Structural and Biochemical Insights into Bis(2-hydroxyethyl) Terephthalate Degrading Carboxylesterase Isolated from Psychrotrophic Bacterium Exiguobacterium antarcticum.

This study aimed to elucidate the crystal structure and biochemically characterize the carboxylesterase EaEst2, a thermotolerant biocatalyst derived from Exiguobacterium antarcticum, a psychrotrophic bacterium. Sequence and phylogenetic analyses showed that EaEst2 belongs to the Family XIII group of carboxylesterases. EaEst2 has a broad range of substrate specificities for short-chain p-nitrophenyl (pNP) esters, 1-naphthyl acetate (1-NA), and 1-naphthyl butyrate (1-NB). Its optimal pH is 7.0, losing its enzymatic activity at temperatures above 50 °C. EaEst2 showed degradation activity toward bis(2-hydroxyethyl) terephthalate (BHET), a polyethylene terephthalate degradation intermediate. We determined the crystal structure of EaEst2 at a 1.74 Å resolution in the ligand-free form to investigate BHET degradation at a molecular level. Finally, the biochemical stability and immobilization of a crosslinked enzyme aggregate (CLEA) were assessed to examine its potential for industrial application. Overall, the structural and biochemical characterization of EaEst2 demonstrates its industrial potency as a biocatalyst.

Feruloyl Esterase (LaFae) from Lactobacillus acidophilus: Structural Insights and Functional Characterization for Application in Ferulic Acid Production.

Ferulic acid and related hydroxycinnamic acids, used as antioxidants and preservatives in the food, cosmetic, pharmaceutical and biotechnology industries, are among the most abundant phenolic compounds present in plant biomass. Identification of novel compounds that can produce ferulic acid and hydroxycinnamic acids, that are safe and can be mass-produced, is critical for the sustainability of these industries. In this study, we aimed to obtain and characterize a feruloyl esterase (LaFae) from Lactobacillus acidophilus. Our results demonstrated that LaFae reacts with ethyl ferulate and can be used to effectively produce ferulic acid from wheat bran, rice bran and corn stalks. In addition, xylanase supplementation was found to enhance LaFae enzymatic hydrolysis, thereby augmenting ferulic acid production. To further investigate the active site configuration of LaFae, crystal structures of unliganded and ethyl ferulate-bound LaFae were determined at 2.3 and 2.19 Å resolutions, respectively. Structural analysis shows that a Phe34 residue, located at the active site entrance, acts as a gatekeeper residue and controls substrate binding. Mutating this Phe34 to Ala produced an approximately 1.6-fold increase in LaFae activity against p-nitrophenyl butyrate. Our results highlight the considerable application potential of LaFae to produce ferulic acid from plant biomass and agricultural by-products.

Structural and biochemical insights into PsEst3, a new GHSR-type esterase obtained from Paenibacillus sp. R4.

PsEst3, a psychrophilic esterase obtained from Paenibacillus sp. R4, which was isolated from the permafrost of Alaska, exhibits relatively high activity at low temperatures. Here, crystal structures of PsEst3 complexed with various ligands were generated and studied at atomic resolution, and biochemical studies were performed to analyze the structure-function relationship of PsEst3. Certain unique characteristics of PsEst3 distinct from those of other classes of lipases/esterases were identified. Firstly, PsEst3 contains a conserved GHSRA/G pentapeptide sequence in the GxSxG motif around the nucleophilic serine. Additionally, it contains a conserved HGFR/K consensus sequence in the oxyanion hole, which is distinct from that in other lipase/esterase families, as well as a specific domain composition (for example a helix-turn-helix motif) and a degenerative lid domain that exposes the active site to the solvent. Secondly, the electrostatic potential of the active site in PsEst3 is positive, which may cause unintended binding of negatively charged chemicals in the active site. Thirdly, the last residue of the oxyanion hole-forming sequence, Arg44, separates the active site from the solvent by sealing the acyl-binding pocket, suggesting that PsEst3 is an enzyme that is customized to sense an unidentified substrate that is distinct from those of classical lipases/esterases. Collectively, this evidence strongly suggests that PsEst3 belongs to a distinct family of esterases.

Crystal structure and biochemical analysis of acetylesterase (LgEstI) from Lactococcus garvieae.

