WO2016162605A1 - Amélioration de production de diacide avec des micro-organismes génétiquement modifiés - Google Patents

Amélioration de production de diacide avec des micro-organismes génétiquement modifiés Download PDF

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WO2016162605A1
WO2016162605A1 PCT/FI2016/050228 FI2016050228W WO2016162605A1 WO 2016162605 A1 WO2016162605 A1 WO 2016162605A1 FI 2016050228 W FI2016050228 W FI 2016050228W WO 2016162605 A1 WO2016162605 A1 WO 2016162605A1
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micro
diacid
diacids
gene
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Kari Koivuranta
Laura Ruohonen
Tiina NAKARI-SETÄLÄ
Merja Penttilä
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Teknologian Tutkimuskeskus Vtt Oy
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    • 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
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C12P7/44—Polycarboxylic acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
    • C07K14/39—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts
    • 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
    • C12N9/14—Hydrolases (3)
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y306/00—Hydrolases acting on acid anhydrides (3.6)
    • C12Y306/03—Hydrolases acting on acid anhydrides (3.6) acting on acid anhydrides; catalysing transmembrane movement of substances (3.6.3)
    • C12Y306/03047—Fatty-acyl-CoA-transporting ATPase (3.6.3.47)

Definitions

  • the present invention relates to a field of diacids, and more precisely to a method of producing diacids.
  • the invention further relates to recombinant microorganisms comprising omega-oxidation and increased diacid production, and uses and methods related thereto.
  • Bioplastics market will likely be increased due to the fact that petroleum based plastics are not allowed in many applications.
  • Polymers are used in the manufacture of plastics, and furthermore, diacids are used as raw materials in production of polymers (e.g. polyamides and polyesters).
  • diacids are used as raw materials in production of polymers (e.g. polyamides and polyesters).
  • diacids are used for manufacturing for example cosmetics, phar- maceuticals, biocides, herbicides and pesticides.
  • Diacids i.e. fatty dicarboxylic acids are produced from fatty acids by oxidation.
  • Yeasts metabolize fatty acids by two types of oxidation reactions: omega-oxidation of fatty acids to diacids and beta-oxidation of fatty acids to CO2 and water.
  • Beta-oxidation ( Figure 1 ) is a process by which fatty acid molecules are broken down in the yeast peroxisomes to generate acetyl-coA, which will be transported into the cytosol for further metabolism. Not only fatty acids but also diacids are degraded via beta-oxidation.
  • Omega-oxidation ( Figure 1 ) is a process where mainly alkanes are oxidized to fatty acids before they are degraded in beta-oxidation.
  • Omega-oxidation pathway involves oxidation of the omega-carbon (the carbon most distant from the carboxyl group of the fatty acid) instead of the beta-carbon.
  • Omega- oxidation process is normally a minor catabolic pathway for medium- or long- chain fatty acids, but becomes more important when beta-oxidation is defective. For example some yeast strains retain omega-oxidation where fatty acids are oxidized to diacids.
  • diacids from the same length fatty acids are not possible. E.g. in chemical cleavage of oleic acid (C18:1 ) one C9 diacid and one C9 fatty acid are produced.
  • diacids from the same length fatty acids can be directly produced by biotechnical utilization of yeasts having omega-oxidation. This biotechnical production of diacids is not very effective because in wild type yeasts fatty acids are degraded before omega-oxidation, or alternatively produced diacids are degraded prior to accumulation. Indeed, the wild- type yeasts produce only little dicarboxylic acids.
  • Diacid production has been enhanced in yeast strains by genetic modification.
  • Diacid production has been enhanced by blocking the first enzyme reaction of beta-oxidation (i.e. POX genes).
  • POX genes beta-oxidation
  • publication WO 00/15828 describes a method wherein beta-oxidation of Candida is blocked by disrupting both copies of the chromosomal POX5, POX4A and POX4B genes encoding distinct subunits of long chain acyl-CoA oxidase.
  • the disruption of the POX4 and POX5 genes effectively blocks the beta-oxidation pathway at its first reaction, thereby redirecting the substrate toward the omega-oxidation pathway.
