Siderophores from Marine Microorganisms and Their Applications

In view of the fact that siderophores from microorganisms in different environments have received much attention in recent years because of their potential applications and diverse physiological functions, this review deals with side rophore producing marine microorganisms and the detection, chemical structure and potential applications of siderophores.

Studies on the bioactivity of marine microorganisms using Pyricularia oryzae model

A total of 2 088 marine microorganisms including 1 392 strains of bacteria and 696 strains of fungi were isolated from diverse marine environment such as the South Pole, the North Pole, and Qingdao tideland, etc. Through a systematic screening process involving the use of Pyricularia oryzae mode, 90 bacteria and 31 fungi strains demonstrating positive bioactivities were identified. The rates of active strains of marine bacteria and marine fungi were 6.5% and 4.5%, respectively. The relationships between the active strains and the sampling locations and hosts were studied. For marine bacteria, the three locations showing the highest rotes of active strains were 8.4% in the Zhujiang Estuary, 7.2% at the North Pole and 6.3% in the Bohai Sea. For marine fungi, the highest rates observed were 7.8% at the South Pole, 7.4% in the South China Sea and 6.8% at the Qingdao tideland. An analysis of the host showed that the three highest rates of active strain for marine bacteria were 8.2% for seawater from the Zhujiang Estuary, 8% for alga from the Qingdao tideland and 7.6% for sea mud from the North Pole. For marine fungi, the highest rates observed were 8.3% for Qingdao actiniae, 7.4% for South China Sea poriferan and 4.5% for soil of Qingdao Suaeda salsa. Four fungi and two bacteria samples were selected for further study because of their high bioactivity. They were found to be active towards several pathogenic microorganisms, and demonstrated stable genetic and thermal characteristics and conservation activities.

Bibliometric analysis of domestic symbiotic and epibiotic marine microorganisms based on CNKI database

The application of overgrowth microorganisms in ocean research was discussed,using the China National Knowledge Infrastructure(CNKI)network database to retrieve papers on the topic of symbiotic microbes(=198)and symbiotic marine microorganisms(n=94).Bibliometric Analysis are conducted in terms of the published trends,subject headings and other aspects.The output of literature in this field has increased rapidly in the past15years.57.4%of the papers were published in the last five years.And the relevance of research topics has increased in recent years.The current research focuses on community diversity,secondary metabolites,biological activities and antibacterial activities.Literature analysis is helpful to understand the overall development trend of overgrowth microorganisms in ocean research.

The intriguing chemistry and biology of sulfur-containing natural products from marine microorganisms(1987-2020)

Natural products derived from marine microorganisms have received great attention as a potential resource of new compound entities for drug discovery.The unique marine environment brings us a large group of sulfur-containing natural products with abundant biological functionality including antitumor,antibiotic,anti-inflammatory and antiviral activities.We reviewed all the 484 sulfur-containing natural products(non-sulfated)isolated from marine microorganisms,of which 59.9%are thioethers,29.8%are thiazole/thiazoline-containing compounds and 10.3%are sulfoxides,sulfones,thioesters and many others.A selection of 133 compounds was further discussed on their structure-activity relationships,mechanisms of action,biosynthesis,and druggability.This is the first systematic review on sulfur-containing natural products from marine microorganisms conducted from January 1987,when the first one was reported,to December 2020.

