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EP1042451A1 - Linoleate isomerase - Google Patents

Linoleate isomerase

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Publication number
EP1042451A1
EP1042451A1 EP98964935A EP98964935A EP1042451A1 EP 1042451 A1 EP1042451 A1 EP 1042451A1 EP 98964935 A EP98964935 A EP 98964935A EP 98964935 A EP98964935 A EP 98964935A EP 1042451 A1 EP1042451 A1 EP 1042451A1
Authority
EP
European Patent Office
Prior art keywords
seq
nucleic acid
linoleate isomerase
isomerase
acid molecule
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP98964935A
Other languages
German (de)
English (en)
Other versions
EP1042451A4 (fr
Inventor
Reinhardt A. Rosson
Alan D. Grund
Ming-De Deng
Fernando Sanchez-Riera
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
DCV Inc
Original Assignee
DCV Inc
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Filing date
Publication date
Application filed by DCV Inc filed Critical DCV Inc
Publication of EP1042451A1 publication Critical patent/EP1042451A1/fr
Publication of EP1042451A4 publication Critical patent/EP1042451A4/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • 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
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6409—Fatty acids
    • C12P7/6427—Polyunsaturated fatty acids [PUFA], i.e. having two or more double bonds in their backbone
    • 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
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • 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
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/90—Isomerases (5.)
    • C—CHEMISTRY; METALLURGY
    • 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
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6409—Fatty acids
    • C12P7/6427—Polyunsaturated fatty acids [PUFA], i.e. having two or more double bonds in their backbone
    • C12P7/6431—Linoleic acids [18:2[n-6]]
    • C—CHEMISTRY; METALLURGY
    • 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
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6436—Fatty acid esters
    • C12P7/6445—Glycerides
    • C12P7/6472—Glycerides containing polyunsaturated fatty acid [PUFA] residues, i.e. having two or more double bonds in their backbone
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y502/00—Cis-trans-isomerases (5.2)
    • C12Y502/01—Cis-trans-Isomerases (5.2.1)
    • C12Y502/01005—Linoleate isomerase (5.2.1.5)

Definitions

  • One method of overcoming the shortcomings of chemical transformation is a whole cell transformation or an enzymatic transformation of linoleic acid, linolenic acid or their derivatives to CLA. It is well known that a biological system can be an effective alternative to chemical synthesis in producing a desired chemical compound where such a biological system is available.
  • the existence of linoleate isomerase enzyme to convert linoleic acid to CLA has been known for over thirty years, however, no one has yet successfully isolated the enzyme. And because it has not yet been isolated, the linoleate isomerase enzyme has not been sequenced.
  • the present invention generally relates to isolated linoleate isomerase nucleic acid molecules, isolated linoleate isomerase proteins, immobilized bacterial cells having a genetic modification that increases the action of linoleate isomerase, and methods of using such nucleic acid molecules, proteins and cells to produce CLA.
  • One embodiment of the invention relates to an isolated linoleate isomerase. Included in the invention are linoleate isomerases from Lactobacillus, Clostridium, Propior ⁇ bacteriwn, Butyrivibrio and Eubacterium, and particularly, from Lactobacillus reuteri, Clostridium sporogenes, Propionibacterium acnes, Butyrivibrio fibrisolvens, Propionibacterium a ⁇ dipropiom ⁇ , Propionibacterium freudenreichii and Eubacterium lent m.
  • linoleate isomerases include linoleate isomerases from Lactobacillus reuteri, Clostridium sporogenes, and Propionibacterium acnes.
  • an isolated linoleate isomerase of the present invention converts linoleic acid and linolenic acid to CLA, including (cis, trans)-9, 11 -linoleic acid and/or (trans, cw)-10,12-linoleic acid.
  • a linoleate isomerase of the present invention includes linoleate isomerases having one or more of the following biochemical characteristics: a size of about 50 kDa or about 67 kDa; an optimum pH of about 6.8 or about 7.5 ; a specific linoleic acid isomerization activity of at least about 1000 nmoles mg "1 min "1 ; a Km of about 8.1 M for linoleic acid, a pH optimum of about 7.5, and a Ki of about 80 M for oleic acid; and/or an initial velocity that decreases at about 60 ⁇ M linoleic acid.
  • a linoleate isomerase of the present invention can be either a membrane bound or a soluble enzyme.
  • the linoleate isomerase of the present invention can include lipid material.
  • an isolated linoleate isomerase is encoded by a nucleic acid molecule comprising a nucleic acid sequence selected from the group of SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16,
  • the linoleate isomerase is bound to a solid support, which includes, but is not limited to organic supports, biopolymer supports and inorganic supports.
  • a solid support which includes, but is not limited to organic supports, biopolymer supports and inorganic supports.
  • Another embodiment of the present invention relates to an isolated antibody that selectively binds to the isolated linoleate isomerase of the present invention.
  • Yet another embodiment of the present invention relates to a method for producing CLA, including contacting an oil, which comprises a compound selected from the group of linoleic acid and linolenic acid, with an isolated linoleate isomerase enzyme of the present invention to convert at least a portion of the compound to CLA.
