Patent Grant
10364447
The present invention provides a method for producing omega-3 polyunsaturated fatty acids (PUFAs) by culturing specific myxobacterial stains suitable for said method. In addition, it provides a method for identification of such omega-3 producing myxobacteria by using phylogenetic analyses based on 16S rDNA sequence data in combination with physico-chemical data based on gas chromatography coupled with mass spectroscopy to discover further producers of the omega-3 polyunsaturated fatty acids.
Long-chain polyunsaturated fatty acids (PUFAs), including those of the omega-3 family [also known as ω-3 (“omega-3”) fatty acids] are interesting fatty acids in nature. They are important constituents of phospholipids that play a role in decreasing membrane rigidity. Eicosapentaenoic acid (EPA) is a major constituent of the human brain's phospholipids and serves as precursor of prostaglandins and resolvins. Another important PUFA of the omega-3 family is docosahexaenoic acid (DHA). Improved cognitive and behavioural function in infant development seems correlated to high levels of this compound. For omega-3 PUFAs, and in particular for DHA and EPA, beneficial health effects have been e.g. the prevention of cancer, rheumatoid arthritis, cardiovascular diseases, the improvement of immune function, and eye and brain health [for recent overview see Teale M C (ed.) (2006) Omega-3 fatty acid research. Nova Science Publishers. New York, and references therein]. Because of these beneficial properties omega-3 PUFAs are being used extensively as nutritional lipids in health and dietary supplements and as functional ingredients in a wide range of foods. Omega-3 PUFAs presently comprise one of the biggest and strongest growing market segments in the food and beverage industry sector, with substantially increasing demand over the past years.
These days, fish oil is the most abundant and widely used natural source for omega-3 fatty acids, but named source suffers from over fishing, lack in high grade oil supply with sufficient content of DHA/EPA, and quality issues (smell, formulation challenges etc.). Alternative processes involving algae and oomycetes as producer organisms are established or under development [see overviews by Hinzpeter I et al. (2006) Grasas y Aceites 57:336-342 and Ward O P, Singh A (2005). Process Biochemistry 40:3627-3652, respectively]. Since the supply of fish oil of high quality is increasingly limited, it was attempted to find alternative, sustainable biological sources. Various groups of marine algae have been explored for over 20 years and some products based on algal biomass have meanwhile entered the market. Some oomycetes belonging to the group of stramenopiles (a group of algae-like eukaryontic organisms previously known as “Chromophyta”) were also occasionally reported to produce the above mentioned compounds (e.g. of the genera Achyla and Pythium; [Aki T et al. (1998) J Ferm Bioengin 86:504-507; Cheng M H et al. (1999) Bioresour Technol 67:101-110; Athalye S K et al. (2009) J Agric Food Chem 57:2739-2744]). In other stramenopiles (e.g. the genera Schizochytrium and Thraustochytrium; as described in U.S. Pat. No. 7,022,512 and WO2007/068997) and in species of the dinoflagellate Amphidinium (US 2006/0099694), DHA may represent up to 48% of the fatty acid content of the cells, which are the highest contents so far known in the Eukaryota. However, the cultivation of these organisms in industrial scale still poses a challenge even after several years of development.
Other alternative biological sources for omega-3 PUFAs hitherto found are prokayotic eubacteria [Nichols D et al. (1999), Curr Opin Biotechnol 10:240-246; Metz J G et al. (2001), Science 293:290-293; Gentile G et al. (2003) J Appl Microbiol 95:1124-1133]. However, the commercial exploitation of these organisms for PUFA production on an industrial scale is hampered by the slow growth characteristics of these psychrophilic microorganisms, as well as their inherently low yields and productivity. In myxobacteria, an unidentified PUFA with 20 carbon atoms and four double bonds was found first in marine genera of Plesiocystis and Enhygromyxa [Iizuka T et al. (2003) Int J Syst Evol Microbiol 53:189-195; Iizuka T. et al. (2003), Syst Appl Microbiol 26:189-196]. Recently, ARA (an omega-6 PUFA) was encountered in Phaselicystis flava, a representative of a novel myxobacterial family [Garcia R O et al. (2009), Int J Syst Evol Microbiol 50(PT12):1524-1530].
The occurrence of omega-3 PUFAs like DHA and EPA is not reported at all for Myxobacteria and so far described processes are insufficient with regard to yields, amount of PUFAs and especially concerning the production of the important omega-3 PUFAs.
Myxobacterial Taxonomy and Phylogeny
The myxobacteria are believed to have evolved as a monophyletic group of organisms in the order Myxococcales, a delta subgroup in proteobacteria. At present, 3 suborders (Cystobacterineae, Nannocystineae, and Sorangiineae) are recognized in myxobacteria [Reichenbach H (2005) Order VIII. Myxococcales Tchan, Pochon and Prevot 1948, 398AL. In Brenner D J, et al. (eds.) Bergey's Manual of Systematic Bacteriology, 2nd edn, vol. 2, part C, pp. 1059-1072, New York: Springer]. These suborders are divided into six families, namely Cystobacteraceae, Myxococcaceae, Nannocystaceae, Kofleriaceae, Polyangiaceae, and Phaselicystidaceae.
The family Myxococcaceae is composed of the genera Myxococcus, Corallococcus and Pyxidicoccus. In the related family Cystobacteraceae, five genera are known (Cystobacter, Archangium, Hyalangium, Melittangium and Stigmatella). Nannocystaceae of the suborder Nannocsytineae are comprised of the Nannocystis and two marine genera (Enhygromyxa and Plesiocystis). Its related family Kofleriaceae is composed of the terrestrial genus Kofleria and the marine genus Heliangium. The family Polyangiaceae encompasses the genera Jahnella, Chondromyces, Polyangium, Byssovorax, and Sorangium. So far, the latter two are the only known genera of cellulose degraders among the order; most of the other taxa are difficult to isolate and cultivate. The recently discovered genus Phaselicystis is the only genus in the recently erected family Phaselicystidaceae [Garcia R O et al. (2009) Int J Syst Evol Microbiol 59:1524-1530]. At present 20 genera are recognizable and validly described in myxobacteria to cover all the known soil and marine isolates.
General Importance of 16S rDNA in Bacterial Taxonomy and Phylogeny.
16S rDNA has been widely and commonly used in bacterial systematics to designate ancestral groupings of the taxa because this gene is highly conserved between species [Weisburg W G et al. (1991) J Bacteriol 173:697-703]. In myxobacteria, the 16S rDNA phylogeny along with morphological characteristics provides a strong evidence for genetic classification [Sp{umlaut over (r)}oer C et al (1999), Int J Syst Bacteriol 49(PT 3):1255-1262]. Those myxobacterial strains assigned to the same genus by morphological classification were found to be tightly clustered in their 16S rDNA gene phylogeny. This method also provides patterns for ancestral relatedness among its member species which are reflected on the degree of phenotypic characteristics [Vellicer G J, Hillesland K (2008) In Myxobacteria: Multicellularity and Differentiation (Whitworth D E, ed.), pp. 17-40, Washington, D.C.:ASM Press].
Importance of Fatty Acid Profiles as Chemotaxonomic Markers in Bacteria
The phylogeny is in accordance with the morphological and physiological characteristics of myxobacteria. Most importantly, fatty acid profiles as inferred from GC-MS analyses of the cellular fatty acid content are generally used and deemed acceptable for taxonomic segregation of Myxobacteria, as well as many other groups of bacterial organisms, since they were found to be a constant feature, at least when standardised methodology is applied. First applications of this technique have been made in the early 1989s [Tornabenet G (1985) Methods in Microbiology 18, 209-234]. Therefore, such GC-MS (or GC-) based fatty acid profiles have been widely used in bacterial phylogeny and taxonomy. Nevertheless, a systematic approach combining both the search for particular economically important fatty acids in combination with other means of investigation to evaluate the taxonomic and phylogenetic positions of the respective fatty acid producers has so far never been carried out.
Importance of PCR-Based Methods to Explore Bacterial Species Diversity and Functional Biodiversity in Ecosystems
It has been discussed for a long time (and meanwhile proven by methods of molecular biology directed at the in situ identification of eubacteria in environmental samples) that the overall diversity of extant bacterial species is much higher than the number of known and well-described, culturable species [Amann R I et al. (1995) Microbiol Rev 59:143-169; Torsvik V et al. (1990) Appl Environ Microbiol 56:782-787]. According to current estimates, as much as 90% of the extant bacteria still remain to be discovered. Using methods such as direct sequencing of 16S rDNA from soil and other environmental samples is increasingly revealing a great diversity of DNA sequences that cannot be correlated to any of the known, culturable bacterial species. However, their phylogenetic affinities may be revealed from a homology comparison of their 16S rDNA with those of reference strains. Metagenomic techniques, which are currently under development, may in future eventually facilitate the direct utilisation of the genes and enzymes of these “unculturable” organisms. At present it remains necessary in most cases to find suitable culture conditions for the hitherto unexplored bacterial organisms and explore them at the stage of their pure cultures. As prerequisite for the characterisation of all the unexplored bacteria, as well as for their biotechnological exploitation, especial isolation techniques need to be established. This is also true in particular with regard to the discovery of novel myxobacterial taxa, as well as for various other groups of eubacteria and other microbial groups with great potential on biotechnology.