Chemistry,Biosynthesis,and Biological Activity of Halogenated Compounds Produced by Marine Microorganisms

Natural products derived from marine microorganisms have been received great attention as a potential source of new compound entities for drug discovery.The unique marine environment brings us a large group of halogen-containing natural products with abundant biological functionality and good drugability.Meanwhile,biosynthetically halogenated reactions are known as a significant strategy used to increase the pharmacological activities and pharmacokinetic properties of compounds.Given that a tremendous increase in the number of new halogenated compounds from marine microorganisms in the last five years,it is necessary to summarize these compounds with their diverse structures and promising bioactivities.In this review,we have summarized the chemistry,biosynthesis(related halogenases),and biological activity of a total of 316 naturally halogenated compounds from marine microorganisms covering the period of 2015 to May 2021.Those reviewed chlorinated and brominated compounds with the ratio of 9:1 were predominantly originated from 36 genera of fungi(62%)and 9 bacterial strains(38%)with cytotoxic,antibacterial,and enzyme inhibitory activities,structural types of which are polyketides(38%),alkaloids(27%),phenols(11%),and others.This review would provide a plenty variety of promising lead halogenated compounds for drug discovery and inspire the development of new pharmaceutical agents.

Viable but nonculturable bacteria and their resuscitation:implications for cultivating uncultured marine microorganisms

Culturing has been the cornerstone of microbiology since Robert Koch first successfully cultured bacteria in the late nineteenth century. However, even today, the majority of microorganisms in the marine environment remain uncultivated. There are various explanations for the inability to culture bacteria in the laboratory, including lack of essential nutrients, osmotic support or incubation conditions, low growth rate, development of micro-colonies, and the presence of senescent or viable but nonculturable (VBNC) cells. In the marine environment, many bacteria have been associated with dormancy, as typified by the VBNC state. VBNC refers to a state where bacteria are metabolically active, but are no longer culturable on routine growth media. It is apparently a unique survival strategy that has been adopted by many microorganisms in response to harsh environmental conditions and the bacterial cells in the VBNC state may regain culturability under favorable conditions. The resuscitation of VBNC cells may well be an important way to cultivate the otherwise uncultured microorganisms in marine environments. Many resuscitation stimuli that promote the restoration of culturability have so far been identified;these include sodium pyruvate, quorum sensing autoinducers, resuscitation-promoting factors Rpfs and YeaZ, and catalase. In this review, we focus on the issues associated with bacterial culturability, the diversity of bacteria entering the VBNC state, mechanisms of induction into the VBNC state, resuscitation factors of VBNC cells and implications of VBNC resuscitation stimuli for cultivating these otherwise uncultured microorganisms. Bringing important microorganisms into culture is still important in the era of high-throughput sequencing as their ecological functions in the marine environment can often only be known through isolation and cultivation.

Recent Researches of Bioactive Metabolites in Marine Organisms-associated Microorganisms

Recent researches have shown that some compounds isolated from marine organisms have striking structural similarities with the metabolites from known microorganisms. It is inferred from the researches that the symbiotic or associated marine microorganisms may be the true sources of those compounds or at least involved in the biosynthesizing process. This view has been further evidenced by the researches for many sponges and sponge-associated microorganisms. Importantly, growing evidence has highlighted that the symbiotic or associated marine microorganisms live in the microenvironment within the hosts, and they also produce secondary metabolites which are new and original in structure and unique in activity. All these suggest that the microorganisms associated with marine organisms are the sources with very high potential to be new natural bioactive agents. This article reviews briefly the research advances in the study of new bioactive metabolites from marine organisms-associated microorganisms since 2000.

Safety assessment of a novel marine multi-stress-tolerant yeast Meyerozyma guilliermondii GXDK6 according to phenotype and whole genome-sequencing analysis

