Muhammad Zubair Siddiqi

Arachidicoccus ginsenosidivorans sp. nov., with ginsenoside-converting activity isolated from ginseng cultivating soil

A Gram-reaction-negative, catalase- and oxidase-positive, aerobic, non-motile, light yellow and rod-shaped bacterium (designated Gsoil 809T) isolated from soil of ginseng field, was characterized by a polyphasic approach to clarify its taxonomic position. Strain Gsoil 809T was observed to grow optimally at 30 °C and at pH 7.0 on nutrient agar medium. Strain Gsoil 809T possessed β-glucosidase activity, which was responsible for its ability to transform protopanaxatriol-type ginsenoside Rg1 to ginsenoside Rh1. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain Gsoil 809T belongs to the genus Arachidicoccus of the family Chitinophagaceae and was most closely related to Arachidicoccusrhizosphaerae Vu-144T (98.1 % 16S rRNA gene sequence similarity). The DNA G+C content was 39.4 mol%. The DNA-DNA hybridization value between strain Gsoil 809T and A.rhizosphaerae Vu-144T was 41.27±1.03 %. The major polar lipids were phosphatidylethanolamine and an unknown polar lipid. The predominant quinone was MK-7. The major fatty acids were iso-C15 : 0, iso-C15 : 1 G, iso-C17 : 0 3-OH and summed feature 3, which supported the affiliation of Gsoil 809T to the genus Arachidicoccus. Strain Gsoil 809T contained homospermidineas the major polyamine. Moreover, the physiological and biochemical test results and low DNA-DNA relatedness value allowed the phenotypic and genotypic differentiation of strain Gsoil 809T from recognized species of the genus Arachidicoccus. Therefore, strain Gsoil 809T represents a novel species of the genus Arachidicoccus, for which the name Arachidicoccus ginsenosidivorans sp. nov. is proposed. The type strain is Gsoil 809T (=KCTC 22820T=JCM 30984T).

Sphingobacterium jejuense sp. nov., with ginsenoside-converting activity, isolated from compost.

A Gram-stain-negative, strictly aerobic, non-motile, light yellow, short-rod bacterium (designated GJ30-7T) isolated from compost, was characterized using a polyphasic approach, in order to clarify its taxonomic position. Strain GJ30-7T was observed to grow optimally at 30 °C and at pH 7.0 on R2A agar medium. Strain Gsoil GJ30-7T possessed β-glucosidase activity, which was responsible for its ability to transform ginsenosides Rb1 and Rc (the two main active components of ginseng) to ginsenoside F2. Phylogenetic analysis, based on 16S rRNA gene sequence similarities, indicated that GJ30-7T belongs to the genus Sphingobacterium of the family Sphingobacteriaceae and was most closely related to Sphingobacterium yanglingense JCM 30166T (92.6 %), Sphingobacterium psychroaquaticum KACC 18188T (92.6 %), and Sphingobacterium thermophilum KCTC 23708T (92.0 %). The DNA G+C content was 43 mol% and MK-7 was the major isoprenoid quinone. The main polar lipids were phosphatidylethanolamine, one unidentified phospolipid and one unknown polar lipid. In contrast to standard and reference strains, unidentified sphingolipid was also present. The predominant fatty acids of strain GJ30-7T were iso-C15 : 0, iso-C17 : 0 3-OH, C16 : 1ω7c and/or C16 : 1ω6c (summed feature 3) and iso-C17 : 1ω9c and/or C16 : 0 10-methyl (summed feature 9), supporting the affiliation of strain GJ30-7T to the genus Sphingobacterium. However, strain GJ30-7T could be distinguished genotypically and phenotypically from species of the genus Sphingobacterium with validly published names. The novel isolate therefore represents a novel species, for which the name Sphingobacterium jejuense sp. nov. is proposed, with the type strain GJ30-7T (=KACC 18625T=JCM 30948T).