  • the problem in this kind of blocking is that there are several POX genes which should be deleted.
  • WO12071439 A1 and W013006730 A2 describe modified host cells for the production of fatty acid molecules and diacids.
  • WO12071439 A1 utilizes different pathways and modifications such as five distinct routes for fatty acid production, namely type I, II and III fatty acid, type I polyketide synthase and 2-ketoacid biosynthetic pathways.
  • the publication also reveals specific genes (e.g. acetyl-CoA carboxylase), which have been genetically engineered to increase the production of fatty acid compounds. Increased fatty acid production leads to increased omega-oxidation and production of diacids.
  • WO13006730 A2 describes a combination of genetic modifications that substantially block beta-oxidation activity and modifications not affecting the beta-oxidation pathway but increasing e.g. monooxygenase activity, monooxy- genase reductase activity, thioesterase activity, acyltransferase activity, iso- citrate dehydrogenase activity, glyceraldehyde-3-phosphate dehydrogenase ac- tivity, glucose-6- phosphate dehydrogenase activity, acyl-CoA oxidase activity, fatty alcohol oxidase activity, acyl-CoA hydrolase activity, alcohol dehydrogenase activity, peroxisomal biogenesis factor activity or fatty aldehyde dehydrogenase activity.
  • the present invention provides a method and microorganisms for diacid production which is not linked to disruption of genes of the beta-oxidation pathway but on the contrary utilizes a specific activity or a gene not taking part in the beta-oxidation pathway.
  • An object of the present invention is to provide a method to solve the above problems i.e. laborious, time consuming, expensive and inefficient production of diacids.
  • the present invention provides a recombinant micro-organism which is not capable of degrading medium chain fatty acids and correspond- ing diacids. Therefore, accumulation of medium chain diacids and/or fatty acids occurs.
  • the invention is based on the idea of blocking fatty acid and diacid degradation by one or at least one genetic modification resulting in accumulation of diacid in cultivations where fatty acid containing substrate has been fed.
  • Degradation of fatty acids (e.g. medium chain fatty acids) and diacids is blocked before they will be activated for degradation in cell organelles called peroxisomes.
  • Peroxisomes contain so called beta-oxidation where fatty acids and corresponding diacids will be degraded into acetyl-CoA which will be used for many purposes in a cell.
  • the gene ANT1 which encodes an ATP transporter into peroxisomes, will be deleted and therefore no ATP will be trans- ported into peroxisomes.
  • ATP is needed in peroxisomes for activation of medium chain fatty acids and diacids into corresponding CoA esters which will subsequently be degraded in beta-oxidation. Indeed, by the present invention it is possible to block fatty acid degradation prior to beta-oxidation.
  • fatty acids and diacids of specific chain length are blocked thus providing a possibility of using fatty acids and/or diacids of other length as carbon sources.
  • ANT1 deletion prevents degradation of diacids and fatty acids more specifically than blocking of beta-oxidation itself.
  • the present invention relates to a method of producing diacids in a micro-organism comprising omega oxidation, said method compris- ing
  • the present invention relates to a recombinant micro-organism capable of producing diacids, wherein the micro-organism has been genetically modified to reduce peroxisomal membrane ATP transporter activity (e.g. for decreasing diacid degradation).
  • the present invention relates to a recombinant micro-organism comprising omega oxidation and genetic modification to reduce or inactivate peroxisomal membrane ATP transporter activity, wherein the recombinant micro- organism comprises or has increased diacid production (e.g. compared to a wild type micro-organism).
  • a further aspect of the present invention relates to use of the recombinant micro-organism of the present invention for producing diacids from a carbon substrate.
  • the present invention relates to a method of producing products selected from the group consisting of polymers, hot-melt ad- hesives, surfactants, lubricants, additives such as grease and corrosion inhibitors, food additives, solvents and cleaning additives, plasticizers, fragrances, insecticides, fungicides, cosmetics, pharmaceuticals, biocides, herbicides or pes- ticides, said method comprising culturing the recombinant micro-organism of the present invention in a carbon substrate containing medium to produce diacids, recovering the resulting diacids and utilizing the recovered diacids in production of polymers, hot-melt adhesives, surfactants, lubricants, additives such as grease and corrosion inhibitors, food additives, solvents and cleaning additives, plasticizers, fragrances, insecticides, fungicides, cosmetics, pharmaceuticals, biocides, herbicides or pesticides.