Esterase, a member of the serine hydrolase family, catalyzes the cleavage and formation of ester bonds with high regio- and stereospecificity, making them attractive biocatalysts for the synthesis of optically pure molecules. In this study, we performed an in-depth biochemical and structural characterization of a novel microbial acetylesterase, LgEstI, from the bacterial fish pathogen Lactococcus garvieae. The dimeric LgEstI displayed substrate preference for the short acyl chain of p-nitrophenyl esters and exhibited increased activity with F207A mutation. Comparative analysis with other esterases indicated that LgEstI has a narrow and shallow active site that may exhibit substrate specificity to short acyl chains. Unlike other esterases, LgEstI contains bulky residues such as Trp89, Phe194, and Trp217, which block the acyl chain channel. Furthermore, immobilized LgEstI retained approximately 90% of its initial activity, indicating its potential in industrial applications. This study expands our understanding of LgEstI and proposes novel ideas for improving its catalytic efficiency and substrate specificity for various applications.

Gene family expansions in Antarctic winged midge as a strategy for adaptation to cold environments.

Parochlus steinenii is the only flying insect native to Antarctica. To elucidate the molecular mechanisms underlying its adaptation to cold environments, we conducted comparative genomic analyses of P. steinenii and closely related lineages. In an analysis of gene family evolution, 68 rapidly evolving gene families, involved in the innate immune system, unfolded protein response, DNA packaging, protein folding, and unsaturated fatty acid biosynthesis were detected. Some gene families were P. steinenii-specific and showed phylogenetic instability. Acyl-CoA delta desaturase and heat shock cognate protein 70 (Hsc70) were representative gene families, showing signatures of positive selection with multiple gene duplication events. Acyl-CoA delta desaturases may play pivotal roles in membrane fluidity, and expanded Hsc70 genes may function as chaperones or thermal sensors in cold environments. These findings suggest that multiple gene family expansions contributed to the adaptation of P. steinenii to cold environments.

Safety evaluation of mycotoxin citrinin production from Monascus ruber through whole-genome sequencing and analytical evaluation.

In this study, the whole genome of Monascus ruber KACC 46666 was generated using the PacBio RSII sequencer with high-quality de novo assembly to obtain trustworthy assembly and annotation using genome assemblies with long reads from PacBio single-molecule real-time sequencing. The whole genome of M. ruber has a total length of 25.9 Mb, divided in 13 contigs with 9639 genes. The functions of genes involved in secondary metabolite production were further analyzed. Gene clusters involved in the production of Monascus pigment, monacolin K, and mycotoxin citrinin were identified. Notably, most of the citrinin gene cluster was lost, as confirmed via high-performance liquid chromatography analysis. This genome-level safety evaluation of industrially important Monascus strains will provide valuable information for genome-based microbial engineering of natural food colorants and production of commercially important secondary metabolites such as monacolin K.

Investigating the Functional Role of the Cysteine Residue in Dehydrin from the Arctic Mouse-Ear Chickweed Cerastium arcticum.

The stress-responsive, SK5 subclass, dehydrin gene, CaDHN, has been identified from the Arctic mouse-ear chickweed Cerastium arcticum. CaDHN contains an unusual single cysteine residue (Cys143), which can form intermolecular disulfide bonds. Mutational analysis and a redox experiment confirmed that the dimerization of CaDHN was the result of an intermolecular disulfide bond between the cysteine residues. The biochemical and physiological functions of the mutant C143A were also investigated by in vitro and in vivo assays using yeast cells, where it enhanced the scavenging of reactive oxygen species (ROS) by neutralizing hydrogen peroxide. Our results show that the cysteine residue in CaDHN helps to enhance C. arcticum tolerance to abiotic stress by regulating the dimerization of the intrinsically disordered CaDHN protein, which acts as a defense mechanism against extreme polar environments.

Bioactive Terphenyls Isolated from the Antarctic Lichen Stereocaulon alpinum.

Three p-terphenyls (2-4)-2-hydroxy-3,5-dimethoxy-p-terphenyl (2), 2-hydroxy-3,6-dimethoxy-p-terphenyl (3), and 2,3,5,6-tetramethoxy-p-terphenyl (4)-were isolated for the first time as natural products along with seven known compounds (1, 5-10) from the Antarctic lichen Stereocaulon alpinum. Structures of the new compounds were elucidated by comprehensive analyses of 1D and 2D NMR and HREIMS experiments. Compound 3 exhibited cytotoxicity against HCT116 cells with the IC50 value of 3.76 ± 0.03 µM and also inhibited NO production in LPS-induced RAW264.7 macrophages with the IC50 value of 22.82 ± 0.015 µM.