Myxobacterial groups can be specifically searched for by PCR using specific 16S rDNA primers. A previous study on a soil niche revealed at least 30 additional unknown phylogenetic groups of myxobacteria that could be detected using this approach. They are not only different from each other, but also their 16S rDNA genes differed from those of the known myxobacterial 16S rDNA gene sequences existing in GenBank and other public domain databases. These results suggested the presence of a vast undiscovered diversity of soil myxobacteria that yet remain to be cultured and explored [Zhi-Hong W. et al. (2005) Env. Microbiol. 7(10): 1602-1610].
Microbial Fermentation, with Particular Emphasis on Myxobacteria
Myxobacterial strains are usually fermented in an aqueous nutrient medium under submerged aerobic conditions. Various examples for the feasibility of large scale fermentation of this group of organisms in pilot and industrial scale are widely known to science, e.g., concerning the discovery and development of the epothilones, which have recently been approved as anticancer drugs.
After the thorough evaluation of their growth and nutrient conditions, these organisms can normally be grown well in laboratory culture and their production be scaled up in a straightforward manner. Typically, microorganisms are fermented in a nutrient medium containing a carbon source and a proteinaceous material. Preferred carbon sources include glucose, brown sugar, sucrose, glycerol, starch, corn starch, lactose, dextrin, molasses, and the like. Preferred nitrogen sources include cottonseed flour, corn steep liquor, yeast, autolysed brewer's yeast with milk solids, soybean meal, cottonseed meal, corn meal, milk solids, pancreatic digest of casein, distillers' solids, animal peptone liquors, meat and bone scraps, and the like. Combinations of these carbon and nitrogen sources can be used advantageously. There is no need to add trace metals, e.g. zinc, magnesium, manganese, cobalt, iron and the like to the fermentation medium since tap water and unpurified ingredients are used as medium components.
Large scale fermentation for production cultures can be induced at any temperature conductive to satisfactory growth of the microorganisms between about 18° and 32° C. and preferably at about 28° C. Ordinarily, optimum production of compounds is obtained in about 2 to 8 days of fermentation, and preferably in about 4 to 5 days of fermentation.
Production can be carried out in shake flasks but also in solid media and stirred fermentors. When growth is carried out in shake flasks or large vessels and tanks, it is preferable to use the vegetative form, rather than the spore form of the microorganism for inoculation. This avoids a pronounced lag in the production of the PUFA compounds and the attendant inefficient utilisation of the equipment. Accordingly, it is desirable to produce a vegetative inoculum in an aqueous nutrient medium by inoculating this medium with an aliquot from a soil or a slant culture. When a young, active vegetative inoculum has thus been secured, it is transferred aseptically to other shake flasks or other suitable devices for fermentation of microorganisms. The medium in which the vegetative inoculum is produced can be the same as, or different from, that utilised for the production of compounds, as long as adequate growth of the microorganism is obtained.
In general, seeding of myxobacterial strains and fermentation and the production of compounds in submerged aerobic fermentation in stirred vessels is utilised. The production is independent of used containers, fermentors and starter proceedings. The compounds can also be obtained by shake-flask culture, or in other specially designed vessels such as airlift or Biowave fermentation tanks. For large volume fermentations it is preferable to use a vegetative inoculum. The vegetative inoculum is prepared by inoculating small volume of culture medium with the spore form or a lyophilised pellet of the organism. The vegetative inoculum is then transferred to a fermentation vessel where, after a suitable incubation time, compounds are produced in optimal yield.
As is customary in aerobic submerged culture process, sterile air is dispersed through the culture medium. For efficient growth of the organism, the volume of the air used is in the range of from about 0.25 to about 0.5 vvm. An optimum rate in a 10 l vessel is about 0.3 vvm with agitation provided by conventional impellers rotating at about 240 rpm. Adding of small amount (i.e. 1 ml/l) of an antifoaming agent such as silicone to fermentations media is necessary if foaming becomes a problem. For microaerophilic organisms it may be favorable to reduce the aeration further in order to support biomass production. The fermentation is usually carried out in batch mode, but to attain better growth and increased product yield, fed-batch fermentations can be carried out by supplying the required nutrient source to a growing culture once it has been depleted in the original culture medium.
The desired products will usually be present mostly in the biomass of the fermented myxobacterial strains, but in case of their overproduction, they may as well be located in the culture filtrate of the fermentation broth. The culture broth can be separated by filtering on a filter press. A variety of procedures can be employed to isolate and purify the PUFA compounds from the fermentation broth, for example, by chromatographic adsorption procedures followed by elution with a suitable solvent, column chromatography, partition chromatography, by supercritical fluid extraction, and combinations of the aforementioned methods.
In the course of the characterization and identification of hitherto unknown myxobacterial strains of the suborder Sorangiineae, all to be classified in a hitherto unknown, newly discovered genus for which the name Aetherobacter is proposed here, were surprisingly found to produce significant amounts of polyunsaturated fatty acids (PUFAs), in particular omega-3 polyunsaturated fatty acids such as EPA and DHA. Three overproducers of EPA and DHA were found, which were designated as Aetherobacter fasciculatus sp. nov. ined, represented by strain DSM 21835, Aetherobacter rufus sp. nov. ined., represented by strain DSM 23122 and Aetherobacter sp. DSM 23098, This invention also relates to the surprising novel discovery that certain other strains of species belonging to class myxobacteria (in particular, strains of the genera Enhygromyxa and Sorangium) were also found to produce of DHA and EPA to a lower extent. At the same time, a screening of phylogenetically related bacteria based in published DNA sequences in public databases revealed several “uncultured” bacteria as closest relatives of the newly obtained omega-3 PUFA overpropducing strains, which are presumably also myxobacteria.
This phylogenetic group of bacteria is believed to be widely unexplored for its biological diversity, hence it appears highly probable that additional new and hitherto unstudied taxa and strains that produce DHA and/or EPA in substantial quantities might be found in the near future. This can be facilitated by using a similar screening approach to that performed in the course of the discovery of the PUFA-producing strains that are subject of this application. This does not only concern additional new strains of the novel, yet inedited genus Aetherobacter, but even novel strains of other orders and families of myxobacteria.
upper line: representative culture sample of Aetherobacter fasciculatus DSM 21835 lower line: purchased reference of DHA methyl ester
upper line: representative culture sample of Aetherobacter fasciculatus DSM 21835 lower line: purchased reference of EPA methyl ester
The myxobacterial strains of the invention are proposed to be classified in a novel genus Atherobacter and its novel species A. fasciculatus, A. rufus and as a yet unnamed Aetherobacter species. All of them were isolated from soil and plant debris samples originally collected in Indonesia in 1962. The samples had been preserved at the Zentrum für Biodokumentation, Landsweiler-Reden, Germany. For the isolation of myxobacteria the soil/plant debris samples were processed using live Escherichia coli as bait, following a previously described method [Reichenbach H & Dworkin M (1992) The Myxobacteria, In The Prokaryotes, 2nd edn, pp. 3416-3487 (Balows A et al., eds) New York: Springer].
Aetherobacter fasciculatus DSM 21835 was isolated in November 2007 from a soil sample containing pieces of roots and other decaying plant materials. The strain A. rufus DSM 23122 was isolated in December 2007 and Aetherobacter sp. DSM 23098 was isolated in February 2009.
The above myxobacterial strains belong to the suborder Sorangiineae of the order Myxococcales and are representatives of the novel inedited genus Aetherobacter and novel inedited species (A. fasciculatus, A. rufus, Aetherobacter sp.) proposed herewith, being aerobic to facultatively aerobic and chemoheterotrophic, having 16S rDNA sequences as shown in SEQ ID NO: 1 (DSM 21835), SEQ ID NO: 2 (DSM 23122) and SEQ ID NO: 4 (DSM 23098) that are about 96% identical with that of strain Byssovorax (syn. Byssophaga) cruenta DSM 14553T (GenBank Acc. No AJ833647, SEQ ID NO: 3) and/or having omega-3 PUFA contents of at least 10%, preferably at least 15% by weight of total cellular fatty acid content. In the following a description of the proposed taxa is provided.
The scientific names of the strains of the invention are still unpublished. There are no other strains known from the art of the genus Aetherobacter. This genus is described for the first time in this invention. Its phylogenetic relations are outlined as follows. The characteristics are outlined below, and, in more detail, in the examples.
Myxococcales include the following exemplified suborders which in turn include the following exemplified families, exemplified genera and exemplified species.