  • additives such as grease and corrosion inhibitors, food additives, solvents and
  • the present invention relates to use of the recombinant micro-organism of the present invention for producing polymers, hot-melt adhesives, surfactants, lubricants, additives such as grease and corrosion inhibitors, food additives, solvents and cleaning additives, plasticizers, fragrances, insecti- cides, fungicides, cosmetics, pharmaceuticals, biocides, herbicides or pesticides.
  • the present invention relates to a method of preparing the recombinant micro-organism of the present invention, said method comprising providing a micro-organism having ANT1 activity and genetically modifying the micro-organism to reduce ANT1 activity.
  • Figure 1 shows omega-oxidation and beta oxidation and the role of ANT1 in fatty acid degradation.
  • Figure 2 shows plasmid map of plasmid pBC10.
  • Figure 3 shows plasmid map of plasmid pBC31 .
  • Figure 4 shows plasmid map of plasmid pBC14.
  • Figure 5 shows plasmid map of plasmid pBC27.
  • Figure 6 shows plasmid map of plasmid pBC94.
  • alkanes i.e. saturated hydrocarbons consisting only of hydrogen and carbon atoms, all bonds being single bonds
  • ER endoplasmic reticulum
  • Conversion of the fatty acids to their corresponding fatty acid CoA esters depends on the length of the fatty acid.
  • the long or medium chain fatty acids formed in the ER are activated to their corresponding acyl-CoA esters by the long-chain length specific cytoplasmic ATP- dependent acyl-CoA synthetase I.
  • Acyl-CoA esters are subsequently transported into the peroxisome for degradation by beta-oxidation.
  • the short- and medium-chain fatty acids (C2-C15) are first transported into the peroxisome as fatty acids or fatty alcohols, which are oxidized there to fatty acids. Free short- or medium-chain fatty acids are activated to their corresponding acyl-CoA esters by the peroxisomal acyl-CoA synthetase II. Activation of short- or medium-chain fatty acids to acyl-CoA esters requires the presence of intra-peroxisomal ATP. ANT1 plays a role in transporting ATP into peroxisomes. Short- and medium- chain acyl-CoAs are also degraded by beta oxidation. (Thevenieau F et al. Fungal genetics and Biology 2007, 44:531 -542)
  • Fatty acids may originate from alkanes but also from other carbon sources as a result of fatty acid synthesis.
  • Fatty acids may be derived from triglycerides or phospholipids.
  • the term "fatty acid” refers to an aliphatic monocar- boxylic acid, a compound obtainable from acetyl-CoA and/or malonyl-CoA precursors through action of enzymes.
  • Fatty acids may be saturated or unsaturated, and when they are not attached to other molecules, they are known as "free" fatty acids.
  • acyl-CoA refers to a fatty acid residue, which has been esterified to a CoA molecule.
  • Fatty acid residues in acyl-CoA can be short, medium or long chain fatty acids with or without double bonds.
  • the present invention provides a method for producing diacids from carbon sources by using genetically modified recombinant organisms, which have reduced ANT1 activity compared to a wild type micro-organism with- out the genetic modification of the present invention.
  • diacids refers to aliphatic fatty dicarboxylic acids, which are organic compounds containing two carboxylic acid functional groups. Diacids may be saturated or unsaturated.
  • ANT1 gene refers to a gene encoding a peroxisomal membrane localized adenine nucleotide transporter protein belonging to the carrier protein family which contains ATP transporter proteins.
  • ANT1 is an adenine nucleotide transporter involved in the uniport of ATP and adenine nucleotide hetero-exchange transport between the cytosol and the peroxisomal lumen. This transport is accompanied by a proton transport from the peroxisomal lumen to the cytosol. Transport of ATP into the peroxisome is required for degradation of short and medium chain diacids and fatty acids.
