xinjiang Kefir and identified as \u003cem\u003eBacillus cereus\u003c/em\u003e ZR72-1,there was 69.39% degradation rate of 20 mg/L Benzo(a)pyrene by strain ZR72-1 after incubation for 72 h. The genome of the test strain \u003cem\u003eBacillus cereus\u003c/em\u003e ZR72-1 consists of a total length of 5,745,611 base pairs, with a GC content of 35.24%. It is predicted to have a total of 5,719 coding genes. Through functional database annotation, it was found that there are 219 genes involved in the transportation and metabolism of hydrocarbons in this strain, as well as 9 metabolic pathways related to the degradation and metabolism of exogenous substances. There are a total of 67 genes encoding these functions. In the genome data of \u003cem\u003eBacillus cereus\u003c/em\u003e ZR72-1, key enzymes related to the degradation of Benzo(a)pyrene, namely catechol 2,3-dioxygenase, were detected. The corresponding genes encoding these enzymes are dmpB and xylE. Additionally, monooxygenases and dehydrogenases were also found, suggesting that this strain mainly degrades Benzo(a)pyrene through the salicylic acid metabolic pathway.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Aofei Jin and Lirong Tan for help with the screening and identifcation of strain TL106. We also thank Yanan Qin and Zhixian Duo for technical assistance. All authors participated in the writing of the manuscript and agreed with the final format.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u003c/strong\u003e\u003cstrong\u003e\u0026rsquo;\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDilibaier Tuerxun and Aofei Jin conceived and designed the experiments. Lirong Tan, Zhuonan Yang,Zhixian Duo contributed to the samples collection and performed the experiments. Dilibaier Tuerxun performed the bioinformatics analysis and wrote the frst draft of manuscript. Rui Zhang and Yanan Qin revised the fnal manuscript. All the authors contributed to the article and approved the submitted version.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by Natural Science Foundation of Xinjiang Uygur Autonomous Region(2022D01D42),, the National Natural Science Foundation of China (31560440),Natural Science Foundation of Xinjiang (grant numbers 2022D01C404).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study were approved and instructed by the Xinjiang Key Laboratory of Special Species Conservation and Regulatory Biology and Normal University, Xinjiang, China\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eMa L, Xing J, Li A, et al. 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Rhizosphere assisted biodegradation of benzo(a)pyrene by cadmium resistant plant-probiotic Serratia marcescens S2I7, and its genomic traits.Scientific reports,10(1), 5279. https://doi.org/10.1038/s41598-020-62285-4 \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Bacillus cereus ZR72-1, Benzo(a)pyrene, Biodegradation, Genomics","lastPublishedDoi":"10.21203/rs.3.rs-3856829/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3856829/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eBenzo (a) pyrene produced by food during high-temperature process enters the body through ingestion, which causes food safety issues to the human body. In order to alleviate the harm of foodborne benzo (a) pyrene to human health, a strain that can degrade benzo (a) pyrene was screened from Kefir, a traditional fermented product in Xinjiang.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003e \u003cem\u003eBacillus cereus\u003c/em\u003e ZR72-1 is a Gram-positive bacteria sourced from XinJiang traditional fermented product Kefir, under Benzo(a)pyrene stress conditions, there was 69.39% degradation rate of 20 mg/L Benzo(a)pyrene by strain ZR72-1 after incubation for 72 h. The whole genome of ZR72-1 sequenced using PacBio sequencing technology was reported in this study. The genome size was 5754801 bp and a GC content was 35.24%, a total of 5719 coding genes were predicted bioinformatically. Through functional database annotation, it was found that the strain has a total of 219 genes involved in the transportation and metabolism of hydrocarbons, a total of 9 metabolic pathways related to the degradation and metabolism of exogenous substances, and a total of 67 coding genes.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eAccording to the KEGG database annotation results, a key enzyme related to Benzo(a)pyrene degradation, catechol 2,3-dioxygenase, was detected in the genome data of \u003cem\u003eBacillus cereus\u003c/em\u003e ZR72-1, encoding genes dmpB and xylE, respectively; There are also monooxygenases and dehydrogenases. Therefore, it can be inferred that this strain mainly degrades Benzo(a)pyrene through Benzoate metabolic.\u003c/p\u003e","manuscriptTitle":"Whole Genome Sequencing and Analysis of Benzo(a)pyrene Degrading Bacteria Bacillus cereus ZR72-1","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-02-07 18:06:36","doi":"10.21203/rs.3.rs-3856829/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"889ee1d9-3f27-4f00-b73f-a31813bfb1e0","owner":[],"postedDate":"February 7th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-02-28T08:34:57+00:00","versionOfRecord":[],"versionCreatedAt":"2024-02-07 18:06:36","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3856829","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3856829","identity":"rs-3856829","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}