Enhygromyxa salina
Byssovorax cruenta
Aetherobacter fasciculatus
Aetherobacter rufus
Aetherobacter sp.
Sorangium cellulosum
The phylogenetic relationships as inferred from a comparison of 16S rDNA sequence data of representative genera and species of which viable cultures are extant, are presented in
The three novel strains are morphologically rather similar. Aetherobacter fasciculatus DSM 21835 shares nearly the same growth stages appearance with Aetherobacter sp. DSM 23098, with regard to the vegetative cells as well as the fruiting bodies. Aetherobacter rufus DSM 23122 differs in having red fruiting bodies and smaller sporangioles. In addition, DSM 23122 produced a whitish swarm that differs in the yellowish orange color of the remaining two strains of Aetherobacter.
Physiological tests also revealed differences among the novel strains and species. Obviously the three novel strains prefer different sugars and nitrogen sources for growth. The antibiotic resistance seems to distinguish isolates, e.g., DSM 21835 and DSM 23098 appear resistant to Hygromycin B, whereas DSM 23122 is sensitive to this compound. Resistance to ampicillin and neomycin differentiates A. fasciculatus DSM 23098 and A. rufus DSM 23122 from strain DSM 23098. It also appears that DSM 21835 and DSM 23098 exhibit a wider range of antibiotic resistance in comparison to DSM 23122.
The 16S rDNA sequences derived from the three novel strains clustered together in the phylogenetic tree, supported by 100% bootstrap value. Aetherobacter fasciculatus DSM 21835 appears not only morphologically similar to A. sp. DSM 23098, but the 16S rDNA sequences also showed a high degree of homology (99.4% identity). A. rufus DSM 23122 was found 98.9% identical with DSM 21835 and 99.2% with DSM 23098. This relationship is notable in the phylogenetic tree where sequences of DSM 21835 clustered together with DSM 23098, rather than with DSM 23122 (
Description of New Genus and Species
1. Aetherobacter R. O. Garcia & R. Müller, Gen. Nov. Ined.
Entymology: Aetherobacter [Ae.the.ro.bac'ter. Gr. masc. n. Aether Greek God of Light (refers to clear and transparent swarming); Gr. fem. n. bacter from Gr. neut. n. bakterium small rod, stick; M.L. masc. n. Aetherobacter clear swarming rod]
Vegetative cells moderately long and slender cylindrical rods with blunt ends; movement by gliding on surface and under the agar. Swarm, film-like to transparent-clear colony. Congo-red-negative, edges characterized by coherent migrating cells, penetrating mostly under the solid medium; agar slightly depressed. Myxospores refractive slender rods with blunted ends, shorter than vegetative cells, enclosed in sporangial wall. Fruiting bodies as tiny ovoid sporangioles, usually compact or clustered. Yeasts are completely degraded. Strongly bacteriolytic. With omega-3 polyunsaturated fatty acids as major components of cellular FA. Percent G+C, 68.0-70%.
The novel genus Atherobacter is clearly different from the most similar accepted genus of Myxobacteria, Byssovorax in many ways. Morphologically, Byssovorax shows an intense red pseudoplasmodia-like swarm and fruiting bodies on agar medium. This independent flocks of migrating cells is regarded as very characteristic of that genus. The sporangioles are large (60-180 μm wide) and also show intense red colors. In contrast, the species of the novel genus Aetherobacter as represented by the strains described in detail further below produce a swarming pattern that deeply penetrates the agar. The swarm created by the burrowing cells of Aetherobacter spp. rather shows a radial (circular) appearance. In addition, the swarming cells exhibit white to light orange colours. The center of the colonies in all these novel strains are usually clear in yeast agar (VY/2). The single sporangiole is small (<20 μm) and arranged in tightly packed clusters or bundles that are often located within the agar.
Another significant difference between Aetherobacter spp. and Byssovorax is the inability of the members of the new genus to degrade cellulose. Both genera also differ significantly in their fatty acid profiles. Byssovorax contains a higher amount of iso-15:0 and straight chain fatty acids and is devoid of DHA and EPA.
The genetically characteristic of a 96% similarity of the 16S rDNA was also used for the recently erected myxobacterial genera Enhygromyxa [Iizuka T. et al. (2003) Syst Appl Microbiol 26:189-196] and Byssovorax [Reichenbach et al., 2006. Int J Syst Evol Microbiol. 56(PT 10): 2357-2363].
Based on the aforementioned data, the novel Aetherobacter strains appear significantly different from the known genus Byssovorax, i.e. the myxobacterial genus that appears most closely related as inferred from a homology comparison of their 16S rDNA. Taken together these differences in morphology, chemo-physiology, topology of the 16S rDNA gene, and phylogenetic analysis suggest in unison that the erection of a new genus is justified.
2. Aetherobacter fasciculatus R. O. Garcia & R. Müller, Sp. Nov. Ined.
Entymology: fasciculatus [fasc.i.cu'la. L. masc. n. fasciculum little bundles or packets (refers to the arrangement of sporangioles)].
With all characteristics of the genus. Vegetative cells fat rods, 1.2-1.3×2.9-5.7 μm in size, and phase dark. Swarms are orange tint which show complete clearing of the yeast cell bait, with shallow depression on surface of the agar, often deeply penetrating the medium to form coherent curtain-like structures. Fruiting bodies are yellow-orange in color, often found under the agar, as sod (30×50 μm) composed 5-20 tiny sporangioles (10.4×11.4 μm) tightly arranged as bunch. Myxospores refractive, stout rods, with rounded ends similar but shorter (1.0-1.2×3.2-4.0 μm) than vegetative cells; enclosed in a sporangial wall. Nutritional type, bacteriolytic, yeast degrader. Cellulose and chitin not degraded. Good growth in saccharose, fructose, D-mannose and L-arabinose. Resistant to broad spectra of antibiotics: gentamycin, apramycin, tobramycin, streptomycin, ampicillin, neomycin and hygromycin B. Sensitive to kanamycin, spectinomycin, tetracycline, oxytetracycline, carbenicillin, and rifampicin. Major cellular fatty acid components are C22:6 (docosahexaenoic acid, DHA) iso-C15:0, C20:5. (eicosapentaenoic acid, EPA). Mol percent G+C is 68.9.
Type Strain:
Aetherobacter fasciculatus was deposited according to the Budapest Treaty at the DMSZ, Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH, Inhoffenstr. 7 B, 38124 Braunschweig, Germany on Aug. 27, 2008 with the deposition number DSM 21835.
A. fasciculatus DSM 21835 has a 16S rDNA sequence that is 96% identical to that of Byssovorax (syn: Byssophaga) cruenta DSM 14553T. It shows an omega-3 PUFA content of at least 10%, preferably at least 15% by weight of the total cellular fatty acid content.
A. fasciculatus DSM 21835 was identified as member of class Myxobacteria, order Myxococcales by showing characteristic swarming of Gram-negative, slender rod-shaped vegetative cells, fruiting body formation, and bacteriolytic activity. The strain is aerobic to facultative aerobic, chemoheterotrophic, and also exhibits resistance to various antibiotics. Major fatty acids are C22:6 (docosahexaenoic acid), iso-C15:0, anti-iso C17:0 and C20:5 (eicosapentaenoic acid). The G+C content of the genomic DNA is 68.9 mol %. The 16S rDNA sequence shows 96% identity to the cellulose-degrading Byssovorax cruenta and 95% to Sorangium cellulosum. This clearly shows that the strain belongs to the suborder Sorangiineae of the order Myxococcales. In addition, uniqueness in morphological growth stages and novel fatty acid profile, clearly implies that DSM 21835 belongs to a new taxon which is proposedly classified as belonging to the novel genus Aetherobacter and in the novel species A. fasciculatus
3. Aetherobacter rufus R. O. Garcia & R. Müller, Sp. Nov. Ined.
Etymology: rufus (ru.fus. L. masc. adj. rufus red).
With all the characteristics of the genus. Vegetative cells fat rods, 1.0-1.2×3.0-6.0 μm in size, longest 15 μm, phase dark. In yeast agar, the swarm moves coherently in the medium to form a ring or circular structure, with white edge color. Colonies on (VY/2 agar) appear clear and transparent as a result of complete yeast degradation. On the surface of the agar, they are often produced as thin sheet or film with mounds of cell aggregates toward the colony edge, exhibiting shallow agar depressions. Fruiting bodies showing red to vermilion color, appearing as a simple mound (120×140 μm), or as very long rolls (340×400 μm-1900×2900 μm), visible to the naked eye, initially developing from white humps of cellular aggregation. Composed of tiny sporangioles (6-12 μm), compacted in a sorus (14×15 μm-16×26 μm). Myxospores refractive, stout and short rods (1.0-2.0 μm), with rounded ends like the vegetative cells; enclosed in the sporangial wall. Nutritional type bacteriolytic. Cellulose and chitin not degraded. Equally good growth in all sugars tested: L-arabinose, fructose, galactose, D-glucose, D-mannose, molasses, sorbitol, xylose, cellobiose, lactose, maltose, saccharose, and soluble starch. Resistant to ampicillin, neomycin and gentamycin. Sensitive to apramycin, tobramycin, kanamycin, spectinomycin, hygromycin B ampicillin, tetracycline, oxytetracycline, streptomycin, carbenicillin, and rifampicin, major cellular fatty acid components are iso-C15:0, C22:6 (DHA), C15:0, C16:0. Also produces C20:5. (EPA). Mol percent G+C is 68.0.