  • the compound is in the form of a triglyceride and the method further includes contacting the oil with a hydrolysis enzyme to convert at least a portion of the triglyceride to free fatty acids.
  • a hydrolysis enzyme can include lipases, phospholipases and esterases.
  • the method of the present invention can also include a step of recovering the CLA.
  • an isolated nucleic acid molecule of the present invention encodes a linoleate isomerase, including a linoleate isomerase homologue.
  • such an isolated nucleic acid molecule hybridizes under stringent conditions to a nucleic acid molecule having a nucleic acid sequence selected from the group of SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO:26, and/or the complement of any of such nucleic acid sequences.
  • an isolated nucleic acid molecule of the present invention includes a nucleic acid sequence having at least about 70% identity with a nucleic acid sequence selected from the group of SEQ ID NO:4, SEQ ID NO: 8, SEQ
  • an isolated nucleic acid molecule of the present invention has a sequence selected from the group of SEQ ID NO:4, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO:
  • recombinant molecules recombinant viruses and recombinant cells which include an isolated nucleic acid molecule of the present invention.
  • recombinant cell of the present invention is from a microorganism which includes, but is not limited to, Lactobacillus reuteri,
  • Another embodiment of the present invention relates to a method for producing CLA, including contacting an oil which comprises a compound selected from the group of linoleic acid and linolenic acid, with an isolated linoleate isomerase enzyme encoded by the isolated nucleic acid molecule of the present invention to convert at least a portion of the compound to CLA.
  • the bacterial cell can be from a microorganism including Lactobacillus, Clostridium, Propionibacterium, Butyrivibrio, Escherichia, Bacillus and Eubacterium cells, preferably Lactobacillus reuteri, Clostridium sporogenes, Propionibacterium acnes, Propionibacterium freudenreichii, Propionibacterium acidipropionici, Escherichia coli,
  • the cell can be a naturally occurring bacterial cell having a linoleate isomerase, or a genetically modified microorganism as described above.
  • a genetically modified microorganism has increased linoleate isomerase action.
  • the compound can include compounds in the form of a triglyceride such that at least a portion of the triglycerides are converted to free fatty acids. Other features of the method are as described above in the method to produce CLA.
  • FIG. IA is a line graph showing whole cell biotransformation of CLA from linoleic acid by Clostridium sporogenes ATCC 25762 under aerobic conditions.
  • Fig. 2A is a line graph illustrating whole cell biotransformation of CLA from linoleic acid by C. bifermentans ATCC 638 under aerobic conditions.
  • Fig. 2B is a line graph illustrating whole cell biotransformation of CLA from linoleic acid by C. bifermentans ATCC 638 under anaerobic conditions.
  • Fig. 3 A is a line graph showing whole cell biotransformation of CLA from linoleic acid by Propionibacterium jensenii ATCC 14073.
  • Fig. 3B is a line graph showing whole cell biotransformation of CLA from linoleic acid by P. acnes ATCC 6919.
  • Fig. 4 is a line graph demonstrating whole cell biotransformation of CLA from linoleic acid by P. acidipropionici ATCC 25562.
  • Fig. 5 is a line graph illustrating whole cell biotransformation of CLA from linoleic acid by L. reuteri PYR8.
  • Fig. 6 is a line graph showing DEAE chromatography of detergent solubilized isomerase by L. reuteri PYR8.
  • Fig. 7 is a line graph demonstrating hydroxyapatite chromatography of isomerase activity by L. reuteri PYR8.
  • Fig. 8 is a line graph illustrating chromatofocusing of linoleic acid isomerase activity by L. reuteri PYR8.
  • Fig. 9 is a schematic illustration of the linoleate isomerase genes and flanking open reading frames in L. reuteri PYR8.
  • Fig. 10 is a schematic illustration of the putative transcription terminator in the linoleate isomerase gene.
  • Fig. 11 is an illustration of several constructs for linoleate isomerase expression in is. coli.
  • Fig. 12 is an illustration of several constructs for linoleate isomerase expression in Bacillus.
  • Fig. 13 is a flow diagram of the experimental protocol for the preparation of different protein fractions of E. coli which have expressed recombinant linoleate isomerase.
  • Fig. 14 is a line graph showing the formation of rlO,cl2-CLA from linoleic acid using whole cells of P. acnes.
  • Fig. 15 is a flow diagram showing the cell fractionation protocol for P. acnes
  • Fig. 16 is a line graph showing the effect of pH on linoleate isomerase activity in crude extracts of P. acnes ATCC 6919.
  • Fig. 17 is a line graph showing the time course of CLA formation in crude extracts of P. acnes ATCC 6919.
  • Fig. 18 is a line graph showing the time course for the formation of CLA in crude extracts of P. acnes ATCC 6919 at different levels of linoleic acid.
  • Fig. 19. is a line graph showing end point for formation of CLA in crude extracts of P. acnes ATCC 6919 at different levels of linoleic acid.
  • Fig. 20 is a graph illustrating DEAE ion exchange chromatography of total soluble protein from P. acnes ATCC 6919.