Niabella aquatica sp. nov., isolated from lake water

A Gram-reaction-negative, strictly aerobic, non-motile, yellow and rod-shaped bacterium (designated RP-2T) isolated from lake water, was characterized by a polyphasic approach to clarify its taxonomic position. Strain RP-2T was observed to grow optimally at 30 °C and at pH 7.0 on R2A medium. Phylogenetic analysis based on 16S rRNA gene sequences indicated that RP-2T represented a member of the genus Niabella of the family Chitinophagaceae and was most closely related to Niabella yanshanensis KACC 14980T (96.6 %), Niabella ginsengisoliKACC 13021T (96.5 %), Niabella drilacis DSM 25811T (95.7 %) and Niabella aurantiaca KACC 11698T (95.6 %). The DNA G+C content was 44.5 mol%. The major polar lipids were diphosphatidylglycerol (DPG) and phosphatidylethanolamine (PE). The predominant quinone was MK-7. The major fatty acids were iso-C15:0, iso-C15:1G, iso-C17:0 3-OH and summed feature 3 (comprising C16:1ω7c and/or C16:1ω6c) supported the affiliation of RP-2T to the genus Niabella. However, RP-2T could be distinguished genotypically and phenotypically from the species of the genus Niabella with validly published names. The novel isolate therefore represents a novel species, for which the name Niabella aquatica sp. nov. is proposed, with the type strain RP-2T (=KACC 18623T =JCM 30952T).

Compostibacter hankyongensis gen. nov., sp. nov., isolated from compost.

A novel bacterial strain, designated strain BS27T, was isolated from mushroom compost and subjected to a taxonomic study using a polyphasic approach. Colonies of BS27T were milky-white, circular with regular fringes and opaque. Cells were short rods, 0.3-0.5 µm wide and 1.2-2.0 µm long. Phylogenetic study based on the 16S rRNA gene sequence placed BS27T in a distinct lineage in the family Chitinophagaceae, sharing 90.1-90.9 % sequence similarity with members of the closely related genera Chitinophaga, Flavitalea, Flavihumibacter, Lacibacter and Flavisolibacter. The novel isolate showed the highest sequence similarities with the members of the genus Chitinophaga. BS27T contained MK-7 as predominant quinone, and iso-C15 : 0, iso-C17 : 0 3-OH, C16 : 1ω7c and/or C16 : 1ω6c (summed feature 3) and iso-C17 : 1I and/or anteiso-C17 : 1B (summed feature 4) as major fatty acids. The DNA G+C content was 53.0 mol%. The major polar lipids of BS27T were phosphatidylethanolamine (PE) and five unidentified polar lipids (L1, L2, L5, L6 and L7). The results of physiological and biochemical tests allowed phenotypic differentiation of BS27T from its closest phylogenetic neighbours. On the basis of the evidence of this polyphasic study, isolate BS27T represents a novel genus and species in the family Chitinophagaceae for which the name Compostibacter hankyongensisgen. nov., sp. nov. is proposed. The type strain is BS27T (=KACC 18745T=JCM 17664T).

Mucilaginibacter hankyongensis sp. nov., isolated from soil of ginseng field Baekdu Mountain

A Gram-negative, non-motile, aerobic, and rod-shaped bacterial strain designated as BR5-28T was isolated from the soil of a ginseng field at Baekdu Mountain Korea, and its taxonomic position was investigated using a polyphasic approach. Strain BR5-28T grew at 10–42°C (optimum temperature, 30°C) and pH 5.5–8.5 (optimum pH, 7.0) on R2A agar medium without additional NaCl supplementation. Strain BR5- 28T exhibited β-glucosidase activity, which was responsible for its ability to transform the ginsenosides Rb1 and Rd (the two dominant active components of ginseng) to compound-K. Based on 16S rRNA gene phylogeny, the novel strain showed a new branch within the genus Mucilaginibacter of the family Sphingobacteriaceae, and formed clusters with Mucilaginibacter frigoritolerans FT22T (95.8%) and Mucilaginibacter gotjawali SA3-7T (95.7%). The G+C content of the genomic DNA was 45.1%. The predominant respiratory quinone was MK-7 and the major fatty acids were summed feature 3 (comprising C16:1ω6c and/or C16:1ω7c), iso-C15:0 and anteiso-C15:0. The major polar lipids were diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine. Strain BR5-28T was differentiated genotypically and phenotypically from the recognized species of the genus Mucilaginibacter. The isolate therefore represents a novel species, for which the name Mucilaginibacter hankyongensis sp. nov. is proposed, with the type strain BR5-28T (=KCTC 22274T =DSM 21151T).