Type Strain:
Aetherobacter rufus was deposited at the Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH (DSMZ), Braunschweig, Germany, at Jul. 10, 2009 under the deposition number DSM 23122.
A. rufus strain DSM 23122 was also found to produce EPA and DHA. Morphologically this strain shares many similarities with A. fasciculatus strain DSM 21835. However, the two species differ in several physiological characteristics such as antibiotic sensitivities, carbon requirement, pH tolerance and in the 16S rDNA sequence. In the analysis of omega-3 fatty acids, A. rufus DSM 23122 also produces both DHA and EPA.
4. Aetherobacter sp. DSM 23098
Aetherobacter sp. DSM 23098 was also found to be related to the above mentioned strains and to contain omega 3-PUFAs in substantial quantities in its cellular biomass. This strain was therefore also deposited according to the Budapest Treaty at the DMSZ, Nov. 12, 2009 with the deposition number DSM 23098.
The general expressions, within the present disclosure, preferably have the following or precedingly mentioned meanings, where in each embodiment on, more than one or all more general expressions may, independently of each other, be replaced with the more specific definitions, thus forming preferred embodiments of the invention, respectively.
Preferably, the following abbreviations are used for the purpose of the invention:
Myxobacteria are preferably scientifically classified in the kingdom: bacteria, phylum: Proteobacteria, class: Delta Proteobacteria, order: Myxococcales.
For the purpose of the invention the terms “myxobycteria” and “myxobacterial strain” preferably refer to any species or other members of microorganisms belonging to the order of Myxococcales.
The used term “members” preferably is used for any myxobacterial strain of the invention which is defined as to be a homolog neighbour of Aetherobacter.
For the purpose of the invention all used taxonomic relations are in accordances to Brenner D J, et al. (eds.) Bergey's Manual of Systematic Bacteriology, 2nd edn, New York: Springer, especially the methods of the invention are referring to this standard.
For the purpose of the invention genetic affinity is described with the term of “homology”. This is based on the use of 16S rDNA sequences for a binary comparison of a FASTA sequence (identified by their accession number as outlined in table 1) against the respective 16S rDNA of a specified strain of the present invention using the BLASTn 2.2.22 algorithm from the url http://blast.ncbi.nlm.nih.gov/Blast.cgi which is further described in Zhang Z et al. (2000) J Comput Biol 7:203-214.
An “omega-3 polyunsaturated fatty acid (PUFA)” according to the present invention includes eicosa-cis-5,8,11,14,17-pentaenoic acid (20:5 EPA) and docosa-cis-4,7,10,13,16,19-hexaenoic acid (22:6 DHA). The method of the first aspect of the invention is particularly useful for the production of EPA and DHA and mixtures of said fatty acids.
A preferred embodiment of the invention relates to the production of omega-3 unsaturated fatty acids which comprises culturing a myxobacterial strain capable of producing one or more omega-3 polyunsaturated fatty acids.
Preferably, a myxobacterial strain “capable” of producing omega-3 PUFAs is regarded to contain of at least 0.5% of omega-3 PUFAs determined as described in example 5, more preferably to contain at least 1%, still more preferably to contain at least 2%, yet more preferably to contain at least 5%, most preferably to contain at least 10% and in particular to contain at least 15% by weight of total cellular fatty acid content.
A preferred embodiment of the invention relates to a method, wherein the myxobacterial strain belongs to the suborder Sorangiineae of the order Myxococcales.
In a preferred embodiment the myxobacterial strain has a 16S rDNA sequence that is at least 84%, more preferably at least 85% or 86%, still more preferably at least 87%, 88%, 89% or 90%, yet more preferably at least 91%, 92% or 93%, most preferably at least 94%, 95%, 96% identical with the Byssovorax (Byssophaga) cruenta DSM 14553T.
A preferred embodiment of the invention relates to a method, wherein the myxobacterial strain belongs to the suborder Polyangiaceae of the order Myxococcales.
In a preferred embodiment the myxobacterial strain has a 16S rDNA sequence that is at least 85% or 86%, more preferably at least 87%, 88%, 89% or 90%, still more preferably at least 91%, 92% or 93%, most preferably at least 94%, 95%, 96% identical with Byssovorax (Byssophaga) cruenta strain DSM 14553T.
A preferred embodiment of the invention is related to a method, wherein the myxobacterial strain has an omega-3 polyunsaturated fatty acid content of at least 10%, preferably at least 15% by weight of total cellular fatty acid content.
In a further preferred embodiment the omega-3 unsaturated fatty acids are selected from eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) and mixtures thereof.
In a preferred embodiment of further methods of the invention said culturing includes a seed culture incubation of said myxobacterial strain and a subsequent main culture for omega-3 PUFA production. It is preferred that the seed culture is performed on a VY/2 (yeast medium) agar medium, most preferably in an MD1G agar. Moreover it is preferred that the culture is performed at a temperature of 25 to 32° C., preferably at about 30° C.
In another preferred embodiment of further methods of the invention the culturing for omega-3 PUFA production is performed in a liquid medium. In particular it is preferred that the culturing is performed in MD1G broth. Again, the culture is preferably performed at a temperature of 25 to 32° C., preferably at about 30° C. Another preferred mode of cultivation is performed in the same medium with pH adjusted to 7.0, under high volume medium, so as to reduce air.
In another preferred embodiment of the methods of the invention the method further comprises
(i) isolating said one or more omega-3 PUFAs from the culture; and/or
(ii) purifying said one or more omega-3 PUFAs; and/or
(iii) isolating an individual omega-3 PUFAs.
In a further preferred embodiment the myxobacterial strain belongs to the suborder Sorangiineae of the order Myxococcales and is a representative of a novel genus (Aetherobacter) and novel species (fasciculatus) proposed herewith, being aerobic to facultative aerobic and chemo heterotrophic, having a 16S rDNA sequence that is about 96% identical with the Byssovorax (Byssophaga) cruenta DSM 145531 as shown in SEQ ID NO:3 and/or having an omega-3 polyunsaturated fatty acid content of at least 10%, preferably at least 15% by weight of total cellular fatty acid content.
In a preferred embodiment the myxobacterial strain has a 16S rDNA sequence that is at least 94%, more preferably at least 94.5% or 95%, still more preferably at least 95.5%, 96%, 96.5% or 97%, yet more preferably at least 97.5%, 98% or 98.5%, most preferably at least 99.0%, 99.2%, 99.4%, 99.6% or 99.8% identical with the 16S rDNA sequence shown in SEQ ID NO:1. In a particularly preferred embodiment the myxobacterial strain is Aetherobacter fasciculatus (DSM 21835) and is characterised by the 16S rDNA sequence shown in SEQ ID NO:1.
In a preferred embodiment the myxobacterial strain has a 16S rDNA sequence that is at least 94%, more preferably at least 94.5% or 95%, still more preferably at least 95.5%, 96%, 96.5% or 97%, yet more preferably at least 97.5%, 98% or 98.5%, most preferably at least 99.0%, 99.2%, 99.4%, 99.6% or 99.8% identical with the 16S rDNA sequence shown in SEQ ID NO:2. In a particularly preferred embodiment the myxobacterial strain is Aetherobacter rufus (DSM 23122) and is characterised by the 16S rDNA sequence shown in SEQ ID NO:2.
In a preferred embodiment the myxobacterial strain has a 16S rDNA sequence that is at least 94%, more preferably at least 94.5% or 95%, still more preferably at least 95.5%, 96%, 96.5% or 97%, yet more preferably at least 97.5%, 98% or 98.5%, most preferably at least 99.0%, 99.2%, 99.4%, 99.6% or 99.8% identical with the 16S rDNA sequence shown in SEQ ID NO:4. In a particularly preferred embodiment the myxobacterial strain is Aetherobacter sp. (DSM 23098) and is characterised by the 16S rDNA sequence shown in SEQ ID NO:4.
The invention is further illustrated by the following examples which are not to be construed as limiting its scope. The skilled person recognizes that the procedures used in the examples may be adapted in order to identify further individual myxobacterial strains capable of producing omega-3 PUFAs. For example, other known chromatographic methods and other known mass spectrometric methods may be employed differing from the exemplified methods that are described hereinafter.