  • ANT1 provides ATP for the activation of short and medium chain diacids and fatty acids by acyl-CoA synthetase II in peroxisomes.
  • the ANT1 enzyme encoded by the ANT1 gene is classified as EC 3.6.3.47.
  • ANT1 refers to not only Yarrowia lipolytica or Pichia guilliermondii ANT1 but also to any other ANT1 homologue from any micro-organism.
  • the enzyme encoded by the Yarrowia lipolytica ANT1 gene is described in the article of Thevenieau F et al. (2007, Fungal Genetics and Biology 44: 531 -542). S.
  • ORF open reading frames
  • polypeptide and “protein” are used interchangeably to refer to polymers of amino acids of any length.
  • An engineered microorganism of the present invention comprises a genetic modification to reduce or inactivate peroxisomal membrane ATP transporter activity. Any of the genetic modifications described below in connection with ANT1 may be alternatively used for any other gene, which is capable of reducing peroxisomal membrane ATP transporter activity.
  • the gene to be modified for reducing peroxisomal membrane ATP transporter activity is ANT1.
  • peroxisomal membrane ATP transporter activity refers to the activity of one or more proteins to transport ATP in peroxisomal membranes.
  • Reduced peroxisomal membrane ATP transporter activity refers to the presence of less peroxisomal membrane ATP transporter activity, if any, in a specific protein or modified microorganism compared to a wild type protein or microorganism, respectively, or lower activity (if any) in a cell or micro-organism compared to an unmodified cell or micro-organism.
  • Methods for studying peroxisomal membrane ATP transporter activity are well known to a person skilled in the art and include but are not limited to methods described e.g. in van Roermund et al (2004 J Cell Sci 1 17:4231 -4237).
  • inactivation refers to a situation wherein peroxisomal membrane ATP transporter activity of a protein or a cell is totally inactivated i.e. a protein or a cell has no peroxisomal membrane ATP transporter activity.
  • ANT1 gene of the micro-organism has been genetically modified.
  • an engineered microorganism of the present invention comprises a genetic modification reducing ANT1 activity.
  • reduced ANT1 activity refers to the presence of less ANT1 activity (i.e.
  • adenine nucleotide transporter activity involved in the uniport of ATP and adenine nucleotide hetero-exchange transport between the cytosol and the peroxisomal lumen if any, in ANT1 protein compared to a wild type ANT1 pro- tein, or lower ANT1 activity (if any) in a cell or micro-organism compared to a cell or micro-organism comprising wild type ANT1 .
  • Reduced ANT1 activity may result from down regulation of the ANT1 polypeptide expression, down regulation of the ANT1 gene expression, lack of at least part of the ANT1 gene, lack of ANT1 protein and/or lowered activity of ANT1 protein.
  • the ANT1 protein is inactivated.
  • the micro-organism of the invention may comprise one or several genetic ANT1 modifications.
  • a genetic modification lowering ANT1 activity may refer to a deletion or substitution of one or more nucleic acids or any fragment of a nucleotide sequence encoding ANT1 having the adenine nucleotide transporter activity or any insertion of one or more nucleic acids or any nucleic acid sequence fragment into a nucleotide sequence encoding ANT1 having the adenine nucleotide transporter activity.
  • the genetic modification includes temporary or permanent silencing of ANT1 gene. Reduced activity of ANT1 may be proved for example by using a method based on the pH in peroxisomes de- scribed by van Roermund et al (2004 J Cell Sci 1 17:4231 -4237).
  • a "fragment" of a given sequence means any part of that sequence, for example one or several nucleotides or amino acids or a truncated form of the sequence.
  • the genetic modification down regulates the expression o ⁇ ANT1 gene or ANT1 polypeptide.
  • down regulated expression refers to either a lack of expression or decreased expression of the gene or polypeptide of interest compared to a wild type micro-organism without the genetic modification. Lack of expression or decreased expression can be proved for example by western, northern or southern blotting or quantitative PCR or any other suitable method known to a person skilled in the art.
