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  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P13/00—Preparation of nitrogen-containing organic compounds
  • C12P13/02—Amides, e.g. chloramphenicol or polyamides; Imides or polyimides; Urethanes, i.e. compounds comprising N-C=O structural element or polyurethanes
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/14—Fungi; Culture media therefor
  • C12N1/16—Yeasts; Culture media therefor
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/14—Fungi; Culture media therefor
  • C12N1/16—Yeasts; Culture media therefor
  • C12N1/165—Yeast isolates
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
  • C12R2001/00—Microorganisms ; Processes using microorganisms
  • C12R2001/645—Fungi ; Processes using fungi
  • C12R2001/84—Pichia

Definitions

• the present invention relates to microbial strains improved in their sphingoid base productivity and to a method for the preparation thereof .
• sphingolipids refers to a group of lipids which are derived from sphingoid bases like sphingosine. Sphingolipids occur frequently in the cellular membranes of animals, plants and microorganisms. The exact function of sphingolipids in humans remains unknown, but it is clear that this group of compounds is involved in the transmission of electrical signals in the nervous system and in the stabilization of cell membranes. It has also been suggested that glycosphingosines have a function in the immune system: specific glycosphingosines function as receptors for bacterial toxins and possibly also as receptors for bacteria and viruses.
• Ceramides are a specific group of sphingolipids containing sphingosine- dihydrosphingosine or phytosphingosine as a base in amide linkage with a fatty acid. Ceramides are the main lipid component of the stratum corneum, the upper layer of the skin. The stratum corneum has an important barrier function, external compounds are generally kept outside of its barrier and the loss of moisture is limited. Topical application of compositions comprising sphingolipids such as ceramides improves for instance the barrier function and moisture-retaining properties of the skin (Curatolo, 1 987, Pharm. Res. 4, 271 -277; Kerscher et al . , 1 991 , Eur. J . Dermatol.
• Sphingoid bases as such are known to mediate several physiological effects by inhibiting the activity of protein kinase C, a key enzyme in the signal transduction pathway. Furthermore, sphingoid bases are included in cosmetic or dermatological compositions for their anti-inflammatory and antimicrobial activity.
• heterogenous sphingolipid preparations for cosmetics are mainly extracted from animal sources. Obviously, this is a rather costly process on an industrial scale. Moreover, it has been found that these materials are potentially unsafe due, for example, to the possible presence of bovine spongiform encephalopathy (BSE) in bovine tissue.
• BSE bovine spongiform encephalopathy
• Microorganisms such as the yeasts Pichia ciferrii (Wickerham and Stodola, 1 960, J. Bacteriol. 80, 484-491 ) have been found to produce sphingolipids as such, as well as sphingosine, phytosphingosine and/or derivatives thereof. This discovery provides sources for sphingolipids themselves and for starting materials for the production of other commercially valuable compounds which could offer a viable alternative to the use of animal sources of these compounds.
• acetylated derivatives of sphingosine, dihydrosphingosine and phytosphingosine as produced by Pichia ciferrii may be deacetylated and the thus-obtained sphingosine, dihydrosphingosine or phytosphingosine may be chemically converted into related compounds such as ceramides, pseudoceramides and/or glycoceramides which in turn may be applied in cosmetic and dermatological products (International patent application WO 93/20038) .
• the present invention discloses Pichia strains displaying a substantially enhanced productivity of a sphingoid base or derivative thereof.
• a substantially enhanced productivity of a sphingoid base or derivative thereof is meant to relate to a productivity which is at least two times higher than the productivity of Pichia ciferrii Y-1 031 F-60-1 0, deposited as CBS 408.94 (see WO 95/1 2683) when measured under identical conditions.
• a substantially enhanced productivity of a sphingoid base or derivative thereof is meant to relate to a productivity which is at least four, more preferably which is at least six, even more preferably which is at least eight, most preferably which is at least ten times higher than the productivity of Pichia ciferrii ' Y-1 031 F-60-1 0, deposited as CBS 408.94.
• the present invention further discloses a novel method to isolate microbial strains displaying an enhanced productivity of sphingoid bases or derivatives thereof.
• the novel method of the invention comprises an initial screening step wherein the sphingoid base productivity of a series of strains is measured by determining the antimicrobial activity of the sphingoid base or derivative thereof as produced by each individual strain being present within said series of strains.