The complete chloroplast genome sequence of a medicinal citrus landrace, Citrus erythrosa Hort. ex Tanaka in Jeju Island, Korea.

Citrus erythrosa (Dongjeongkyool in Korean) is a medicinal citrus landrace that grows in Korea. In this study, we characterized the complete chloroplast (Cp) genome (160,120 bp) of C. erythrosa. The Cp genome was consisted of 4 distinct regions: a large single copy (87,731 bp), a small single copy (18,393 bp), and a pair of inverted repeat regions (26,998 bp). The Cp genome encodes a total of 133 genes including 88 protein-coding genes, 37 tRNA genes and 8 rRNA genes. The phylogenetic analysis reveals that C. erythrosa is a sister group to the clade of species including C. reticulata within the genus Citrus.

Hymenobacter siberiensis sp. nov., isolated from a marine sediment of the East Siberian Sea and Hymenobacter psoromatis sp. nov., isolated from an Antarctic lichen.

Gram-stain-negative, strictly aerobic, red-pink-coloured, rod-shaped and non-motile bacterial strains PAMC 29290, PAMC 29294T and PAMC 29296 were isolated from marine surface sediment sampled in the East Siberian Sea and strains PAMC 26553 and PAMC 26554T were obtained from an Antarctic lichen. Strains PAMC 29290, PAMC 29294T and PAMC 29296 were closely related to Hymenobacter artigasi (98.8 % 16S rRNA gene similarity), Hymenobacter antarcticus (97.3 %) and Hymenobacter glaciei (96.9 %), and PAMC 26553 and PAMC 26554T showed high similarity to Hymenobacter ginsengisoli (97.0 %), Hymenobacter rivuli (96.1 %) and Hymenobacter setariae (95.9 %). Genomic relatedness analyses showed that strains PAMC 29290, PAMC 29294T and PAMC 29296 could be distinguished from H. artigasi by average nucleotide identity (ANI; 93.1-93.2 %) and digital DNA-DNA hybridization (dDDH; 50.3-51.0 %) values. Strains PAMC 26553 and PAMC 26554T could be clearly distinguished from H. ginsengisoli with ANI values <79.8 % and dDDH values <23.3 %. The major fatty acids of strains PAMC 29290, PAMC 29294T and PAMC 29296 were C15 : 0 iso (21.0-26.0 %), summed feature 3 (C16 : 1 ω7c and/or C16 : 1 ω6c; 17.4-18.2 %), C15 : 0 anteiso (12.7-19.1 %) and summed feature 4 (C17 : 1 iso I and/or anteiso B; 8.6-16.1 %) and those of strains PAMC 26553 and PAMC 26554T were summed feature 3 (C16 : 1 ω7c and/or C16 : 1 ω6c; 20.7-22.2 %), C15 : 0 anteiso (17.5-19.7 %) and summed feature 4 (C17 : 1 iso I and/or anteiso B; 15.5-18.1 %). The major respiratory quinone was MK-7. The genomic DNA G+C contents were 60.6-60.8 mol%. The polar lipids of PAMC 29294T were found to consist of phosphatidylethanolamine, four unidentified aminolipids, an unidentified aminophospholipid and five unidentified lipids; those of PAMC 26554T were phosphatidylethanolamine, three unidentified aminolipids, four unidentified aminophospholipid and two unidentified lipids. The distinct phylogenetic position and some physiological characteristics distinguished the novel strains from closely related type strains in the genus Hymenobacter. Thus, two novel species are proposed, with the names Hymenobacter siberiensis sp. nov. (type strain, PAMC 29294T=KCTC 82466T=JCM 34574T) and Hymenobacter psoromatis sp. nov. (type strain, PAMC 26554T=KCTC 82464T=JCM 34572T), respectively.

Complete genome sequence of Rhodoferax sp. PAMC 29310 from a marine sediment of the East Siberian Sea.