  • Fig. 21 is a graph illustrating hydrophobic interaction chromatography of total soluble protein from P. acnes ATCC 6919.
  • Fig. 22 is a graph illustrating chromatofocusing of isomerase activity from P. acnes ATCC 6919.
  • Fig. 23A is a graph showing a time course of CLA formation by C. sporogenes
  • Fig. 23B is a graph showing a time course of CLA formation by C. sporogenes ATCC 25762 under anaerobic conditions at room temperature.
  • Fig. 24 is a flow diagram showing an extraction protocol for C. sporogenes ATCC 25762.
  • Fig. 25 is a line graph showing linoleate isomerase optimum pH and temperature in C. sporogenes ATCC 25762.
  • Fig. 26 is a line graph showing optimum linoleic acid concentration for C. sporogenes ATCC 25762 linoleate isomerase.
  • Fig. 27 is a graph showing the time course for CLA formation by C. sporogenes ATCC 25762 linoleate isomerase.
  • Fig. 28 is a bar graph illustrating the stability of C. sporogenes ATCC 25762 linoleate isomerase in Tris and phosphate buffers.
  • Fig. 29 is an elution profile of fresh C. sporogenes ATCC 25762 linoleate isomerase extracts from DEAE-5PW.
  • Fig. 30 is a bar graph demonstrating the effect of culture medium on C. sporogenes ATCC 25762 growth and linoleate isomerase activity.
  • Fig. 31 is a bar graph showing the effect of CaCl 2 on C. sporogenes ATCC 25762 linoleate isomerase activity.
  • Fig. 36 is a bar graph showing the effect of buffer system on the activity of linoleate isomerase in C. sporogenes ATCC 25762.
  • Fig. 37 is a line graph illustrating the effect of glycerol and salt concentration on the stability of crude extracts of linoleate isomerase in C. sporogenes ATCC 25762.
  • Fig. 38 is a line graph showing the stability of detergent solubilized linoleate isomerase in C. sporogenes ATCC 25762.
  • Fig. 39 is an elution profile of C. sporogenes ATCC 25762 linoleate isomerase on DEAE Mono Q.
  • Fig. 40 is an elution profile of C. sporogenes ATCC 25762 detergent solubilized linoleate isomerase on DEAE-5PW column.
  • Fig. 41 is an elution profile showing separation of partially purified C. sporogenes ATCC 25762 linoleate isomerase by chromatofocusing.
  • the isolated linoleate isomerase can be used to produce CLA from linoleic acid, linolenic acid or their derivatives. More specifically, isolated linoleate isomerase can convert linoleic acid to conjugated linoleic acid and/or linolenic acid to conjugated linolenic acid.
  • conjugated refers to a molecule which has two or more double bonds which alternate with single bonds in an unsaturated compound.
  • Linoleate isomerase is a part of a biohydrogenation pathway in microorganisms which convert linoleic acid and other unsaturated fatty acids containing a 9,12-diene moiety into a 9, 11 -conjugate diene moiety which is then further metabolized to other fatty acids containing a 9-11 monoene moiety.
  • most linoleate isomerase converts (cis,cis)-9,12-linoleic acid to (cis,trans)-9,ll-linoleic acid as an intermediate in the biohydrogenation pathway.
  • the formation of CLA is followed by metabolism to other CLA isomers as well as metabolism to non-CLA compounds, such as a monoene fatty acid. Lactobacillus reuteri, however, produces and accumulates
  • CLA as an end product.
  • Other microorganisms such as Propionibacterium acnes convert (cis,cis)-9,12-linoleic acid to (trans,cis)-10,12-linoleic acid.
  • isolated linoleate isomerase refers to a linoleate isomerase outside of its natural environment in a pure enough form to achieve a significant increase in activity over crude extracts having linoleate isomerase activity.
  • a linoleate isomerase can include, but is not limited to, purified linoleate isomerase, recombinantly produced linoleate isomerase, membrane bound linoleate isomerase, linoleate isomerase complexed with lipids, linoleate isomerase having an artificial membrane, soluble linoleate isomerase and isolated linoleate isomerase containing other proteins.
  • An “artificial membrane” refers to any membrane-like structure that is not part of the natural membrane which contain linoleate isomerase.
  • An isolated linoleate isomerase of the present invention can be characterized by its specific activity.
  • a "specific activity” refers to the rate of conversion of linoleic acid to CLA by the enzyme. More specifically, it refers to the number of molecules of linoleic acid converted to CLA per mg of the enzyme per time unit.
  • the isolated linoleate isomerase of the present invention has a specific activity of at least about 1000 nmoles mg "1 min "1 , more preferably at least about 10,000 nmoles mg "x min "1 , and most preferably at least about 100,000 nmoles mg "1 min "1 .
  • Menten constant (IC- . K,- is a kinetic (i.e. , rate) constant of the enzyme-linoleic acid complex under conditions of the steady state.
  • the isolated linoleate isomerase of the present invention has K-, of at least about 8.1 ⁇ M at a pH of about 7.5 and at a temperature of about 20° C.