• This initial screening step advantageously allows the screening of a substantial amount of individual strains for their sphingoid base productivity and the selection of a subset of strains enriched in strains displaying an enhanced productivity as compared to a reference strain.
• the method of the invention is directed to the selection or
• Said reference strain preferably is Pichia ciferrii Y- 1 031
• the method of the invention comprises essentially the steps of : ⁇ o • screening the sphingoid base productivity , of a series of strains by determining the antimicrobial activity of the sphingoid base as produced by each individual strain being present within said series of strains, selecting a subset of strains for at least one further passaging of cells, further passaging of cells by transferring an amount of cells from each 15 individual strain within said selected subset to a next medium and culturing said amount of cells in said next medium under conditions conducive to the production of a sphingoid base, selecting a strain with an enhanced productivity of a sphingoid base as compared to the productivity of a reference strain when cultured under 20 identical conditions.
• sphingoid base as used in this invention includes sphingoid base derivatives as specified hereinbelow ⁇ although the term derivatives may not always be specifically mentioned .
• the method of the invention as 25 described hereinabove represents one cycle of screening and selection. However, repeated cycles of screening and selection also are applicable to obtain microbial strains displaying an enhanced sphingoid base productivity.
• the cells are cultured under conditions conducive to the production of a sphingoid base compound.
• the sphingoid base productivity of each individual culture is measured by determining the antimicrobial activity of the sphingoid base-containing culture supernatant.
• the culturing of cells to obtain a culture supernatant for determining the antimicrobial activity thereof conveniently may be performed in any suitable system. For instance, the culturing may be performed in a shake flask or in a microtiterplate.
• the antimicrobial activity of a sphingoid base-containing culture supernatant is conveniently determined by measuring the extent of the inhibiton of the growth of a suitable indicator strain being produced by incubation of said indicator strain in the presence of said culture supernatant.
• a suitable indicator strain may be any microbial strain which is susceptible to the antimicrobial activity of a sphingoid base or derivative thereof.
• the indicator strain is a bacterial strain, more preferably a gram-positive bacterial strain.
• a suitable dilution of the indicator strain is incubated with culture supernatant which is obtained from individual strains to be screened for sphingoid base productivity.
• a suitable dilution may be obtained by diluting a stationary culture, for instance an overnight culture, of the indicator strain.
• a suitable dilution of an overnight culture typically is about 1 00 to about 500 times.
• the extent of growth inhibition as produced by the sphingoid base-containing culture supernatant is determined by measuring the optical density of the incubated samples.
• the incubation conditions may be dependent on the indicator strain used and the sphingoid base productivity. For instance, the time period for incubation may vary from 4-8 hours to an overnight incubation, depending on the extent of growth inhibition which is obtained.
• the incubation temperature may be dependent on the optimal conditions for growth of the indicator strain. In general, the incubation temperature is 37 ° C. For prolonged incubations, the temperature may be lowered to about 25 ° C.
• the indicator strain is a bacterial strain of the species Staphylococcus aureus or Streptococcus pyogenes.
• the initial screening step is performed using a microtiterplate system for the incubation and analysis steps.
• a microtiterplate system for the incubation and analysis steps.
• a subset of strains is selected for at least one further passaging of cells.
• Said further passaging of cells comprises the transfer of cells from each individual strain being present within said selected subset of strains to a next medium and a subsequent culturing of each individual strain under conditions conducive to the production of the sphingoid base compound.
• the culture medium or a suitable dilution thereof is then analyzed for sphingoid base productivity using a suitable assay.
• cells from each individual strain being present within the selected subset are cultured in shake flasks or tube cultures.
• the sphingoid base productivity of each culture is determined by analysis, preferably a quantitative analysis, of the culture medium or a suitable dilution thereof. Strains are then identified which fulfil a certain increase in productivity as compared to a reference strain.
• Said series of strains to be subjected to the screening and selection method of the invention may be obtained in several ways, said ways being not critical to the invention.
• said series of strains may comprise a series of unrelated strains to be analyzed for sphingoid base productivity, such as strains from different species, different genera, different families, etc.
• Strains which may be subjected to the screening and selection method according to the present invention include yeast, bacterial and fungal strains capable of naturally producing sphingoid bases and/or derivatives thereof.
• strains which are selected by the method of the invention are strains which demonstrate the highest inherent production levels of the desired products.