Rhodoferax sp. PAMC 29310 was isolated from a surface marine sediment of the East Siberian Sea, Arctic. Whole-genome sequencing of the strain Rhodoferax sp. PAMC 29310 was achieved using PacBio RS II and Illumina platform. The resulting complete genome comprised of 4,593,249 base pairs (G + C content of 58.0%) with a single chromosome, 4546 protein-coding genes, 57 tRNAs and 6 rRNA operons. A complete set of genes encoding the enzymes of glycolysis and citric acid cycle were identified. No genes encoding ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) and nitrogenase reductase (nif) were present indicating that strain PAMC 29310 is not capable of fixing of carbon and nitrogen. PAMC 29310 genome contains genes for dissimilatory and assimilatory nitrate reduction. Gene encoding choline dehydrogenase enzyme which functions at the first step in the synthesis of betaine, one of the most effective osmoprotectants, was detected. In particular, among the genomes of the genus Rhodoferax strains, gene encoding nitrite reductase (nirK), which reduces nitrite to nitric oxide and tetA gene encoding tetracycline resistance protein involved in the resistance to tetracycline were identified only in the genome of Rhodoferax sp. PAMC 29310. As the first genome from the strain which was isolated from marine sediment in the genus Rhodoferax, investigation of physiological characteristics based on the complete genome sequences will help understand the adaptation of Rhodoferax sp. PAMC 29310 in the marine sediment.

Characterization of antimicrobial resistance genes and virulence factor genes in an Arctic permafrost region revealed by metagenomics.

Antimicrobial resistance genes (ARGs) and virulence factor genes (VFGs) constitute a serious threat to public health, and climate change has been predicted to affect the increase in bacterial pathogens harboring ARGs and VFGs. However, studies on bacterial pathogens and their ARGs and VFGs in permafrost region have received limited attention. In this study, a metagenomic approach was applied to a comprehensive survey to detect potential ARGs, VFGs, and pathogenic antibiotic resistant bacteria (PARB) carrying both ARGs and VFGs in the active layer and permafrost. Overall, 70 unique ARGs against 18 antimicrobial drug classes and 599 VFGs classified as 38 virulence factors were detected in the Arctic permafrost region. Eight genes with mobile genetic elements (MGEs) carrying ARGs were identified; most MGEs were classified as phages. In the metagenome-assembled genomes, the presence of 15 PARB was confirmed. The soil profile showed that the transcripts per million (TPM) values of ARGs and VFGs in the sub-soil horizon were significantly lower than those in the top soil horizon. Based on the TPM value of each gene, major ARGs, VFGs, and these genes in PARB from the Arctic permafrost region were identified and their distribution was confirmed. The major host bacteria for ARGs and VFGs and PARB were identified. A comparison of the percentage identity distribution of ARGs and VFGs to reference databases indicated that ARGs and VFGs in the Arctic soils differ from previously identified genes. Our results may help understand the characteristics and distribution of ARGs, VFGs, and these genes in PARB in the Arctic permafrost region. This findings suggest that the Arctic permafrost region may serve as potential reservoirs for ARGs, VFGs, and PARB. These genes could pose a new threat to human health if they are released by permafrost thawing owing to global warming and propagate to other regions.

CRISPR/Cas9-mediated Inactivation of arginase in a yeast strain isolated from Nuruk and its impact on the whole genome.

Despite the advantages of CRISPR/Cas9 technology in the food industry, controversy over its off-target effects exists. We engineered an industrial Saccharomyces cerevisiae strain isolated from a Korean rice wine starter, Nuruk, using CRISPR/Cas9 to decrease ethyl carbamate (EC) formation. We disrupted the CAR1 gene encoding arginase, which plays a key role in EC formation. Subsequently, we compared the whole genome of the engineered strain to that of the wild type by analyzing heterozygous and homozygous mutations through variant calling. Homozygous mutations in the genome of the engineered strains were identified as the target mutations in CAR1 induced by CRISPR/Cas9, and no other off-target effects were observed. Our findings have critical implications for the use of CRISRP/Cas9 technology in yeasts in the food industry.

Crystal structure of a MarR family protein from the psychrophilic bacterium Paenisporosarcina sp. TG-14 in complex with a lipid-like molecule.