General Experimental Procedures
Materials and Methods
List of Media Used in the Cultivation of Myxobacteria
CY-SWS
Sea Water Salts Solution (SWS)
Trace Element Solution SL-4
Trace Element Solution SL-6
HS-Medium
Supplement after Autoclaving with the Following:
Soluble Medium M
MD1 Liquid Medium
MD1G Liquid Medium
VY/2 Liquid Medium
VY/2 Maltose
VY/2-SWS
LB Medium
VY/2 Agar Medium
MD1G Agar Medium
Buffered Water Agar
Mineral Salt Agar (ST21 Agar)
Solution A
Make up in about two-thirds of the water volume in distilled water.
Solution B
Make up in the remaining water volume. Autoclave separately. Combine solutions A and B
CT 7Agar
Top Agar Layer
Adjust the pH to 7.5 with KOH, then poured as a thin layer on top of the base agar after autoclaving.
Base Agar Layer
Cel3 Agar
Separately autoclave the KNO3, and add to the medium after the medium has cooled to about 50° C. Pour as a thin layer on top of mineral salt agar (ST21 agar).
Reference Strains
Several strains were used as references for the work performed in examples 5 and 6, and their reference DNA sequence data have been retrieved for comparison from public databases such as GenBank (a database provided by the National Center for Biotechnology Information; http://www.ncbi.nlm.nih.gov/) and/or respective databases generated by EMBL (European Molecular Biology Laboratory, http://www.embl.de/) They are all listed in table 1. Wherever possible, publications referring to these sequences were cited, and the sequence data are not depicted here.
Archangium gephyra DSM 2261T
Byssovorax cruenta DSM 14553T
Chondromyces apiculatus
Chondromyces crocatus DSM14714T
Chondromyces lanuginosus DSM
Chondromyces pediculatus DSM
Chondromyces robustus
Corallococcus coralloides DSM 2259T
Corallococcus exiguus DSM 14696T
Corallococcus macrosporus DSM
Cystobacter armeniaca DSM 14710T
Cystobacter badius DSM 14723T
Cystobacter disciformis DSM52716T
Cystobacter ferrugineus DSM 14716T
Cystobacter fuscus DSM 2262T
Cystobacter gracilis DSM 14753T
Cystobacter miniatus DSM 14712T
Cystobacter minus DSM 14751T
Cystobacter velatus DSM 14718T
Cystobacter violaceus DSM 14727T
Enhygromyxa salina DSM 15217T
Haliangium ochraceum DSM 14365T
Haliangium tepidum DSM 14436T
Hyalangium minutum DSM 14724T
Jahnella thaxteri DSM 14626T
Kofleria flava DSM 14601T
Melittangium alboraceum Me b7T
Melittangium boletus DSM 14713T
Melittangium lichenicola DSM 2275T
Myxococcus fulvus DSM 16525T
Myxococcus stipitatus DSM 14675T
Myxococcus virescens DSM 2260T
Myxococcus xanthus DSM 16526T
Nannocystis exedens DSM 71T
Nannocystis pusilla DSM 14622T
Phaselicystis flava DSM21295T
Plesiocystis pacifica DSM 14875T
Polyangium fumosum DSM14668T
Polyangium sorediatum DSM 14670T
Polyangium spumosum DSM 14734T
Pyxidicoccus fallax DSM 14698 T
Sorangium cellulosum DSM 14627T
Stigmatella aurantiaca DSM 17044T
Stigmatella erecta DSM 16858T
Stigmatella hybrida DSM 14722T
Production of Omega-3 PUFA, Notably DHA, EPA:
Actively swarming cells from MD1G agar were scraped off from a well-grown agar plate and inoculated into 100 ml flask containing 20 ml MD1G broth. This culture was incubated on a rotary shaker for 7 days at 160 rpm and 30° C. as seed culture. An aliquot of 2 ml taken from the seed culture was introduced into three 250 ml flask containing 50 ml of liquid MD1G medium (i.e., the production medium for fatty acid analysis). Flasks were shaken for ten days at the same speed and temperature as those of the seed culture. Aliquots of 2 ml which contained mainly clumping cells were taken from each flask after five and 10 days of incubation. Wet and dried cell weights were then calculated.
Extraction of the Cellular Fatty Acids:
The cellular fatty acids were extracted using the FAME method [Bode H B et al. (2006) J. Bacteriol 188:6524-6528; Ring M W et al. (2006), J Biol Chem 281:36691-36700 (2006)]. Aliquots (5 μl) of the extracts were analysed by GC-MS.
Identification of cellular fatty acids including EPA and DHA:
Cellular fatty acids including the omega-3 PUFA (EPA and DHA) were identified based on the fragmentation patterns and retention time. These fatty acids (FA) were compared to FAME mix reference standard (Sigma-Aldrich) which contains 37 fatty acid methyl esters. The presence of DHA and EPA were confirmed using reference standards from Sigma-Aldrich (cis-4,7,10,13,16,19-DHA, cis-5,8,11,14,17-EPA)
Quantification of EPA and DHA:
GC-MS was carried out on an Agilent 6890N gas chromatograph with a 5973 electron impact mass selective detector and a 7683B injector (Agilent, Waldbronn, Germany) using a dimethyl-(5% phenyl)-polysiloxane capillary column (Agilent HP-5 ms, 0.25 mm by 30 m by 0.25_m) and helium as the carrier gas at a flow rate of 1 ml/min. Samples were injected in split mode (split ratio, 10:1). The column temperature was kept at 130° C. for 2.5 min, increased to 240° C. at a rate of 5° C./min, and then ramped to 300° C. at 30° C./min and held at 300° C. for 5 min. Other temperatures were as follows: inlet, 275° C.; GC-MS transfer line, 280° C.; ion source, 230° C.; and quadrupole, 150° C. The mass selective detector was operated in scan mode, scanning the mass range from m/z 40 to 500. Data analysis was carried out with AMDIS software, version 2.64 (NIST, Gaithersburg, Md., USA), using the value “integrated signal” for quantification. Amounts were calculated in percentages relative to the sum of integrals of the total fatty acid methyl esters.
DHA and EPA were estimated based on the amount present (2% or 0.2 μg/μl each, respectively) in the reference FAME mixture. An aliquot of 5 μl was taken from the reference mixture and mixed with 95 μl chloroform to give a final volume of 100 μl. From this mixture, an aliquot was injected into the column for GC-MS analysis as specified above.
DHA and EPA were first determined in the FAME mix by analysis of fragmentation pattern and retention time. These peaks areas were then calculated using the integrated signal area which represents the amount present in the standards.
After the cellular fatty acid extracts were analyzed in the GC-MS, peaks corresponding to DHA and EPA were measured using the integrated signal which represents the amount present in the sample (retention time range: EPA 17.5-18.5 min., DHA 20.5-21.5 min).
DHA and EPA amounts were calculated by taking the average of the sample integrated signal divided by the standard integrated signal. It was then multiplied by the concentration of the standard fatty acid and total volume of the fatty acid extract, see the following formula:
SFA=Standard FA concentration [mass per volume]
AIS=Average Integrated signal of the sample
IS=Integrated signal of the standard
TVFAE=total volume of FA extract [volume]
The total percentages of polyunsaturated omega-3 PUFAs were finally obtained from the summation of average EPA and DHA. The percentage of cellular fatty acids was determined from the average of triplicate samples.
Maintenance of Strains:
All myxobacterial strains producing omega-3 PUFAs were routinely cultured and maintained in VY/2 agar, where they were found to grow fairly. Long term preservation was carried out in 10% glycerol under liquid nitrogen.
Morphological Observations:
Swarming colonies and fruiting bodies were observed under a Olympus (Hamburg, Germany) SH-ILLB stereoscopic microscope and photographed using Axiocam MRC (Zeiss, Göttingen, Germany) camera. Fruiting bodies were also analysed using laser scanning fluorescent microscope (Zeiss). Vegetative cells and myxospores morphology were studied using a phase-contrast microscope (Zeiss). All growth stages were also observed on VY/2 agar.
Physiological Tests:
Reactions of vegetative cells to Gram and Congo Red stains were determined; staining with the latter was after the method of McCurdy, H. D., Can. J. Microbiol. 15:1453-1461 (1969). Catalase activity was tested with 3% H2O2. Cellulose degradation was performed on VY/2 and ST21 agar, all overlaid with filter paper (2×1 cm), and in parallel test on Cel-3 agar [Reichenbach H. & Dworkin M (1992) The Myxobacteria, In The Prokaryotes. 2nd edn, pp. 3416-3487 (Balows A et al., eds.) New York: Springer] to determine degradation of cellulose powder. Degradation assay for chitin was done as described by Reichenbach H et al. (2006) Int J Syst Evol Microbiol 56:2357-2363.
Microbial Predation Tests:
Overnight cultures of the gramnegative bacterium Escherichia coli were spot-inoculated (ca. 10 mm diameter) on the water agar and air-dried before inoculation of the environmental samples. The culture plates were sealed with parafilm and incubated at 30° C. for 1 week. Clearing of the microbial food baits indicated the lytic action by the spreading myxobacterial colonies.