• yeast strains for use in the present invention may be selected from species of the genera Saccharomyces, Kluveromyces, Debaromyces, PicTiia, Hansenula, Lipomyces, Sporobolomyces, Cryptococcus, Torulopsis, Endomycopsis, Candida, Trichosporon, Hanseniaspora and Rhodotorula.
• Preferred yeast strains belong to the genus Pichia, Hansenula, Endomycopsis, Candida, Saccharomyces and Hanseniaspora and particularly the species Pichia ciferrii, Candida utilis and Saccharomyces cerevisiae. Most preferred are yeast strains which belong to the genus Pichia and most preferably to the species Pichia ciferrii (especially Pichia ciferrii NRRL Y- 1 031 F-60-1 0) .
• Preferred fungi are of the genera Aspergillus and Penicillium and are more preferably of the species Aspergillus sydowi and Penicillium notatum.
• Preferred bacteria are of the genera Sphingobacterium (especially S. versatilis, S. multivorum and S. mizutae), Acetobacter (especially A. xy/inum), Bacteroides (especially B. melaninogenicus , B. fragilis, B. ruminicola and B.
• Bdellovibrio especially Bdellovibrio bacteriovus
• Xanthomonas especially Xanthomonas campestris
• Flavobacterium especially Flavobacterium devorans
• said series of strains is obtained via mutagenesis of a suitable parent strain.
• a parent population of cells is subjected to a mutagenic treatment with the aim to introduce genetic variation into said population.
• the type of mutagenic treatment which is applied is not critical to the invention.
• a mutagenic treatment typically may comprise a so-called classical treatment, but also may include DNA-mediated transformation.
• a classical mutagenic treatment includes a treatment with a chemical mutagenic agent, such as an alkylating agent like N-methyl, N'-nitro, N-nitrosoguanidine (NTG) or ethyl methane sulfonate (EMS), or a physical treatment, such as UV irradiation, or a combination of both treatments.
• a chemical mutagenic agent such as an alkylating agent like N-methyl, N'-nitro, N-nitrosoguanidine (NTG) or ethyl methane sulfonate (EMS), or a physical treatment, such as UV irradiation, or a combination of both treatments.
• the treatment preferably is performed in such a way that a population of mutagenized cells is obtained wherein a substantial amount of cells has received a single mutation and/or that a mutant strain is provided which is capable of producing enhanced levels of the desired product as compared-to its parent strain when cultured under identical conditions.
• DNA-mediated transformation includes a transformation with random fragments of homologous or heterologous nucleic acid and/or a transformation with specifically selected DNA fragments, for instance DNA fragments comprising one or more genes involved in the biosynthesis of a sphingoid base or derivative thereof.
• LCB 1 and LCB2 encode the subunits of the serine palmitoyl transferase
• SUR2 encoding the hydroxylase that introduces the 4-OH group of phytosphingosine
• a suitable parent strain to be subjected to a mutagenic treatment is a strain of the genus Pichia, more preferably a strain of the species Pichia ciferrii, most preferably a strain of the species Pichia ciferrii Y-1 031 F-60-1 0 or a derivative thereof.
• the present invention thus provides a method for the isolation of a microbial strain capable of producing enhanced levels of a sphingoid base or a derivative thereof as compared to a reference strain.
• microbial strains are provided which are capable of producing enhanced levels of sphingosines and/or keto, glycosylated or acetylated derivatives thereof including 3-ketosphingosine, triacetylsphingosines, diacetylsphingosines and N-acetylsphingosine; dihydrosphingosines and/or keto, glycosylated or acetylated derivatives thereof including 3-ketodihydrosphingosine, triacetyldihydrosphingosines, diacetyldihydrosphingosines and N-acetyldihydrosphingosine; and/or phytosphingosines and/or keto, glycosylated or acetylated derivatives thereof including tetraacetylphytosphingosine, triacetylphytosphingosines, diacetylphytosphingosines and N-acetylphy
• Preferred strains are strains capable of producing enhanced levels of phytosphingosines and partially or fully acetylated derivatives thereof such as triacetylphytosphingosines (TriAPS) and tetraacetylphytosphingosine (TAPS) .
• TriAPS triacetylphytosphingosines
• TAPS tetraacetylphytosphingosine
• strains displaying a certain enhancement in sphingoid base productivity as compared to a reference strain when analyzed in a shake flask culture typically display a substantially similar enhancement when measured on a larger scale.