  • Kj is a dissociation rate of the oleic acid- enzyme complex.
  • the isolated linoleate isomerase of the present invention has Kj of from about 50 ⁇ M to about 100 ⁇ M at a pH of about 7.5 and at a temperature of about 20°C, and more preferably, greater than 100 ⁇ M, with no inhibition being most preferred.
  • the initial velocity (v 0 ) refers to the initial conversion rate of linoleic acid to CLA by the enzyme. Specifically, it refers to the number of molecules of linoleic acid converted to CLA per mg of the enzyme per time unit.
  • the maximum initial velocity rate of the isolated linoleate isomerase at a pH of about 7.5 is least about 100 nmoles/(sec-mg of protein), more preferably at least about 1,000 nmoles/(sec-mg of protein), and most preferably at least about 10,000 nmoles/(sec-mg of protein).
  • the isolated linoleate isomerase can be further characterized by its optimum pH.
  • the optimum pH refers to the pH at which the linoleate isomerase has a maximum initial velocity.
  • the optimum pH is between about 5 and about 10, more preferably between about 6 and about 8, and most preferably from about 6.8 to about 7.5.
  • Further embodiments of the isolated linoleate isomerase of the present invention include proteins which are encoded by any of the nucleic acid molecules which are described below. Further embodiments of the present invention include nucleic acid molecules that encode linoleate isomerases.
  • nucleic acid molecules include isolated nucleic acid molecules that hybridize under stringent hybridization conditions with: the complement of a gene encoding a naturally occurring linoleate isomerase, a nucleic acid molecule comprising the complement of a nucleic acid molecule encoding an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO: 11 and/or SEQ ID NO: 18, or a nucleic acid molecule comprising the complement of a nucleic acid molecule having a nucleic acid sequence SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 or SEQ ED NO:26.
  • the present invention includes an isolated nucleic acid molecule that encodes a protein comprising amino acid sequence selected from the group consisting of SEQ ID NO: l, SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO: 11 and/or SEQ ID NO: 18 and an isolated nucleic acid molecule having a nucleic acid sequence of SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 or SEQ ID NO:26.
  • a linoleate isomerase gene i.e.
  • nucleic acid molecule which encodes a linoleate isomerase can include an isolated natural linoleate isomerase gene or a homologue thereof, the latter of which is described in more detail below.
  • a nucleic acid molecule of the present invention can include one or more regulatory regions that control production of the linoleate isomerase protein encoded by that gene (such as, but not limited to, transcription, translation or post- translation control regions) as well as a full-length or partial coding region itself. It is to be noted that an isolated linoleate isomerase nucleic acid molecule of the present invention need not encode a protein having linoleate isomerase activity.
  • a linoleate isomerase nucleic acid molecule can encode a truncated, mutated or inactive protein, for example.
  • Such genes and the proteins encoded by such genes are useful in diagnostic assays, for example, or for other purposes such as antibody production, as is described in the Examples below.
  • stringent hybridization conditions refer to standard hybridization conditions under which nucleic acid molecules are used to identify similar nucleic acid molecules. Such standard conditions are disclosed, for example, in
  • stringent hybridization and washing conditions refer to conditions which permit isolation of nucleic acid molecules having at least about 70% nucleic acid sequence identity with the nucleic acid molecule being used to probe in the hybridization reaction, more particularly at least about 75 % , and most particularly at least about 80% .
  • Such conditions will vary, depending on whether DNA:RNA or DNA:DNA hybrids are being formed. Calculated melting temperatures for DNA:DNA hybrids are 10°C less than for DNA:RNA hybrids.
  • stringent hybridization conditions for DNA:DNA hybrids include hybridization at an ionic strength of 6X SSC (0.9 M Na + ) at a temperature of between about 20°C and about 35°C, more preferably, between about 28°C and about 40°C, and even more preferably, between about 35 °C and about 45 °C.
  • stringent hybridization conditions for DNA:RNA hybrids include hybridization at an ionic strength of 6X SSC (0.9 M Na + ) at a temperature of between about 30°C and about 45°C, more preferably, between about 38°C and about 50°C, and even more preferably, between about 45°C and about 55°C.
  • Preferred linoleate isomerase nucleic acid molecules of the present invention include nucleic acid molecules which comprise a nucleic acid sequence having at least about 70%, more preferably, at least about 80% and most preferably, at least about 90% identity with a nucleic acid sequence selected from SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 and/or SEQ ID NO:26.
  • nucleic acid molecules can be modified using a variety of techniques including, but not limited to, by classic mutagenesis and recombinant DNA techniques (e.g., site-directed mutagenesis, chemical treatment, restriction enzyme cleavage, ligation of nucleic acid fragments and/or PCR amplification), or synthesis of oligonucleotide mixtures and ligation of mixture groups to "build" a mixture of nucleic acid molecules and combinations thereof.
  • Nucleic acid molecule homologues can be selected by hybridization with a linoleate isomerase gene or by screening the function of a protein encoded by a nucleic acid molecule (e.g., ability to convert linoleic acid to CLA).