The application of microorganisms as probiotics is limited due to lack of safety evaluation.Here,a novel multi-stress-tolerant yeast Meyerozyma guilliermondii GXDK6 with aroma-producing properties was identified from marine mangrove microorganisms.Its safety and probiotic properties were assessed in accordance with phenotype and whole-genome sequencing analysis.Results showed that the genes and phenotypic expression of related virulence,antibiotic resistance and retroelement were rarely found.Hyphal morphogenesis genes(SIT4,HOG1,SPA2,ERK1,ICL1,CST20,HSP104,TPS1,and RHO1)and phospholipase secretion gene(VPS4)were annotated.True hyphae and phospholipase were absent.Only one retroelement(Tad1-65_BG)was found.Major biogenic amines(BAs)encoding genes were absent,except for spermidine synthase(JA9_002594),spermine synthase(JA9_004690),and tyrosine decarboxylase(inx).The production of single BAs and total BAs was far below the food-defined thresholds.GXDK6 had no resistance to common antifungal drugs.Virulence enzymes,such as gelatinase,DNase,hemolytic,lecithinase,and thrombin were absent.Acute toxicity test with mice demonstrated that GXDK6 is safe.GXDK6 has a good reproduction ability in the simulation gastrointestinal tract.GXDK6 also has a strong antioxidant ability,β-glucosidase,and inulinase activity.To sum up,GXDK6 is considered as a safe probiotic for human consumption and food fermentation.

Characterization of a novel marine microbial esterase and its use to make D-methyl lactate

A novel marine microbial esterase PHE14 was cloned from the genome of Pseudomonas oryzihabit‐ans HUP022 isolated from the deep sea of the western Pacific Ocean. Esterase PHE14 exhibited very good tolerance to most organic solvents, surfactants and metal ions tested, thus making it a good esterase candidate for organic synthesis that requires an organic solvent, surfactants or metal ions. Esterase PHE14 was utilized as a biocatalyst in the asymmetric synthesis of D‐methyl lactate by enzymatic kinetic resolution. D‐methyl lactate is a key chiral chemical. Contrary to some previous reports, the addition of an organic solvent and surfactants in the enzymatic reaction did not have a beneficial effect on the kinetic resolution catalyzed by esterase PHE14. Our study is the first report on the preparation of the enantiomerically enriched product D‐methyl lactate by enzymatic kinetic resolution. The desired enantiomerically enriched product D‐methyl lactate was obtained with a high enantiomeric excess of 99%and yield of 88.7%after process optimization. The deep sea mi‐crobial esterase PHE14 is a green biocatalyst with very good potential in asymmetric synthesis in industry and can replace the traditional organic synthesis that causes pollution to the environment.

Enhancement of Laccase Activity by Marine-derived Deuteromycete Pestalotiopsis sp. J63 with Agricultural Residues and Inducers

Pestalotiopsis sp. J63, producing a high activity of laccase, is a new marine-derived fungus isolated from the oceanic sediment of the East China Sea. Since the marine environment is oligotrophic nutrient, marine derived fungi may use small amount of nutrients to grow and produce laccases. Agricultural residues that are mainly composed of lignin, cellulose and hemicellulose are difficult to be degraded and few microbes can take them as substrates, so they are considered as oligotrophic nutrient and have the potential to be used to produce value added products. In this study, the ability of Pestalotiopsis sp. J63 to use agricultural residues to produce laccases was tested in the submerged fermentation. The combination of 3 g·L 1maltose and 20 g·L 1rice straw was the best carbon sources and 8 g·L 1ammonium sulfate was the best nitrogen source under the condition without inducers. The effects of five inducers, the feeding time and concentration of inducer on laccase production were investigated.Adding 0.09 mmol·L 1phenol after 24 h of incubation led to high laccase activity(5089 U·L 1), while with 0.09mmol·L 1phenol in the medium and wheat bran as the nitrogen source, the laccase activity could reach 5791.7U·L 1. Native-PAGE results showed that two laccase isozymes were present in the cultures. One existed in both induced and non-induced culture filtrates, while the other was only found in the fermentation with the addition of phenol, guaiacol and veratryl alcohol.