MarR family proteins regulate the transcription of multiple antibiotic-resistance genes and are widely found in bacteria and archaea. Recently, a new MarR family gene was identified by genome analysis of the psychrophilic bacterium Paenisporosarcina sp. TG-14, which was isolated from sediment-laden basal ice in Antarctica. In this study, the crystal structure of the MarR protein from Paenisporosarcina sp. TG-14 (PaMarR) was determined at 1.6 Šresolution. In the crystal structure, a novel lipid-type compound (palmitic acid) was found in a deep cavity, which was assumed to be an effector-binding site. Comparative structural analysis of homologous MarR family proteins from a mesophile and a hyperthermophile showed that the DNA-binding domain of PaMarR exhibited relatively high mobility, with a disordered region between the ß1 and ß2 strands. In addition, structural comparison with other homologous complex structures suggests that this structure constitutes a conformer transformed by palmitic acid. Biochemical analysis also demonstrated that PaMarR binds to cognate DNA, where PaMarR is known to recognize two putative binding sites depending on its molar concentration, indicating that PaMarR binds to its cognate DNA in a stoichiometric manner. The present study provides structural information on the cold-adaptive MarR protein with an aliphatic compound as its putative effector, extending the scope of MarR family protein research.

Disproportionate microbial responses to decadal drainage on a Siberian floodplain.

Permafrost thaw induces soil hydrological changes which in turn affects carbon cycle processes in the Arctic terrestrial ecosystems. However, hydrological impacts of thawing permafrost on microbial processes and greenhouse gas (GHG) dynamics are poorly understood. This study examined changes in microbial communities using gene and genome-centric metagenomics on an Arctic floodplain subject to decadal drainage, and linked them to CO2 and CH4 flux and soil chemistry. Decadal drainage led to significant changes in the abundance, taxonomy, and functional potential of microbial communities, and these modifications well explained the changes in CO2 and CH4 fluxes between ecosystem and atmosphere-increased fungal abundances potentially increased net CO2 emission rates and highly reduced CH4 emissions in drained sites corroborated the marked decrease in the abundance of methanogens and methanotrophs. Interestingly, various microbial taxa disproportionately responded to drainage: Methanoregula, one of the key players in methanogenesis under saturated conditions, almost disappeared, and also Methylococcales methanotrophs were markedly reduced in response to drainage. Seven novel methanogen population genomes were recovered, and the metabolic reconstruction of highly correlated population genomes revealed novel syntrophic relationships between methanogenic archaea and syntrophic partners. These results provide a mechanistic view of microbial processes regulating GHG dynamics in the terrestrial carbon cycle, and disproportionate microbial responses to long-term drainage provide key information for understanding the effects of warming-induced soil drying on microbial processes in Arctic wetland ecosystems.

Mitochondrial genome of the Antarctic microalga Micractinium simplicissimum KSF0127 (Chlorellaceae, Trebouxiophyceae).

We report the first mitochondrial genome of the Antarctic microalga Micractinium simplicissimum KSF0127. The circular mitochondrial genome was 67,923 bp in length and contained 45 protein-coding genes, one ribosomal RNA gene, and 60 transfer RNA genes. The phylogenetic tree was constructed with eight previously reported mitogenome sequences and showed the phylogenetic position of M. simplicissimum KSF0127 within the Chlorellaceae family.

Screening and Genetic Network Analysis of Genes Involved in Freezing and Thawing Resistance in DaMDHAR-Expressing Saccharomyces cerevisiae Using Gene Expression Profiling.