  • Transcription control sequences are sequences which control the imtiation, elongation, and termination of transcription. Particularly important transcription control sequences are those which control transcription initiation, such as promoter, enhancer, operator and represser sequences. Suitable transcription control sequences include any transcription control sequence that can function in at least one of the recombinant cells useful for expressing a linoleate isomerase of the present invention. A variety of such transcription control sequences are known to those skilled in the art. Preferred transcription control sequences include those which fimction in bacterial, fungal (e.g., yeast), insect, plant or animal cells.
  • One or more recombinant molecules of the present invention can be used to produce an encoded product (i.e., a linoleate isomerase protein) of the present invention.
  • an encoded product is produced by expressing a nucleic acid molecule as described herein under conditions effective to produce the protein.
  • a preferred method to produce an encoded protein is by transfecting a host cell with one or more recombinant molecules to form a recombinant cell. Suitable host cells to transfect include any bacterial, fungal (e.g., yeast), insect, plant or animal cell that can be transfected. Host cells can be either untransfected cells or cells that are already transformed with at least one nucleic acid molecule.
  • an isolated linoleate isomerase protein of the present invention is produced by culturing a cell that expresses the protein under conditions effective to produce the protein, and recovering the protein.
  • a preferred cell to culture is a recombinant cell of the present invention.
  • Effective culture conditions include, but are not limited to, effective media, bioreactor, temperature, pH and oxygen conditions that permit protein production.
  • An effective, medium refers to any medium in which a cell is cultured to produce a linoleate isomerase protein of the present invention.
  • Such medium typically comprises an aqueous medium having assimilable carbon, nitrogen and phosphate sources, and appropriate salts, minerals, metals and other nutrients, such as vitamins. Examples of suitable media and culture conditions are discussed in detail in the Examples section.
  • Cells of the present invention can be cultured in conventional fermentation bioreactors, shake flasks, test tubes, microtiter dishes, and petri plates. Culturing can be.carried out at a temperature, pH and oxygen content appropriate for a recombinant cell. Such culturing conditions are within the expertise of one of ordinary skill in the art. Depending on the vector and host system used for production, resultant proteins of the present invention may either remain within the recombinant cell; be secreted into the fermentation medium; be secreted into a space between two cellular membranes, such as the periplasmic space in E. coli; or be retained on the outer surface of a cell or viral membrane.
  • a recombinant virus includes a recombinant molecule of the present invention that is packaged in a viral coat and that can be expressed in a cell after delivery of the virus to the cell.
  • a number of recombinant virus particles can be used, including, but not limited to, those based on alphaviruses, baculoviruses, poxviruses, adenoviruses, herpesviruses, and retroviruses.
  • a truncated version of the protein such as a peptide
  • a truncated version of the protein such as a peptide
  • inserted, inverted, substituted and/or derivatized e.g., by glycosylation, phosphorylation, acetylation, myristoylation, prenylation, palmitation, amidation and/or addition of glycosylphosphatidyl inositol.
  • a linoleate isomerase protein homologue includes proteins having an amino acid sequence comprising at least
  • a linoleate isomerase protein homologue includes proteins encoded by a nucleic acid sequence comprising at least 24, and preferably at least 30, and more preferably at least
  • a linoleate isomerase protein homologue has measurable linoleate isomerase enzymatic activity.
  • a homologue of a linoleate isomerase is a protein having an amino acid sequence that is sufficiently similar to a natural linoleate isomerase amino acid sequence that a nucleic acid sequence encoding the homologue is capable of hybridizing under stringent conditions to (i.e. , with) a nucleic acid molecule encoding the natural linoleate isomerase (i.e. , to the complement of the nucleic acid strand encoding the natural linoleate isomerase amino acid sequence).
  • a nucleic acid sequence complement of nucleic acid sequence encoding linoleate isomerase of the present invention refers to the nucleic acid sequence of the nucleic acid strand that is complementary to (i.e. , can form a complete double helix with) the strand for which the nucleic acid sequence encodes linoleate isomerase. It will be appreciated that a double stranded DNA which encodes a given amino acid sequence comprises a single strand DNA and its complementary strand having a sequence that is a complement to the single strand DNA.
  • nucleic acid molecules which encode the linoleate isomerase of the present invention can be either double-stranded or single-stranded, and include those nucleic acid molecules that form stable hybrids under stringent hybridization conditions with a nucleic acid sequence that encodes the amino acid sequence selected from the group consisting of SEQ ID NO:l, SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO: 11 and/or SEQ ID NO: 18, and/or with the complement of the nucleic acid that encodes amino acid sequence selected from the group consisting of SEQ ID NO:l, SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO: 11 and/or SEQ ID NO: 18, and/or with the complement of the nucleic acid that encodes amino acid sequence selected from the group consisting of SEQ ID NO:l, SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO: 11 and/or SEQ ID NO: 18, and/or with the complement of the nucleic acid that encodes amino acid sequence selected from the group
  • allelic variant of a nucleic acid encoding linoleate isomerase is a gene that occurs at essentially the same locus (or loci) in the genome as the gene which encodes an amino acid sequence selected from the group consisting of SEQ ID NO:l, SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO: 11 and or SEQ ID NO: 18, but which, due to natural variations caused by, for example, mutation or recombination, has a similar but not identical sequence.