• Quantitative analysis may be performed by various methods known in the art. For instance, quantitative analysis on the basis of TAPS production levels may be performed on culture supernatants of shake flasks cultures, said culture supernatants being extracted with a suitable solvent, such as CDCI 3, and analyzed by NMR. TAPS is preferably used as a standard for NMR analysis.
• Pichia strains preferably Pichia ciferrii strains, are isolated which produce an enhanced amount of TriAPS as compared to the reference strain Y-1 031 F-60-1 0.
• Pichia strains are isolated which produce TriAPS as the main sphingoid base, i.e. with a low or zero level of TAPS being formed.
• a further aspect of the present invention relates to a method for the production of a sphingoid base or a derivative thereof comprising the fermentation of a strain obtainable by the method of the invention under conditions conducive to the production of said sphingoid base or derivative thereof.
• the sphingoid base or derivative thereof thus-produced may be recovered from the fermentation broth, preferably from the fermentation supernatant, using common extraction and recovery technology. Once the desired product is obtained it may be used directly or may be further processed depending on the desired use. For example, acetylated derivatives of sphingosine, dihydrosphingosine and/or phytosphingosine may be deacetylated either enzymatically or chemically.
• Deacetylated sphingoid base derivatives e.g. sphingosine, dihydrosphingosine and/or phytosphingosine, may be used directly or may subsequently be used as starting materials in the synthesis of commercially valuable sphingolipid products such as ceramides (see WO93/20038), glycoceramides and pseudoceramides.
• Direct use of the sphingoid base compounds obtained according to the invention includes the use of said compounds in cosmetic and dermatological compositions.
• the indicator strain Staphy/ococcus aureus ATCC 1 4458, was stored in deep frozen condition.
• One freeze tube was used to inoculate a 1 00 Tnl shake flask containing 25 ml BHI medium.
• the culture was incubated overnight ( 1 6-1 7 h) at 37 °C. Subsequently, the overnight culture was diluted
• the production strains were inoculated from an agar surface into 1 00 ml shake flasks containing 25 ml of a medium .containing 33 g/l dextrose, 1 g/l yeast extract, 4.83 g/l NH 4 CI, 1 .0 g/l KH 2 P0 4 , 0.88 g/l MgS0 4 .7aq, 0.06 g/l NaCl, 0.2 g/l CaCI 2 .2aq, 20 g/l KH-phthalate, 0.3 ml/l of a concentrated trace element solution, 1 .5 ml/l of a concentrated vitamin solution and 0.1 6 ml/l Structol antifoaming agent. After 72h of growth on a rotary shaker (25 °C) the flasks were sampled, and the samples were centrifuged for 1 0 minutes at 4000 rpm.
• each selected strain was inoculated from a deep-frozen glycerol suspension into 500 ml baffled shake flasks, containing 1 00 ml of the medium described in Example 2. After 40h of growth on a rotary shaker at 25 °C, this seed culture was used to inoculate three 500 ml baffled shake flasks containing 1 00 ml of the same medium, at a level of 5% by volume. Subsequently these production cultures were incubated for another 48h on the shaker, and sampled. The samples were treated as described in Example 2.
• the supernatant fractions were subjected to the bioassay as described in Example 2, but this time the TAPS concentration was also measured directly, using NMR spectroscopy. Strains were selected on the basis of the NMR results, which were also used to assess whether the bioassay was still able to disciminate between the higher production levels. It was found that this was always the case, provided that the sample supernatant was diluted in proportion to the TAPS concentration. Dilutions up to 5-fold were used to tune the bioassay to the higher TAPS-concentrations found in cultures of the more advanced strains.
• COS1 A which is a working cell bank of the haploid wild-type strain Pichia ciferri F-60-1 0, and COS 1 0, which ⁇ s a working cell bank of COS 1 06, one of the strains obtained by chemical mutagenesis and selected by the primary selection described in Example 2.
• COS 1 0 has been deposited at the "Centraal Bureau voor Schimmelcultures" at July 23, 1 998, as CBS 1 01 070.
• strain COS 1 44 showed an increased TAPS production over COS 1 7, by about 25%, which gives a COS 1 44 a production level of 302% compared to COS1 A. Therefore a working cell bank was prepared of this strain, designated COS1 9A, which was shown to have the same TAPS production as did COS 1 44.
• the bioassay can also be used to select TriAPS producers, since the growth inhibition caused by this compound seems to be of the same order of magnitude as for TAPS.

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