Mucilaginibacter panaciglaebae sp. nov., isolated from soil of a ginseng field

A Gram-reaction-negative, strictly aerobic, non-motile and rod-shaped bacterium, designated strain BXN5-31T, was isolated from soil of a ginseng field, and its taxonomic position was investigated using a polyphasic approach. Strain BXN5-31T grew at 18-37 °C and at pH 6.0-8.0 on R2A medium. Based on 16S rRNA gene sequence similarity, strain BXN5-31T was shown to belong to the genus Mucilaginibacter and was closely related to Mucilaginibactersoyangensis HME6664T, Mucilaginibacterximonensis XM-003T and Mucilaginibacterpuniceus WS71T. The DNA G+C content was 43.6 %. The predominant respiratory quinone was menaquinone 7 (MK-7) and the major fatty acids were iso-C15 : 0, iso-C17 : 0 3-OH and summed feature 3 (comprising C16 : 1ω6c and/or C16 : 1ω7c). The major polar lipids were phosphatidylglycerol, diphosphatidylglycerol and phosphatidylethanolamine. The DNA-DNA hybridization values between strain BXN5-31T and three reference strains (M. soyangensis HME6664T, M. ximonensis XM-003T and M. puniceus WS71T) were 9.4±1.9, 8.2±1.3 and 5.7±0.7 %, respectively. The DNA G+C content and chemotaxonomic data supported the affiliation of strain BXN5-31T to the genus Mucilaginibacter. Moreover, the physiological and biochemical results and low level of DNA-DNA relatedness allowed the phenotypic and genotypic differentiation of strain BXN5-31T from recognized species of the genus Mucilaginibacter. The isolate therefore represents a novel species, for which the name Mucilaginibacter panaciglaebae sp. nov. is proposed. The type strain is BXN5-31T (=KACC 14957T=JCM 17085T).

Complete genome sequencing of Arachidicoccus ginsenosidimutans sp. nov., and its application for production of minor ginsenosides by finding a novel ginsenoside-transforming β-glucosidase

A novel bacterial strain (BS20T), which has ginsenoside-transforming ability, was whole genome sequenced for the identification of a target gene. After complete genome sequencing, phylogenetic, phenotypic and chemotaxonomic analyses, the strain BS20T (Arachidicoccus ginsenosidimutans sp. nov.) was placed within the genus Arachidicoccus of family Chitinophagaceae. The complete genome of strain BS20T comprised a circular chromosome of 4 138 017 bp. To find the target functional gene, 17 sets of four different glycoside hydrolases were cloned in E. coli BL21 (DE3) using the pGEX4T-1 vector and were characterized. Among these 17 sets of clones, only one, BglAg-762, exhibited ginsenoside-conversion ability. The BglAg-762 comprised 762 amino acid residues and belonged to the glycoside hydrolase family 3. The recombinant enzyme (GST-BglAg-762) was able to convert major ginsenosides Rb1 to F2 via gypenoside-XVII (Gyp-XVII), Rb2 to C-O, and Rb3, Rc, Rd, and Gyp-XVII to C-Mx1, C-Mc1, and F2, respectively. Finally, ginsenoside F2 was transformed into compound K (C-K). Besides, these pilot data demonstrate the identification of 17 sets of target/functional genes of 4 different glycoside hydrolases from a novel bacterial species via whole genome sequencing. Our results have shown that the recombinant BglAg-762 very quickly converts the major ginsenosides into minor ginsenosides, which can be used for the enhanced production of target minor ginsenosides. Furthermore, the web service of NCBI is suitable for any targeted gene identification, but based on our experimental analysis we concluded that the hypothetical protein present in NCBI should be considered as a putative or uncharacterized protein.