Gel Microbead Cultivation with a Subenrichment Procedure Can Yield Better Bacterial Cultivability from a Seawater Sample than Standard Plating Method

A gel microbead (GMD) cultivation method was employed to cultivate microorganisms from an amphioxus breeding zone in Qingdao, P. R. China. The culture results were compared with those by standard plating method. In the GMD-based method, the microcolony-forming GMDs were sorted by fluorescence-activated cell sorting (FACS). To further get pure cultures, a subsequent enrichment culture and a streaking purification procedure were conducted on marine R2A medium. Eighty bacterial strains isolated by the GMD-based method were randomly selected for sequencing. These isolates belonged to Alphaproteobacteria (33%), Gammaproteobacteria (44%), Bacteroidetes (11%), Actinobacteria (5%), Firmicutes (5%), Epsilonproteobacteria (1%), and Verrucomicrobia (1%), the last two groups being usually difficult to culture. The 16S rRNA gene sequences revealed a diverse community with 91.1%-100% of the bacterial rRNAs similarities. Thirteen strains were sharing 16S rRNA gene sequence which was less than 97% similar to any other rRNA genes currently deposited in TYP16S database. Seventy isolates derived from the standard plating method fell into 4 different taxonomic groups: Alphaproteobacteria (9%), Gammaproteobacteria (81%), Bacteroidetes (7%) and Firmicutes (3%) with a 16S rRNA gene sequence similarities between 95.8%-100%, in which only 3 strains were sharing 16S rRNA gene sequence of less than 97%. The results indicated that the GMD-based method with subenrichment culture yielded more taxonomic groups and more novel microbial strains, including members of previously rarely cultured groups, when compared with the standard plating method, and that this method markedly improved the bacterial cultivability.

Resazurin as an indicator of reducing capacity for analyzing the physiologic status of deep-sea bacterium Photobacterium phosphoreum ANT-2200

Resazurin(RZ)is a weakly fl uorescent blue dye and can be reduced irreversibly to highly fl uorescent pink resorufi n(RF)that is reduced reversibly to colorless dihydroresorufi n(hRF)by photodeoxygenation,chemical reaction and reductive organic compounds produced through cell metabolism.Because of the reliable and sensitive fl uorescence-color change and noninvasive features,RZ has been used widely as a redox indicator in cell viability/proliferation assays for bacteria,yeast,and mammalian cells.However,RZ is used rarely for physiological characterization of marine microorganisms.Here,we developed a custom-made irradiation and absorption-analysis device to assess the reducing capacity and physiologic status of marine bacterial cultures.We measured the absorption spectra of RZ,RF,and hRF in the presence of the reducing compound Na 2 S and under visible-light irradiation.After establishing appropriate parameters,we monitored the color changes of RZ and its reduced derivatives to evaluate the coherence between reducing capacity,bioluminescence and growth of the deep-sea bacterium Photobacterium phosphoreum strain ANT-2200 under various conditions.Emission of bioluminescence is an oxidation process dependent upon cellular reducing capacity.Growth and bioluminescence of ANT-2200 cell cultures were impeded progressively with increasing concentrations of RZ,which suggested competition for reducing molecules between RZ at high concentration with reductive metabolism.Therefore,caution should be applied upon direct addition of RZ to growth media to monitor redox reactions in cell cultures.Analyses of the instantaneous reduction velocity of RZ in ANT-2200 cell cultures showed a detrimental eff ect of high hydrostatic pressure and high coherence between the reducing capacity and bioluminescence of cultures.These data clearly demonstrate the potential of using RZ to characterize the microbial metabolism and physiology of marine bacteria.

Pyridapeptides F–I,cyclohexapeptides from marine sponge-derived Streptomyces sp.OUCMDZ-4539

Four new cyclohexapeptides,pyridapeptides F–I(1–4),were isolated from the fermentation broth of marine sponge-derived Streptomyces sp.OUCMDZ-4539.The pyridapeptides F–H(1–3)are composed ofβ-hydroxyleucine,alanine,O-methylthreonine,hexahydropyridazine-3-carboxylic acid,5-hydroxytetrahydropyridazine-3-carboxylic acid,and(2S,3R,4E,6E)-2-amino-3–hydroxy-4,6-dienoic acid residues.Pyridapeptide I(4)contains(2S,3R,4E,6E)-2-amino-3–hydroxy-8-methylnona-4,6-dienoic acid residue and a very rare glycose residue,aculose.Their structures were determined based on spectroscopic analysis and chemical methods.Pyridapeptides G–I(2–4)have the 2,3,6-trideoxyhexose units glycosylated at theγ-OH-TPDA residue,displayed significant antiproliferative activity against four(PC9,MKN45,Hep G2,K562)or two(PC9,MKN45)human cancer cell lines.