Growth Responses to Temperature, pH, Carbon & Nitrogen Sources, and Antibiotics:
Tests for growth responses to different levels of temperature and antibiotic resistance were also performed in VY/2 agar. Vegetative cell inocula came from the actively growing swarm taken from the same medium. Antibiotics used were apramycin (Fluka, Buchs, Switzerland), ampicillin, kanamycin, hygromycin B (Roth, Karlsruhe, Germany), tobramycin (Sigma-Aldrich), spectinomycin (Serva, Heidelberg, Germany), tetracycline, oxytetracycline, streptomycin (all from Synopharm, Barsbüttel, Germany), carbenicillin, neomycin, rifampicin and gentamycin (Applichem. Darmstadt, Germany), all filter-sterilized and were added after cooling (50° C.) of the autoclaved medium.
Utilization of nitrogenous compounds was analysed in water agar supplemented with 10 mM of L-aspartic acid, L-glutamic acid, urea, KNO3, and (NH4)2SO4. Also tested in the same medium at 0.3% concentration was casamino acid (BD) and different peptone sources [tryptone, casitone, peptone, neopeptone, phytone (BD)].
Carbon source utilization was determined in water agar supplemented with 5 mM HEPES. Final pH was adjusted before autoclaving to 7.0 using KOH. Fructose, D-mannose, saccharose, L-arabinose, D-glucose, D-galactose, sorbitol, cellobiose, soluble starch, molasses, maltose, xylose, and sorbitol were supplemented at concentration of each 0.35%.
G+C Content and 16S rDNA Analysis:
Genomic DNA was extracted from actively growing cells using the protocol for Gram-negative bacteria of the Qiagen Genomic DNA Purification Kit. The DNA G+C content of the novel bacteria was determined by HPLC [Li G et al. (2003) Bio Techniques 34:908-909 (2003); Shimelis O & Giese R (2006) J. Chrom. 1117:132-136]. The 16S rDNA analysis was performed according to Garcia R O et al. (2009) Int J Syst Evol Microbiol 50(PT12):1524-1530. The obtained 16S rDNA sequences were also compared to the NCBI-BLAST nucleotide-nucleotide data bank. This service is provided by the National Center for Biotechnology Information (NCBI) located at the U.S. National Library of Medicine 8600, Rockville Pike, Bethesda Md., 20894 USA as Basic Local Alignment Search Tool (BLAST). In particular the Nucleotide database (BLASTn) was searched.
Aetherobacter fasciculatus DSM 21835, Aetherobacter rufus DSM 23122 and Aetherobacter sp. DSM 23098
A. Production of Omega-3 PUFA (DHA, EPA):
From actively swarming cells on surface of MD1 G agar culture, Aetherobacter fasciculatus DSM 21835, A. rufus DSM 23122 and Aetherobacter sp. DSM 23098 grew as clumps on MD1G liquid culture. This condition made it impossible to quantify the cells by optical density. Cells harvested on the fifth day of shaken culture showed yellowish color which were microscopically revealed as long slender rods (vegetative cells). On the tenth day of growth, cells became red which after microscopic examination revealed structures, which presumably represent fruiting bodies (
Both strains grew well if inoculated in 1 l flasks containing 750 ml MD1G which was inoculated with 50 ml seed culture, but more clumpy cell pellets were produced, and cells were not converted to fruiting bodies even after 10 or more days of incubation.
The observation that better growth in high-volume medium presumably reflects the nature of both strains to burrow under the agar which suggests a lesser demand for air might be required for growth. In addition, the weak catalase reaction observed would also support this hypothesis. Analysis of these data implied that all Aetherobacter strains are potentially microaerophilic or facultative aerobic. This basic information appears important in the cultivation of the strain and consequently for the analysis of PUFA production.
Tables 2-7 show the triplicate cell weight measurements of Aetherobacter fasciculatus DSM 21835, A. rufus DSM 23122 and Aetherobacter sp. DSM 23098 taken after 5 and 10 days of cultivation, respectively, in MD1G shake flasks. DHA and EPA peaks were measured from triplicate samples using the integrated signal.
B. Identification of Cellular Fatty Acids Including EPA and DHA:
Cellular fatty acids were identified as described above in the chapter general methods and materials.
Fragmentation pattern of the samples revealed the identity of the mass units to the standard methyl esters of DHA and EPA as outlined exemplarily in
C. Extraction and Quantification of the Cellular Fatty Acids:
As presented in the tables 2-7 all three strains contain at least 15% total PUFAs (EPA and DHA taken together) of dry cell mass. The highest observed amount was more than 25%. The data further indicate that optimum production will be reached within the period of 10 days.
Strain DSM 21835 was isolated in November 2007 from an Indonesian soil sample containing pieces of roots and other decaying plant materials. The strain was discovered because it formed yellowish-orange fruiting bodies growing in bacterial bait on the surface of buffered water agar. Morphological observations under the dissecting microscope revealed clusters of sporangioles packed into sod. Inoculation of the washed fruiting body material onto fresh VY/2 agar led to production of a thin and delicate swarm that coherently migrated on the surface and inner part of the agar. A series of repetitive transfers of the swarming material lead to the isolation of the pure strain.
Morphological and Cultural Characteristics:
Swarm: On VY/2 agar bacterial colonies or swarms typically spread under producing a curtain-like appearance (
Vegetative cells: The vegetative cells in slide mounts are long and slender rods, measuring 1.2-1.3×2.9-5.7 μm (
Fruiting bodies: On VY/2 agar, fruiting bodies are composed of sporangioles which are packed in bundles or sori that are commonly arranged in a cluster or chained fashion (
Myxospores: Crushed sporangioles release tightly packed, optically refractive rod cells which are presumably myxospores (
Physiological Characteristics
Staining, degradation, and lytic properties: Vegetative cells are Gram-negative and catalase-positive. The swarm colony remains unstained with Congo Red, as typical for Sorangiineae. Live bacterial food bait (E. coli) on water agar is completely degraded and cleared after 6 days of incubation at room temperature, suggesting strongly bacteriolytic properties. The strain is unable to degrade cellulose on Cel-3 agar and ST21 agar overlaid with filter paper, and chitin powder on CT-7 agar is not degraded either. Agar degradation is commonly observed in cultures grown in VY/2 and MD1 agar.
Growth responses to temperature and pH. On yeast agar, the growth optimum is observed at 30° C. incubation, but fair growth is still attained at 18° C. on the same agar. No growth occurred at 37° C. In the same medium, the strain can grow in the range of pH 5-9 with the largest swarm diameter becoming evident at pH 7.0. However, no evidence of growth is found at pH below 4.0 and above pH 10.
Utilisation of carbon sources. The best carbon source to support growth of the strain is lactose. Fair growth is also observed in molasses, maltose, and xylose; moderate growth on saccharose, fructose, D-mannose, L-arabinose, galactose, sorbitol, mannose, and glucose. Cellobiose and poor growth on soluble starch. Fruiting bodies could be seen in the presence of monosaccharide sugars (e.g. glucose, mannose) and sorbitol.
Utilisation of nitrogen sources The largest swarm colony diameter is attained with glutamic acid, potassium nitrate, and aspartic acid. Urea and ammonium sulfate only allow for poor growth, resulting in rather thin and scattered swarms. Among complex nitrogen sources, Casitone, Peptone, and Neopeptone are best suited; tryptone and phytone still exhibit fair growth, but the strain grows only poorly on casamino acids.
Antibiotic resistance: The strain is sensitive to kanamycin, spectinomycin, tetracycline, oxytetracycline, carbenicillin, and rifampicin. Resistance is observed against gentamycin, apramycin, tobramycin, streptomycin, ampicillin, neomycin and hygromycin B.
Mol % G+C Analysis The DNA G+C content of the is 68.9 mol %
The myxobacterial strain DSM23122 was isolated in November 2007 from an Indonesian soil sample containing pieces of roots and other decaying plant materials. The sample had originally been collected in 1962 and preserved at the Zentrum für Biodokumentation, Landsweiler-Reden, Germany. The soil was processed for isolation using standard baiting methods [Reichenbach H & Dworkin M (1992) The Myxobacteria, In The Prokaryotes, 2nd edn, pp. 3416-3487 (Balows A et al., eds) New York: Springer]. The strain was noticed by the reddish-colour of its fruiting bodies on the surface of the filter paper on mineral salts agar [Shimkets et al., 2006. The myxobacteria. In The Prokaryotes: a Handbook on the Biology of Bacteria, 3rd edn, vol. 7, pp. 31-115 (Dworkin M et al., eds) New York: Springer]. Inoculation of the fruiting body onto the same fresh medium resulted in the appearance of a thin and delicate, swarm which coherently migrates on surface of the agar. The strain was isolated to purity after repetitive transfer of the swarming material onto new culture media.