  • Genetic modification leading to down-regulation of a gene or polypeptide refers to a deletion of ANT1 gene, one or more nucleotides or a fragment thereof, or one or more nucleotides or a fragment of a regulatory sequence (i.e.
  • any nucleotide insertions or sub- stitutions (one or more nucleotides including long nucleotide sequences) in ANT1 gene or in a regulatory sequence of this gene or polypeptide may have an effect of decreasing the expression of a gene or polypeptide and thus, may be utilized in the present invention.
  • changing or modifying the promoter or any other regulatory sequence may decrease expression of the gene.
  • epigenetic modifications such as DNA methylation are known to block expression of genes and can be utilized in the present invention.
  • RNA silencing is known to down-regulate translation of polypeptides.
  • RNA silencing is associated with the concept of post-transcriptional gene silencing or RNA interference, wherein the expression of a gene is down-regulated by small RNAs.
  • Small RNAs may be encoded by either endogenous or exogenous nucleotides and in specific embodiments small RNAs are expressed by the microorganism in question. Also, it is possible to provide the micro-organism with small RNAs such as synthetic antisense RNA molecules in order to control the gene expression specifically.
  • the knowledge of the DNA sequence of ANT1 can be used to inactivate the corresponding gene or genes in a suitable microorganism.
  • the gene can be inactivated e.g. by preventing its expression or by mutation or deletion of the gene or part thereof.
  • the ANT1 gene or any fragment thereof has been deleted.
  • the recombinant micro-organism has been genetically modified by deleting at least part of the ANT1 gene.
  • part of the gene refers to one or several nucleotides of the gene or any fragment thereof.
  • gene knockout methods are suitable for deleting the nucleotide sequence that encodes a polypeptide having ANT1 activity of any part thereof. These methods are described for example in the articles of DiCarlo et al . (2013 Nucleic Acid Res 2013:1 -8) and Koivuranta et al. (2014 Microbial Cell Fact. 13:107) and are well- known to a person skilled in the art.
  • An example of an integration construct, configured to generate a deletion mutant for ANT1 is provided in the Example 1 .
  • a recombinant microorganism of the present invention may also comprise genetic modifications in other genes.
  • genetic modifications include any genetic modifications e.g. insertions, deletions or disruptions of one or more genes or a fragment(s) thereof or insertions, deletions or disruptions of one or more nucleotides, or ad- dition of plasmids.
  • disruption refers to insertion of one or several nucleotides into the gene resulting in lack of the corresponding protein or presence of non-functional proteins or protein with lowered activity.
  • genes may be selected from one or several modifications causing down regulation and/or over-expression of a gene or not affecting the expression of a gene.
  • over-expression refers to excessive expression of a gene by producing more products (e.g. protein) than an unmodified micro-organism.
  • products e.g. protein
  • one or more copies of a gene or genes may be transformed to a cell for overexpression.
  • the term also encompasses embodiments, where a promoter or promoter region has been modified or a promoter not nat- urally present in the micro-organism has been inserted to allow the over-expression of the gene.
  • epigenetic modifications such as DNA methylation and histone modifications are included in "genetic modifications”.
  • suitable micro-organisms for ANT1 modification are micro-organisms already modified or to be modified for diacid production, e.g. com- prising or having modifications in a gene or genes participating in transportation of fatty acids, beta-oxidation and/or omega oxidation.
  • overexpression of one or more polypeptides taking part in omega oxidation or down regulation of one or more polypeptides involved in beta oxidation may be utilized.
  • the recombinant micro-organism comprises a genetic modification of one or more genes selected from the group consisting of POX, MFE, CYP52A13, CYP52A17, NCP1, FA01, FAOT, PXA1, HFD1, HFD2, HFD3, HFD4 and PXA2 in addition to the ANTI modification. These strains may provide the most efficient way for diacid production.
  • the recombinant micro-organism comprises a genetic modification of one or more genes selected from the group consisting of MFE, CYP52A17, NCP1, FAOT and PXA2, i.e.