Fibrinolytic Evaluation of Compounds Isolated from a Marine Fungus Stachybotrys Iongispora FG216

As a part of our continuing work to discover bioactive leading molecules from marine microorganism, ethyl acetate fraction of organic extract of the train Stachybotrys longispora FG216 showed fibrinolytic activity in our primary screen. The bioassay-guided purification of the active fractions resulted in isolation of a new isoindolone, FGFC2 （1） （FGFC2, Fungi fibrinolytic compound 2）, together with two known compounds, LL-Zl272β （2） and ergosterol （3）. The structure of compound 1 was elucidated by the spectral analysis of 1D （^1H, ^13C） NMR, 2D （COSY, HSQC, and HMBC） and ESI-MS. Three compounds were evaluated for fibrinolytic activities in vitro. Compared to FGFC1 （EC50=47 μmol/L） as a reference drug, compound 1 and ergosterol （3） showed moderate fibrinolytic activities in vitro with EC50 values of 108.16 and 156.30 μmol/L, respectively. LL-Z127213 （2） had no fibrinolytic activity.

Identification and Fibrinolytic Evaluation of an Isoindolone Derivative Isolated from a Rare Marine Fungus Stachybotrys Iongispora FG216

An isoindolone derivative, Fungi fibrinolytic compound （R）-2,5-bis（（2R,3R）-2-（（E）-4,8-dimethylnona-3,7-dien- 1-y1）-3,5-dihydroxy-2-methy--7-oxo-3,4,7,9-tetrahydr pyrano[2,3-e]isoindol-8（2H）-yl）pentanoic acid （FGFC1, Fungi fibrinolytic compound 1）, was isolated from a rare marine microorganism strain Stachybotrys longispora FG216. The structure of FGFC1 was elucidated by 1H NMR, 13C NMR, IR, and MS data; moreover, it was also evaluated for fibrinolytic activity in vitro and in vivo. The results showed that 0.1--0.4 mmol/L of FGFC1 could stimulate generation of plasmin activity （increased by 2.05--11.44 folds） by measuring Glu-plasminogen and Lys-plasminogen activation in vitro. The experiment of fluorescein isothiocyanate （FITC）-fibrinogen degradation indicated that the effect of FGFCI on fibrinolytic activity was mediated by plasminogen and scuPA. In addition, FGFC 1 （10 mg/kg） could dissolve most of pulmonary thrombus of Wistar rat in vivo. It is possible that FGFC 1 is a potential thrombolytic agent in the future.

Functional genomic analysis of Hawaii marine metagenomes

Using high-throughput sequencing on metagenome to analyze marine microbial community, it is one of current main issues in the field of environmental microbe research. In this paper, we conducted the functional analysis on seven samples of metagenomic data from different depth seawater in Hawaii. The results of gene prediction and function annotation indicate that there are large amounts of potential novel genes of which functions remain unknown at present. Based on the gene annotation, codon usage bias is studied on ribosomal protein-related genes and shows an evident influence by the marine extreme environment. Furthermore, focusing on the marine environmental differences such as light intensity, dissolved oxygen, temperature and pressure among various depths, comparative analysis is carried out on related genes and metabolic pathways. Thus, the understanding as well as new insights into the correlation between marine environment and microbes are proposed at molecular level. Therefore, the studies herein afford a clue to reveal the special living strategies of microbial community from sea surface to deep sea.