Morphological and Cultural Characteristics:
Swarm. On solid VY/2 medium (
Vegetative cells. The vegetative cells (
Fruiting bodies. Completely developed fruiting bodies were observed after two weeks of incubation. In VY/2 and water agar, fruiting bodies (
Myxospores. Crushed sporangioles release tightly packed, slightly optically refractive rod cells which are presumably myxospores (
Physiological Characteristics
Staining and lytic properties. The swarm colony remains unstained with Congo red, as typical for Sorangiineae; Vegetative cells are Gram-negative and catalase-positive. The strain differed from related genera by its inability to degrade cellulose powder on Cel-3 and filter paper on ST21 agar. Chitin powder on CT-7 agar was, also not degraded. Partial agar degradation was commonly observed on VY/2 and MD1 agar.
Growth responses to temperature and pH. On yeast agar, the growth optimum is observed at 30° C. incubation, but fair growth is still attained at 18° C. on the same agar. No growth occurred at 37° C. In the same medium, growth is observed in the range of pH 5-8 with pH 7.0 being optimal. No evidence of growth is found at pH 4.0 and below and pH 9.0 and above.
Utilisation of carbon sources. All carbon sources tested were equally utilised.
Utilisation of nitrogen sources. The largest swarm colony diameter is attained with glutamic acid, aspartic acid, ammonium sulfate and potassium nitrate, while the strain grows poorly in urea. Among complex nitrogen sources, casitone shows the optimal growth; peptone, neopeptone and tryptone still exhibited fair growth, phytone exhibits only poor growth, and no growth at all is observed with casamino acids.
Antibiotic resistance. The strain is resistant to gentamycin, ampicillin and neomycin. Sensitivity was observed to apramycin, tobramycin, kanamycin, spectinomycin, hygromycin B, tetracycline, oxytetracycline, streptomycin, carbenicillin, and rifampicin.
Mol % G+C Analysis. The DNA G+C content is 68.0 mol %.
Strain DSM 23098 was isolated in February 2009 from an Indonesian soil sample. The sample had originally been collected in 1962 and preserved at the Zentrum für Biodokumentation, Landsweiler-Reden, Germany. The soil was processed for isolation using live bait Escherichia coli according to the described method [Shimkets et al., 2006. The myxobacteria. In The Prokaryotes: a Handbook on the Biology of Bacteria, 3rd edn, vol. 7, pp. 31-115 (Dworkin M et al., eds) New York: Springer]. The bacterium was recognized for its almost transparent colony forming independent swarms on the surface of the agar and islated to purity by repetitive transfer onto new culture media.
Morphological and Cultural Characteristics
Swarm. In yeast medium (VY/2), the colonies (
Vegetative cells. The vegetative cells (
Fruiting bodies. In VY/2 agar, the fruiting bodies (
Myxospores: Crushed sporangioles release tightly packed and slightly optically refractive rod cells which are presumably myxospores (
Physiological Characteristics
Staining and lytic properties: The vegetative cells are Gram-negative and catalase-positive. The swarm colony remains unstained with Congo red, as in other Sorangiineae. The strain differed from related cellulose degrading genera by its inability to degrade cellulose powder on Cel-3 and filter paper on ST21 agar. Chitin powder on CT-7 agar was also not degraded by the swarming cells. Shallow agar depression are also be seen in most solid culture media, indicating that the strain is able to degrade agar as well.
Microbial predation test: The baited bacterium was cleared out in the course of 5-7 days incubation at 30° C., suggesting bacteriolytic activity.
Growth responses to temperature and pH: On yeast agar, fair growth is attained in a range from 18-30° C., but no growth occurred at 37° C. In the same medium, growth was observed within a range from pH 6-8, with the largest swarm diameter observed at pH 7.0.
Utilisation of carbon sources: None of the tested carbon sources except for soluble starch, supported well the growth of the strain.
Utilisation of nitrogen sources: Growth is poor in all tested inorganic nitrogen sources Among different complex organic nitrogen sources, tryptone is suited best. However, the strain also grows well in peptone, neopeptone and casitone; and even phytone and casamino acids were comparably better suited than inorganic nitrogen sources.
Antibiotic resistance. The strain is sensitive to all antibiotics tested except for tobramycin and hygromycin B.
To establish correlations between taxonomy and phylogeny in the one hand and production of omega-3 PUFAs on the other hand, a series of type strains and other well-characterised representatives from various taxonomic groups of myxobacterial and allied gliding bacteria were studied for comparison for production of DHA, EPA and other fatty acids. The majority of these strains are listed in Table 1, their 16S rDNA sequences were also taken for the phylogenetic study (Example 6). In addition, some further strains from the SB collection were also studied for FA profiles by GC-MS, which were morphologically in agreement with Sorangium cellulosum but so far not included in the molecular phylogeny.
For this purpose, authentic and type strains of various myxobacteria and related phylogenetic groups were obtained from DSMZ (Braunschweig; Germany), the Helmholtz Center for Infectious Diseases (HZI, dto.) or from the culture collection of Universität des Saarlandes (Saarbrücken, Germany; SB). Most bacteria were cultivated as shake cultures in 300 ml Erlenmeyer flasks containing 50 ml of the respective culture media (as specified below) at 170 rpm. The incubation temperature was 30° C., except for Haliangium tepidum (which was incubated at 37° C.). Members of the genera Polyangium, Chondromyces, Jahnella, and Byssovorax were cultivated in VY/2 medium, to which maltose was added in case of Byssovorax, following the protocol proposed by Kunze B et al. (2006) J Antibiotics 59:664-668. Species of Archangium, Corallococcus, Hyalangium, Kofleria, Melittangium, Myxococcus, Nannocystis, Pyxidicoccus, Stigmatella and Angiococcus (presently classified in Cystobacter), as well as Cystobacter armeniaca and C. disciformis, were cultivated in MD1 medium (Shimkets et al, 2006). All other Cystobacter spp. were grown in M-med [Müller R & Gerth K (2006) J Biotechnol 121:192-200], and HS medium [Kopp M et al (2004) J Biotechnol 107:29-40] was used for Sorangium species. Marine myxobacteria belonging to Enhygromyxa and Plesiocystis were grown in VY/4-SWS [Iizuka T et al. (2003a) Int J Syst Evol Microbiol 53:189-195; Iizuka T et al. (2003b) Syst Appl Microbiol 26:189-196], while Haliangium was cultivated in CY-SWS [Iizuka T et al. (1998) FEMS Microbiol Lett 169:317-322]. Fatty acid profiles of Phaselicystis flava were taken from Garcia R O et al. (2009) Int J Syst Evol Microbiol 59:1524-1530: Gliding bacteria belonging to Herpetosiphon were obtained from HZI, and Flexibacter from SB. The later bacteria were cultivated in LB medium, while Herpetosiphon strains were grown on a solid VY/2 agar medium.
Extraction of Fatty Acids for Analyses
For the majority of strains, cell pellets were obtained from 2 ml liquid culture aliquots of the shake cultures, which were completely dried at 60° C. for 30 min in a vacuum centrifuge. In case of Herpetosiphon spp., which did not grow in liquid medium, a loopful of cells were scraped off from the surface of the agar of well-grown solid media.
Dried cells were then extracted overnight with 500 uL FAME solution (methanol, toluene, sulfuric acid; 50:50:2 v/v). Thereafter, an aliquot of 400 uL R2 reagent (0.5 M NH4HCO3, 2 M KCl) was added. After mixing the sample in a vortex, the solution was centrifuged at 5000 rpm for 4 min and 75 ul extract taken from the upper phase of the solution were derivatized with 25 ul MSTFA. The sample was then incubated at 37° C. for 30 min before being subjected to GC-MS analysis.
Results are depicted in tables 8 and 9, revealing large amounts of EPA and DHA are particularly restricted to the members of the new genus Aetherobacter. Various groups of Myxobacteria showed no production of omega-3-PUFA at all. Among other Sorangiiinae, all type strains studied were found devoid of omega-3 PUFAs. Surprisingly, two recently isolated strains of Sorangium cellulosum from the SB collection show the general morphological and physiological characteristics of the genus and species, revealed production of EPA in small amounts when studied by GC-MS as shown in table 8. This observation gives rise to assume that a closer examination of further Sorangium spp. and other species of suborder Sorangiineae will inadvertently reveal additional producer strains for these omega-3 PUFAs.