  • Natural allelic variants typically encode proteins having similar activity to that of the protein encoded by the gene to which they are being compared.
  • One class of allelic variants can encode the same protein but have different nucleic acid sequences due to the degeneracy of the genetic code.
  • Allelic variants can also comprise alterations in the 5' or 3' untranslated regions of the gene (e.g., in regulatory control regions). Allelic variants are well known to those skilled in the art and would be expected to be found within a given bacterial species since the genome is haploid and/or among a group of two or more bacterial species.
  • Linoleate isomerase proteins also include expression products of gene fusions (for example, used to overexpress soluble, active forms of the recombinant enzyme), of mutagenized genes (such as genes having codon modifications to enhance gene transcription and translation), and of truncated genes (such as genes having membrane binding domains removed to generate soluble forms of a membrane enzyme, or genes having signal sequences removed which are poorly tolerated in a particular recombinant host). It is noted that linoleate isomerase proteins and protein homologues of the present invention include proteins which do not have linoleate isomerase enzymatic activity. Such proteins are useful, for example, for the production of antibodies and for diagnostic assays.
  • An isolated linoleate isomerase of the present invention can be identified in a straight-forward manner by: the proteins' ability to convert linoleic acid and/or linolenic acid to CLA, such as is illustrated in the Examples; the biochemical properties of the protein as described in the Examples; by selective binding to an antibody against a linoleate isomerase; and/or by homology with other linoleate isomerase amino acid and nucleic acid sequences as disclosed in the Examples.
  • the minimal size of a nucleic acid molecule used to encode linoleate isomerase protein or homologue of the present invention is from about 12 to about 18 nucleotides in length. There is no limit, other than a practical limit, on the maximal size of such a nucleic acid molecule in that the nucleic acid molecule can include a portion of a gene, an entire gene, or multiple genes, or portions thereof.
  • the minimal size of linoleate isomerase protein or homologue of the present invention is from about 4 to about 6 amino acids in length, with preferred sizes depending on whether a full-length, multivalent (i.e., fusion protein having more than one domain each of which has a function), or functional portions of such proteins are desired.
  • Preferred linoleate isomerases of the present invention also include proteins which comprise a protein selected from PCLA 35 , PCLA 2g , PCLA 158 , PCLA 324 and/or PCLA 591 .
  • the present invention also includes a fusion protein that includes a linoleate isomerase-containing domain attached to one or more fusion segments.
  • Suitable fusion segments for use with the present invention include, but are not limited to, segments that can: enhance a protein's stability; provide other enzymatic activity (e.g. , lipase, phospholipase, or esterase to hydrolyze esters of 9, 12-diene fatty acids to 9, 12-fatty acids); and/or assist purification of a linoleate isomerase (e.g., by affinity chromatography).
  • a suitable fusion segment can be a domain of any size that has the desired function (e.g., imparts increased stability, solubility, action or activity; provides other enzymatic activity such as hydrolysis of esters; and/or simplifies purification of a protein). Fusion segments can be joined to amino and/or carboxyl termini of the linoleate isomerase-containing domain of the protein and can be susceptible to cleavage in order to enable straight-forward recovery of a linoleate isomerase.
  • Propionibacterium, Butyrivibrio, and Eubacterium have linoleate isomerase activity.
  • bacterial species such as Lactobacillus reuteri, Clostridium sporogenes, Propionibacterium acnes, Butyrivibrio fibrisolvens, Propionibacterium acidipropionici, Propionibacterium freudenreichii and Eubacterium lentum contain linoleate isomerase.
  • a particularly preferred linoleate isomerase is Lactobacillus reuteri linoleate isomerase.
  • a microorganism is genetically modified to increase the action of linoleate isomerase, and preferably, to enhance production of linoleate isomerase, and thereby,
  • a genetically modified microorganism can include a microorganism in which nucleic acid molecules have been inserted, deleted or modified (i.e., mutated; e.g., by insertion, deletion, substitution, and/or inversion of nucleotides), in such a manner that such modifications provide the desired effect within the microorganism.
  • a genetically modified microorganism includes a microorganism that has been modified using recombinant technology.
  • genetic modifications which result in a decrease in gene expression, in the function of the gene, or in the function of the gene product i.e.
  • a genetic modification of a microorganism increases or decreases the action of a linoleate isomerase.
  • Such a genetic modification includes any type of modification and specifically includes modifications made by recombinant technology and by classical mutagenesis. It should be noted that reference to increasing the action (or activity) of linoleate isomerase refers to any genetic modification in the microorganism in question which results in increased functionality of the enzyme and includes higher activity of the enzymes (e.g.
  • the gene is amplified under conditions that lead to a high frequency of misincorporation errors by the DNA polymerase used for the amplification.
  • a high frequency of mutations are obtained in the PCR products.