Comparative analysis of the expression level of recombinant ginsenoside-transforming β-glucosidase in GRAS hosts and mass production of the ginsenoside Rh2-Mix

The ginsenoside Rh2, a pharmaceutically active component of ginseng, is known to have anticancer and antitumor effects. However, white ginseng and red ginseng have extremely low concentrations of Rh2 or Rh2-Mix [20(S)-Rh2, 20(R)-Rh2, Rk2, and Rh3]. To enhance the production of food-grade ginsenoside Rh2, an edible enzymatic bioconversion technique was developed adopting GRAS host strains. A β-glucosidase (BglPm), which has ginsenoside conversion ability, was expressed in three GRAS host strains (Corynebacterium glutamicum, Saccharomyces cerevisiae and Lactococus lactis) by using a different vector system. Enzyme activity in these three GRAS hosts were 75.4%, 11.5%, and 9.3%, respectively, compared to that in the E. coli pGEX 4T-1 expression system. The highly expressed BglPm_C in C. glutamicum can effectively transform the ginsenoside Rg3-Mix [20(S)-Rg3, 20(R)-Rg3, Rk1, Rg5] to Rh2-Mix [20(S)-Rh2, 20(R)-Rh2, Rk2, Rh3] using a scaled-up biotransformation reaction, which was performed in a 10-L jar fermenter at pH 6.5/7.0 and 37°C for 24 h. To our knowledge, this is the first report in which 50 g of PPD-Mix (Rb1, Rb2, Rb3, Rc, and Rd) as a starting substrate was converted to ginsenoside Rg3-Mix by acid heat treatment and then 24.5-g Rh2-Mix was obtained by enzymatic transformation of Rg3-Mix through by BglPm_C. Utilization of this enzymatic method adopting a GRAS host could be usefully exploited in the preparation of ginsenoside Rh2-Mix in cosmetics, functional food, and pharmaceutical industries, thereby replacing the E. coli expression system.

Brevibacterium anseongense sp. nov., isolated from soil of ginseng field

Gram-positive, aerobic, non-motile, pale-yellow, and rodshaped bacterium, designated as Gsoil 188T, was isolated from the soil of a ginseng field in Pocheon, South Korea. A phylogenetic analysis based on 16S rRNA gene sequence comparison revealed that the strain formed a distinct lineage within the genus Brevibacterium and was most closely related to B. epidermidis NBRC 14811T (98.4%), B. sediminis FXJ8.269T (98.2%), B. avium NCFB 3055T (98.1%), and B. oceani BBH7T (98.1%), while it shared less than 98.1% identity with the other species of this genus. The DNA G + C content was 68.1 mol%. The predominant quinone was MK-8(H2). The major fatty acids were anteiso-C15:0 and anteiso-C17:0. The cell wall peptidoglycan of strain Gsoil 188T contained meso-diaminopimelic acid. The major polar lipids were phosphatidylglycerol, diphosphatidylglycerol, and an unidentified aminolipid. The physiological and biochemical characteristics, low DNA-DNA relatedness values, and taxonomic analysis allowed the differentiation of strain Gsoil 188T from the other recognized species of the genus Brevibacterium. Therefore, strain Gsoil 188T represents a novel species of the genus Brevibacterium, for which the name Brevibacterium anseongense sp. nov. is proposed, with the type strain Gsoil 188T (= KACC 19439T = LMG 30331T).

Baekduia soli gen. nov., sp. nov., a novel bacterium isolated from the soil of Baekdu Mountain and proposal of a novel family name, Baekduiaceae fam. nov.

A taxonomic study was conducted on BR7-21T, a bacterial strain isolated from the soil of a ginseng field in Baekdu Mountain. Comparative studies of the 16S rRNA gene sequence showed that the isolate was most closely related to Conexibacter woesei DSM 14684T, Solirubrobacter pauli ATCC BAA-492T, Patulibacter minatonensis JCM 12834T, with 93.8%, 92.4%, and 91.5% sequence similarity, respectively; each genus represented a family in the order Solirubrobacterales. Strain BR7-21T was Gram-reaction positive, non-spore forming, aerobic, non-motile, and short rod-shaped. It grew well on half-strength R2A medium. The G + C content of the genomic DNA was 73.9%. It contained meso-diaminopimelic acid in the cell wall and the major menaquinones were MK-7(H4) and MK-8(H4). The major fatty acids were summarized as (C16:1ω7c/iso-C15:0 2-OH), iso-C16:0, and C17:0 cyclo. On the basis of polyphasic evidence, it was proposed that strain BR7-21T should be placed in a new genus and species, for which the name Baekduia soli gen. nov., sp. nov. was proposed with the type strain BR7-21T (= KCTC 22257T = LMG 24797T). The family Baekduiaceae fam. nov. is proposed to encompass the genus Baekduia gen. nov.