In the type strain of the marine species Enhygromyxa salina (suborder Nannocystineae), EPA was also found in small amounts (table 9), revealing the general potential of this suborder to produce omega-3 PUFAs. The said compounds were not encountered in the gliding non-fruiting eubacteria (Flexibacter, Herpetosiphon) studied concurrently, and they were not found in any of the other taxa of grampositive and gramnegative eubacteria that have so far been widely studied for their fatty acid profiles in the course of chemotaxonomic studies, except for the examples cited above in “State of the Art.” Even the discovery of EPA in Enhygromyxa and Sorangium has never before reported, possibly owing to the fact that only the major FA components of bacteria were so far regarded during their chemotaxonomic evaluation by GC-MS. Myxobacteria therefore remain the only class of eubacteria that has many species that can be easily cultured and produce significant amounts of the commercially valuable omega-3 PUFA (i.e., DHA and EPA) at the same time, as discovered in the course of this study. Using a modification of the well-established chemotaxonomic technique for FA-profiling by GC-MS with special emphasis on DHA and EPA has lead in our study to the identification of additional producers of these compounds. From these data it is concluded that certain taxa of Myxobacteria differ from the remainder of culturable eubacteria by having the ability to produce omega-3 PUFA. As up to 90% of all existing eubacterial species remain to be discovered and described, the chances are good to find additional producers, in addition to the novel genus Aetherobacter that can also produce omega-3 PUFAs. A novel strategy, including phylogenetic and chemotaxonomic data, following the examples provided in this patent application, is therefore proposed to discover additional myxobacterial producers of omega-3 PUFAs.
Enhygromyxa salina
Myxobacteria are currently divided into six families, twenty genera and 46 species, which can be segregated based on morphological, biochemical and physiological character. This taxonomy was also reflected by molecular phylogenetic studies, based on similarity analyses of their 16S rDNA, revealing Myxobacteria as a monophyletic group [Velicer G, Hillesland K (2008) In Myxobacteria: multicellularity and differentiation (Whitworth D, ed), pp. 17-40. ASM Press:Washington D.C.; Spröer C et al. (1999) Int J Syst Bacteriol 49, 1255-1262; Garcia R O et al. (2009) Int J Syst Evol Microbiol 59:1524-1530].
Following the surprising discovery that the members of the novel, undescribed genus Aetherobacter were capable of producing omega-3 PUFAs as major fatty acids in their biomass, a panel of representative strains of class Myxobacteria were selected to study their fatty acid profiles in order to investigate the distribution of PUFAs and other fatty acids and in particular, the specificity of the overproduction of PUFAs by the novel genus Aetherobacter. Preferably, the type strains of culturable species, including terrestrial as well as marine species, were selected.
Reference 16S rDNA gene sequences used in this study were downloaded from GenBank. Corrected sequences of the type and novel strains were also included (table 1). Sequence alignments were created using the software ClustalW version 2.0. [Larkin et al. (2007) Bioinformatics Applications Note 23(21): 2947-2948].
Distance matrices between sequences were calculated using the Jukes-Cantor model [Jukes T H, Cantor C R (1969) Evolution of protein molecules. pp. 21-123 in H N Munro (ed) Mammalian protein metabolism. New York: Academic Press]. From the distance matrices, a neighbour-joining tree was constructed as described by Saitou N & Nei M (1987) Mol Biol Evol 4:406-425. A bootstrap of 1000 replicates was designed [Felsenstein J (1985) Evolution 39:783-791], and a consensus tree was performed using the Geneious tree builder. All these programs are packed in the Geneious Pro 4.7.6 software, available from Geneious (Auckland, New Zealand).
Genomic DNA was extracted from actively growing cultures using the protocol for Gram-negative bacteria of the Qiagen genomic DNA purification kit (Gentra Systems Inc., Minneapolis, USA). Only strains with gaps and ‘N’ sequences were prepared for repeated 16S rDNA gene sequencing. All type strains without 16S rDNA gene sequence were also included (
Homology analyses of 16S rDNA sequence data: According to the widely used and well established BLASTn analysis [Zhang Z et al. (2000) J Comput Biol 7:203-214, the newly obtained 16S rDNA sequences of the Aetherobacter strains were checked for homology to published sequence data. tables 10-12 show the results of the BLASTn searches for each individual strain. The 16S rDNA sequence of A. fasciculatus DSM 21835 showed 96% identity to the cellulose-degrading Byssovorax (Byssophaga) cruenta DSM 14553T and 95% to Sorangium (syn: Polyangium) cellulosum strains (table 10). Surprisingly, the 16S rDNA sequence of A. rufus DSM 23122 and Aetherobacter sp. DSM 23098 also showed 96% identity with Byssovorax cruenta (table 11) and 95% to Sorangium cellulosum strains (table 12).
The above data reveal that as inferred from the homologies of 16S rDNA, the identification of all three Aetherobacter strains as members of the Mycococcales is further confirmed. The novel bacterial strains are most closely related to members of the family Polyangiaceae, as indicated by homologies to Byssovorax (Byssophaga) cruenta and Sorangium (syn: Polyangium) cellulosum. The closest similarity (96-98%) were found to bacterial clones which may perhaps represent further hitherto uncultured species of myxobacteria that are more or less immediately allied to the genus Aetherobacter. This relationship is illustrated for Aetherobacter fasciculatus DSM 21835 in
Byssophaga cruenta partial 16S rDNA gene, strain DSM 14553T
Polyangium cellulosum strain So9857 16S ribosomal DNA gene,
Sorangium cellulosum strain KYC3093 16S ribosomal DNA gene,
Sorangium cellulosum strain KYC3074 16S ribosomal DNA gene,
Polyangium cellulosum strain So9881 16S ribosomal DNA gene,
Polyangium cellulosum strain So ce26 16S ribosomal DNA gene,
Sorangium cellulosum strain KYC3047 16S ribosomal DNA gene,
Sorangium cellulosum strain KYC3139 16S ribosomal DNA gene,
Sorangium cellulosum strain KYC3059 16S ribosomal DNA gene,
Sorangium cellulosum strain KYC3076 16S ribosomal DNA gene,
Polyangium cellulosum strain So9735-22 16S ribosomal DNA gene,
Sorangium cellulosum ‘So ce 56’ complete genome
Byssophaga cruenta partial 16S rDNA gene, strain DSM 14553T
Polyangium cellulosum strain So9857 16S ribosomal DNA gene,
Sorangium cellulosum ‘So ce 56’ complete genome
Polyangium cellulosum strain So9881 16S ribosomal DNA gene,
Polyangium cellulosum strain So ce26 16S ribosomal DNA gene,
Sorangium cellulosum strain KYC3093 16S ribosomal DNA gene,
Sorangium cellulosum strain KYC3074 16S ribosomal DNA gene,
Sorangium cellulosum strain KYC3047 16S ribosomal DNA gene,
Sorangium cellulosum strain KYC3466 16S ribosomal DNA gene,
Sorangium cellulosum strain KYC3139 16S ribosomal DNA gene,
Sorangium cellulosum strain KYC3059 16S ribosomal DNA gene,
Byssophaga cruenta partial 16S rDNA gene, strain DSM 14553T
Polyangium cellulosum strain So9857 16S ribosomal DNA gene,
Polyangium cellulosum strain So ce26 16S ribosomal DNA gene,
Sorangium cellulosum strain KYC3093 16S ribosomal DNA gene,
Sorangium cellulosum strain KYC3074 16S ribosomal DNA gene,
Polyangium cellulosum strain So9881 16S ribosomal DNA gene,
Sorangium cellulosum strain KYC3047 16S ribosomal DNA gene,
Sorangium cellulosum ‘So ce 56’ complete genome
Sorangium cellulosum strain KYC3139 16S ribosomal DNA gene,
Sorangium cellulosum strain DSM14627 16S ribosomal DNA
Sorangium cellulosum strain KYC3025 16S ribosomal DNA gene,
16S rDNA sequence of Flexibacter flexilis (Gen Bank Acc. no AB078050), derived from strain IFO 15060 showed 76% similarity to that of Aerherobacter fasciculatus, and other Aetherobacter species, while the 16S rDNA sequence of Herpetosiphon geysericola (GenBank Acc. No. AF039293) showed 75% similarity upon a BLAST comparison, determined with the same procedure outlined in the above examples.
These two species are gliding bacteria and belong to the delta subgroup of proteobacteria; they are deemed to be closely related to the myxobacteria but do not produce fruiting bodies and omega-3 PUFAs.
| Number | Date | Country | Kind |
|---|---|---|---|
| 08170390 | Dec 2008 | EP | regional |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP2009/008560 | 12/1/2009 | WO | 00 | 7/15/2011 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2010/063451 | 6/10/2010 | WO | A |
| Number | Name | Date | Kind |
|---|---|---|---|
| 7022512 | Barclay | Apr 2006 | B2 |
| 20040203121 | Barclay | Oct 2004 | A1 |
| 20060099694 | Tremblay et al. | May 2006 | A1 |
| 20130196391 | Wenzel et al. | Aug 2013 | A1 |
| Number | Date | Country |
|---|---|---|
| 2006-513713 | Apr 2006 | JP |
| 2004083442 | Sep 2004 | WO |
| WO 2007-068997 | Jun 2007 | WO |
| 2011151298 | Dec 2011 | WO |
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