The cryoprotection of cell activity is a key determinant in frozen-dough technology. Although several factors that contribute to freezing tolerance have been reported, the mechanism underlying the manner in which yeast cells respond to freezing and thawing (FT) stress is not well established. Therefore, the present study demonstrated the relationship between DaMDHAR encoding monodehydroascorbate reductase from Antarctic hairgrass Deschampsia antarctica and stress tolerance to repeated FT cycles (FT2) in transgenic yeast Saccharomyces cerevisiae. DaMDHAR-expressing yeast (DM) cells identified by immunoblotting analysis showed high tolerance to FT stress conditions, thereby causing lower damage for yeast cells than wild-type (WT) cells with empty vector alone. To detect FT2 tolerance-associated genes, 3'-quant RNA sequencing was employed using mRNA isolated from DM and WT cells exposed to FT (FT2) conditions. Approximately 332 genes showed ≥2-fold changes in DM cells and were classified into various groups according to their gene expression. The expressions of the changed genes were further confirmed using western blot analysis and biochemical assay. The upregulated expression of 197 genes was associated with pentose phosphate pathway, NADP metabolic process, metal ion homeostasis, sulfate assimilation, ß-alanine metabolism, glycerol synthesis, and integral component of mitochondrial and plasma membrane (PM) in DM cells under FT2 stress, whereas the expression of the remaining 135 genes was partially related to protein processing, selenocompound metabolism, cell cycle arrest, oxidative phosphorylation, and α-glucoside transport under the same condition. With regard to transcription factors in DM cells, MSN4 and CIN5 were activated, but MSN2 and MGA1 were not. Regarding antioxidant systems and protein kinases in DM cells under FT stress, CTT1, GTO, GEX1, and YOL024W were upregulated, whereas AIF1, COX2, and TRX3 were not. Gene activation represented by transcription factors and enzymatic antioxidants appears to be associated with FT2-stress tolerance in transgenic yeast cells. RCK1, MET14, and SIP18, but not YPK2, have been known to be involved in the protein kinase-mediated signalling pathway and glycogen synthesis. Moreover, SPI18 and HSP12 encoding hydrophilin in the PM were detected. Therefore, it was concluded that the genetic network via the change of gene expression levels of multiple genes contributing to the stabilization and functionality of the mitochondria and PM, not of a single gene, might be the crucial determinant for FT tolerance in DaMDAHR-expressing transgenic yeast. These findings provide a foundation for elucidating the DaMDHAR-dependent molecular mechanism of the complex functional resistance in the cellular response to FT stress.

Complete genome sequence of Paenibacillus xylanexedens PAMC 22703, a xylan-degrading bacterium.

Paenibacillus is widely distributed in various environments and has the potential for use as a biotechnological agent in industrial processes. Here, we report the complete genome sequence of the marine bacterium, Paenibacillus xylanexedens PAMC 22703, which utilizes xylan. The P. xylanexedens PAMC 22703 strain was isolated from marine sediments. P. xylanexedens PAMC 22703 utilizes xylan as a carbon source to grow. The genome sequence clarified that this strain possesses genes for utilizing xylan. The complete genome sequence contained one chromosome (7,053,622 bp with 46.0% GC content) and one plasmid (44,617 bp with 44.1% C + G content). The genome harbored genes that fully deploy the xylan assimilation pathway. The complete genome sequence of P. xylanexedens PAMC 22703 would prove useful in acquiring information for its application with xylan in various industries.

Continuous body impedance measurement to detect bladder volume changes during urodynamic study: A prospective study in pediatric patients.

AIMS: To assess the possibility of the body impedance (BI) reflecting bladder volumes (BV) in pediatric patients, the BI signals are measured continuously with the equipment that we have developed and reported previously, during the filling phase of urodynamic study (UDS). METHODS: A total of 30 children (5-12 years old) are included in this prospective study. The equipment uses two dry electrodes embedded inside a strap to collect impedance and electrocardiogram signals. The factors affecting baseline BI and its decreases during UDS have been investigated. RESULTS: The median age is 6.1 years and BI is accurately measured in 27 out of 30 patients (90.0% accuracy). The median value of baseline BI is 1958 Ω. It is higher when they are older, equal to or taller than 125 cm, or non-neurogenic bladder patients. BI decreases as the bladder is filled with saline in 21 patients (77.8%), and remains constant in 6 patients (22.2%). The median age of the Decreased Group is significantly higher than that of Nondecreased Group (p = .036). Height of 125 cm or more is significant in the Decreased Group (p = .020). Heart rates also have been simultaneously measured and revealed a mild decrease during the filling phase. CONCLUSIONS: The baseline BI is affected by the height and age of the children. BI is effectively measured and reflects a change in the BV in older children who are taller than 125 cm, with a small device using a smartphone and a strap.

Complete chloroplast genome sequence of a medicinal landrace citrus Jinkyool (Citrus sunki Hort. ex Tanaka) in Jeju Island, Korea.

Citrus sunki (Jinkyool) is a medicinal landrace citrus belonging to the Rutaceae family. We determined the complete chloroplast genome (160,699 bp) of C. sunki CRS0085 in Jeju Island, Korea. The genome is composed of four distinct parts; a large single copy of 87,918 bp, a small single copy of 21,355 bp, and a pair of inverted repeat regions of 25,713 bp. A total of 134 genes including 89 protein-coding genes, 37 tRNA genes, and eight rRNA genes were identified. The phylogenetic tree showed that C. sunki CRS0085 has the closest relationship with C. reticulata within genus Citrus.