Isolation of intestinal symbiotic or epiphytic microbes from

Gammarus sp. and chemical characterization 

of Streptomyces xiamenensis YF008

DAI Ying1， LU Jingrong2， ZHOU Meihong1， YANG Fan2， LIN Houwen2

(1. College of Food Science and Technology， Shanghai Ocean University， Shanghai201306，China；

2. Department of Pharmacy，Renji Hospital，School of Medicine，Shanghai Jiao Tong University，Shanghai200127，China)

Abstract: The extreme habitats of the Antarctica have great potential to discover special biologicallyactive natural products. Polar microorganisms have formed survival mechanisms and biosynthetic pathways different from terrestrial strains to adapt to the unique environment of polar regions. Polar actinomycetes have metabolized a series of novel microbial natural products with interesting structures. Polar microbes have greater potential for exploitation as sources of novel active secondary metabolites than terrestrial microbes. The microbiota within the NorthSouth polar circle, a powerful producer of novel natural products, has become the target of scholars in natural product research. Gammarid (Gammarus sp.) is a kind of crustaceans belonging order Amphipoda, suborder Gammaridea, family Gammaridae that can live in the Antarctic deep sea. At present, there are few reports on the intestinal symbiotic or epiphytic microbes of Gammarus sp., and there is large space for innovative development on the isolation of intestinal intestinal symbiotic or epiphytic microbes of Gammarus sp. and the study of their secondary metabolites. This study is aim to discover and identify the intestinal symbiotic or epiphytic microbes from Gammarus sp. 6 000 m deepth in the Antarctic sea, and the chemical structural diversity of metabolites of a valuable actinomycete strain obtained from Gammarus sp. were studied. The strain was isolated by gradient dilution coating method with inverted cultivation under 28 ℃ for 221 days. After the growth of colony, the single colony was obtained by lineation purification. The isolation medium was R2A, SC, M7, SIM2, F, 2216E and M10 solid medium, and the purification medium was Gao’s No.1, SFM and LB solid medium. The isolated strains were cultured on the fresh medium plates and identified by morphological observation and 16S rRNA sequencing. Genomic DNA of the strains was extracted, PCR amplified with bacterial universal primers 27F and 1492R, and the species genera of the strains were identified by 16S rRNA sequencing after agarose electrophoresis showed the target band at 1.5 kb. Strain sequences were submitted to GenBank for blast alignment, and neighborjoining (NJ) method in MEGA 7.0 software was selected to construct a phylogenetic tree for actinomycetes. The selected strains were subjected to fermentation. The comprehensive application of thinlayer chromatography (TLC), silica gel column chromatography, Sephadex LH20 column chromatography, ODS column chromatography and semipreparative high performance liquid chromatography (HPLC) methods were used to separate and purify the compounds. The chemical structure of the compound was identified by spectral data such as proton nuclear magnetic resonance spectroscopy (1HNMR), carbon nuclear magnetic resonance spectroscopy (13CNMR), mass spectrometry (ESIMS) techniques, and combined with the reference data. Seventeen strains were isolated from Gammarus sp. and analysis of 16S rRNA sequences indicated that their taxonomic status was assigned to nine genera, including 11 strains of bacteria and 6 strains of actinomycetes. Among these isolated strains, one strain was Rothia sp., two were Kocuria sp., two were Brachybacterium sp., two were Dietzi sp., one was Carnobacterium sp., two were Psychrobacter sp., one was Staphylococcus sp., one was Streptomyces sp., five were Nocardiopsis sp. The strain YF008 was 100% similar to the model strain Streptomyces sp. ZJ306 (KF954540.1), and in the same evolutionary branch with Streptomyces xiamenensis strain XY111 (MH432665.1), and finally was identified as Streptomyces xiamenensis based on the colony morphological characteristics. The two strains of psychrophilic bacteria (Psychrobacter sp.) isolated in this study were psychrophilic bacteria from polar oceans. The fermentation metabolites of Gao’s No.1 liquid medium from the actinomycete Streptomyces xiamenensis YF008 was of research value, and the whole genome sequencing results of this strain showed that it contained 25 BGCs covering various types of functional gene clusters including PKS, NRPS, terpene, butyrolactone and many others. Among these BGCs, BGC15 had no similar gene cluster, which was speculated to possibly encode a new skeleton type of secondary metabolites. So the strain Streptomyces xiamenensis YF008 was selected for secondary metabolite studies. The strain YF008 was activated on the fresh Gao’s No.1 solid plates. The single colony was put into a 100 mL conical flask containing 25 mL TSBY liquid medium (with stainless steel springs), and shaken for 3 days at 28 ℃, 220 r·min-1 to obtain the seed fluid. Then the seed fluid was inoculated at 5% into a 500 mL conical flask (with stainless steel springs) containing 150 mL of Gao’s No.1 liquid medium and fermented in a 220 r·min-1 oscillating incubator at 28 ℃ for 5 days. 18.5 L fermentation broth of strain Streptomyces xiamenensis YF008 was extracted with equal volume of ethyl acetate (ultrasonic extraction for 2030 min) for 4 times until colorless. 16.537 g crude extract was obtained by combining extraction layers and concentrating under reduced pressure. After several means of separation, eight small molecular secondary metabolites were isolated from the crude extract obtained from the fermentation broth of actinomycete Streptomyces xiamenensis YF008 and identified as: 1(10)E,5Egermacradiene-2,11-diol (1), N［2(4hydroxyphenyl)］ethylacetamide (2), ［cyclo(LPheGly)］(3), ［cyclo(LPheAla)］(4), ［cyclo(DPheLIle)］(5), ［cyclo(LHypLPhe)］(6), ［cyclo(DLeuLPro)］(7), ［cyclo(LPheLPro)］(8). There are mainly one germacranetype sesquiterpene, one tyramine derivative, and six cyclic dipeptides. Compound 1 and compounds 36 were isolated from Streptomyces xiamenensis for the first time, and compound 1 was a special germacranetype sesquiterpene with ten membered ring system. In addition, these compounds have been reported to exhibit various pharmacological activities, such as antiinflammatory, antitumor, and antibacterial activities. Subsequent indepth studies will continue to be carried out on the activity of the isolated compounds. The secondary metabolites isolated from Streptomyces xiamenensis YF008 are known compounds. In order to obtain microbial secondary metabolites with novel structure and activity, it is necessary to further optimize the culture medium and fermentation conditions, and to add metal ions, antibiotics (such as chloramphenicol, tetracycline, kanamycin), inducers (such as arabinose), exogenous small molecules or through microbial coculture, ribosome engineering and other methods are used for fermentation optimization to activate the silent gene of the strain as much as possible and stimulate it to produce more secondary metabolites with rare structure and good activity. We can also try to stimulate the production of novel and special secondary metabolites by heterologous expression of novel BGCs through genome mining. Other separation methods can also be tried. The result of the study contributes to the understanding of the intestinal symbiotic or epiphytic microbes of Gammarus sp. in the deep sea of the Antarctica., enriches the chemical structural diversity of secondary metabolites of polar marine microorganisms, and provides a reference for the comprehensive utilization of Antarctic gammarid and polar actinomycetes.

Keywords: Gammarus sp.; actinomycetes; Streptomyces xiamenensis; metabolites; isolation and identification