  • the resulting linoleate isomerase gene mutants can then be screened for reduced substrate and/or product inhibition by testing the mutant genes for the ability to confer increased CLA production onto a test microorganism, as compared to a microorganism carrying the non-mutated recombinant linoleate isomerase nucleic acid molecule.
  • the method can be operated in batch or continuous mode using a stirred tank, a plug-flow column reactor or other apparatus known to those skilled in the art.
  • the oil comprises a compound selected from the group consisting of free fatty acids, salts of free fatty acids (e.g. , soaps), and mixtures containing linoleic acid, linolenic acid and mixtures thereof.
  • the oil comprises at least about 50% by weight of the compound, more preferably at least about 60% by weight, and most preferably at least about 80% by weight.
  • the method of the present invention converts at least a portion of the compound to CLA.
  • at least about 50% of the oil is converted to CLA, more preferably at least about 70% , and most preferably at least about 95% .
  • the oil is selected from the group consisting of sunflower oil, safflower oil, corn oil, linseed oil, palm oil, rapeseed oil, sardine oil, herring oil, mustard seed oil, peanut oil, sesame oil, perilla oil, cottonseed oil, soybean oil, dehydrated castor oil and walnut oil.
  • the linoleate isomerase is bound to a solid support, i.e. , an immobilized enzyme.
  • a linoleate isomerase bound to a solid support includes immobilized isolated linoleate isomerases, immobilized bacterial cells which contain a linoleate isomerase enzyme, stabilized intact cells and stabilized cell/membrane homogenates. Stabilized intact cells and stabilized cell membrane homogenates include cells and homogenates from naturally occurring microorganisms expressing linoleate isomerase or from genetically modified microorganisms as disclosed elsewhere herein.
  • Stabilized intact cells and or cell/membrane homogenates can be produced, for example, by using bifunctional crosslinkers (e.g. , glutaraldehyde) to stabilize cells and cell homogenates.
  • bifunctional crosslinkers e.g. , glutaraldehyde
  • the cell wall and membranes act as immobilizing supports.
  • integral membrane proteins are in the "best" lipid membrane environment.
  • the cells are either no longer “alive” or “metabolizing", or alternatively, are “resting” (i.e., the cells maintain metabolic potential and active linoleate isomerase, but under the culture conditions are not growing); in either case, the immobilized cells or membranes serve as biocatalysts.
  • Linoleate isomerase can be bound to a solid support by a variety of methods including adsorption, cross-linking (including covalent bonding), and entrapment. Adsorption can be through van del Waal's forces, hydrogen bonding, ionic bonding, or hydrophobic binding.
  • Exemplary solid supports for adsorption immobilization include polymeric adsorbents and ion-exchange resins. Solid supports in a bead form are particularly well-suited. The particle size of an adsorption solid support can be selected such that the immobilized enzyme is retained in the reactor by a mesh filter while the substrate (e.g., the oil) is allowed to flow through the reactor at a desired rate. With porous particulate supports it is possible to control the adsorption process to allow linoleate isomerases or bacterial cells to be embedded within the cavity of the particle, thus providing protection without an unacceptable loss of activity.
  • Cross-linking of a linoleate isomerase to a solid support involves forming a chemical bond between a solid support and a linoleate isomerase. It will be appreciated that although cross-linking generally involves linking a linoleate isomerase to a solid support using an intermediary compound, it is also possible to achieve a covalent bonding between the enzyme and the solid support directly without the use of an intermediary compound. Cross-linking commonly uses a bifunctional or multifunctional reagent to activate and attach a carboxyl group, amino group, sulfur group, hydroxy group or other functional group of the enzyme to the solid support. The term "activate" refers to a chemical transformation of a functional group which allows a formation of a bond at the functional group.
  • Aerobic biotransformations were carried out in baffled 250 mL shake flasks agitated at 200 rpm on a shaker at room temperature.
  • Detergent soluble protein fractions were dialyzed overnight against low salt buffer (0.1 M Bis-Tris pH 5.8, 10 mM NaCI, 2 mM dithiothreitol, 10% glycerol, 0.3% OTGP), and applied to a 2.1 X 15 cm DEAE-5PW column (TosoHaas) previously equilibrated with low salt buffer. The column was washed (4 mL/min) with the same buffer containing 1 M NaCI (high salt buffer). The results of this step are shown in Fig. 6. Protein concentration was monitored continuously at 280 nm. About 4 mL fractions were collected and assayed for isomerase activity. Isomerase activity in the extracts was measured at 20 ppm linoleic acid. Fractions with significant isomerase activity were combined and concentrated using an Amicon ultrafiltration cell.
  • low salt buffer 0.1 M Bis-Tris pH 5.8, 10 mM NaCI, 2 mM dithiothreitol, 10% gly
  • the supernatant generated by low speed centrifugation of the total cell lysate was subjected to an ultra centrifugation step to separate membrane (pellet) from soluble proteins.
  • Detergent was used to solubilize membrane proteins, which were then separated from other insoluble membrane components by ultra-centrifugation.
  • the total cell lysate and different protein fractions were analyzed on SDS gel and by Western blot. In the total cell lysate of E. coli cells expressing the isomerase gene, only the protein band between 60 and 70 kD can be seen after Coomassie staining. This protein band was recovered in the inclusion body fraction and was confirmed to be the isomerase-His tag fusion protein by Western blot.
  • Additional strategies for expressing a linoleate isomerase of the present invention include, but are not limited to: (1) deleting the single hydrophobic domain of the sequence to try to convert the isomerase into a functional soluble protein for use in determination of fusion protein synthesis, solubility and isomerase activity; (2) developing constructs for production of the isomerase in L. reuteri using both the native promoter and non-native inducible or constitutive promoters, including an isomerase-His tag fusion gene under the control of the isomerase native promoter; (3) cloning the promoter from the erythromycin resistance gene for control of isomerase gene expression in L.
  • this unknown product may be an intermediate of linoleic acid conjugation.
  • this product was incubated with . reuteri PYR8 cells or crude enzyme extracts, however, it could not be converted to CLA. It is possible that the intermediate has to be bound with the enzyme or membrane during the conjugation, and once it is released the conjugation could not be completed.
  • Further experiments include developing a series of constructs based on the vector pLATlO to explore the advantage of including the His tag (Fig. 12).
  • pLATlO is a plasmid that can be used to directly transform B. subtilis and B. licheniformis.
  • the effect of pH on enzyme activity in crude extracts was examined.
  • the isomerase activity exhibits a pH optimum centered around 6.8 (Fig. 16).

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Abstract

La présente invention concerne une linoléate isomérase isolée, ainsi que son acide nucléique et sa séquence d'acide aminé. La présente invention concerne également une méthode permettant de produire de l'acide CLA à partir d'une huile, au moyen d'une cellule bactérienne immobilisée ou d'une linoléate isomérase isolée.
EP98964935A 1997-12-23 1998-12-23 Linoleate isomerase Withdrawn EP1042451A4 (fr)

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BR0012873A (pt) * 1999-07-30 2002-04-16 Monsanto Technology Llc Sequências de ácido nucléico que codificam isomerase de ácido graxo polienóico e usos das mesmas
FI108730B (fi) 1999-11-19 2002-03-15 Valio Oy Menetelmä konjugoidun linolihapon valmistamiseksi
EP1178118A1 (fr) 2000-08-02 2002-02-06 Dsm N.V. Isolation d'huiles microbiennes
EP1264893A1 (fr) 2001-06-08 2002-12-11 Teagasc Dairy Products Research Centre Biosynthèse de CLA par bifidobactéries
US7700833B2 (en) 2002-03-01 2010-04-20 Cornell University Process for the production of unsaturated fatty acids
FI115842B (fi) * 2002-03-27 2005-07-29 Valio Oy Menetelmä konjugoidun linolihapon valmistamiseksi
WO2004005442A1 (fr) * 2002-07-03 2004-01-15 Basf Plant Science Gmbh Procede de production d'acides gras polyinsatures conjugues comportant au moins deux liaisons doubles dans des plantes
US7960599B2 (en) 2003-01-13 2011-06-14 Elevance Renewable Sciences, Inc. Method for making industrial chemicals
CN101341254A (zh) 2005-12-24 2009-01-07 蒂加斯克乳制品研究中心 反-10，顺-12十八碳二烯酸的生产方法
JP2008162928A (ja) * 2006-12-27 2008-07-17 Nagasakiken Koritsu Daigaku Hojin 脂肪蓄積抑制剤
WO2008119735A1 (fr) * 2007-04-02 2008-10-09 Georg-August-Universität Göttingen Procédé de fabrication d'acide gras hydroxy
ES2758782T3 (es) 2010-06-01 2020-05-06 Dsm Ip Assets Bv Extracción de lípido de células y productos del mismo
CN102093994A (zh) * 2010-12-07 2011-06-15 淮阴工学院 一种同时纯化和固定化亚油酸异构酶的方法
CN106029624B (zh) 2013-12-20 2021-12-07 帝斯曼知识产权资产管理有限公司 用于从微生物细胞获得微生物油的方法
WO2015095693A2 (fr) 2013-12-20 2015-06-25 Dsm Ip Assets B.V. Procédés d'obtention d'huile microbienne à partir de cellules microbiennes
AU2014369048B2 (en) 2013-12-20 2018-12-13 Dsm Ip Assets B.V. Processes for obtaining microbial oil from microbial cells
AU2014369040B2 (en) 2013-12-20 2019-12-05 Dsm Ip Assets B.V. Processes for obtaining microbial oil from microbial cells
IL278961B2 (en) 2013-12-20 2024-01-01 Dsm Ip Assets Bv Processes for obtaining microbial oil from microbial cells
AU2014369339B2 (en) 2013-12-20 2018-10-18 Dsm Nutritional Products Ag Methods of recovering oil from microorganisms
BR112016014262B1 (pt) 2013-12-20 2022-04-05 MARA Renewables Corporation Método para recuperar lipídeos de uma população de micro-organismos

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