  • the micro-organism comprises or has at least genetic modifications of ANT1 and MFE, ANT1 and CYP52A17, ANT1 and NCP1, ANT1 and FAOT or ANT1 and PXA2.
  • the recombinant micro-organism comprises a genetic modification of ANT1, NCP1, MFE and CYP52A17; ANT1, NCP1, CYP52A17 and F/3 ⁇ 4OT; or ANT1, NCP1, MFE, CYP52A17 an6 FAOT
  • a POX gene refers to a gene encoding any subunit of long chain acyl-CoA oxidase (EC 1 .3.3.6)
  • MFE gene refers to a gene encoding multifunctional enzyme 2 (EC 4.2.1 .1 19)
  • CYP52A13 gene refers to a gene encoding cytochrome P450 52A13 (EC 1 .14.14.1 )
  • CYP52A17 gene refers to a gene encoding cytochrome P450 52A17 (EC 1 .14.14.1 )
  • NCP1 gene refers to a gene encoding cytochrome P-450 reductase (EC 1 .6.2.4)
  • FA01 gene refers to a gene encoding fatty alcohol oxidase (EC 1 .1 .3.13)
  • FAOT gene refers to a gene encoding long-chain-alcohol oxidase (EC 1 .1 .3.20), "HFD1, H
  • PXA1 gene refers to a gene encoding peroxisomal long-chain fatty acid import protein 2 i.e. peroxisomal ABC transporter 1 (TC 3.A.1 .203)
  • PXA2 gene refers to a gene encoding peroxisomal long-chain fatty acid import protein 1 i.e. peroxisomal ABC transporter 2 (TC 3.A.1 .203).
  • Said enzymes encoded by the mentioned genes refer to not only Candida trop- icalis, Yarrowia lipolytica or Pichia guilliermondii enzymes but also to any other homologue from any micro-organism such as Saccharomyces cerevisiae. Enzymes encoded by said genes are described in scientific articles and are well known to a skilled person.
  • the recombinant organism may also contain other genetic modifications than those specifically described herein. "Genetic modifications in other genes" may be present in the micro-organism prior to further modifying the micro-organism according to the present invention for reducing ANT1 activity, or may be done simultaneously with or after such further modifications. Indeed, the micro-organism may be genetically modified to produce, not to produce, increase production or decrease production of other compounds than diacids and/or fatty acids.
  • the engineered microorganism includes a heterologous polynucleotide.
  • the micro-organism can be genetically modified by transforming it with a heterologous nucleic acid that encodes a heterologous protein.
  • heterologous promoters or other regulating sequences can be utilized in the micro-organisms of the invention.
  • heterologous polynucleotide refers to a not naturally occurring polynucleotide.
  • DNA isolation, enzymatic treatment and genetic modifications may be carried out using conventional molecular biology methods. Genetic modification of the micro-organism is accomplished in one or more steps via the design and construction of appropriate vectors and transformation of the micro-organism cell with those vectors. For example electroporation, protoplast-PEG and/or chemical (such as calcium chloride- or lithium acetate-based) transformation methods can be used. Also any commercial transformation methods such as Frozen-EZ Yeast Transformation II kit are appropriate. Suitable transformation methods are well known to a person skilled in the art.
  • vector refers to a nucleic acid compound and/or composition that transduces, transforms, or infects a microorganism, thereby causing the cell to express nucleic acids and/or proteins other than those native to the cell, or in a manner not native to the cell.
  • An "expression vector” contains a sequence of nucleic acids (ordinarily RNA or DNA) to be expressed by the modified microorganism.
  • the expression vector also comprises materials to aid in achieving entry of the nucleic acid into the microorganism, such as a virus, liposome, protein coating, or the like.
  • the expression vectors contemplated for use in the present invention include those into which a nucleic acid sequence can be inserted, along with any preferred or required operational elements.
  • expression vector must be one that can be transferred into a microorganism and replicated therein.
  • Vectors can be circularized or linearized and may contain restriction sites of various types for linearization or fragmentation.
  • expression vectors are plasmids, particularly those with restriction sites that have been well documented and that contain the operational elements preferred or required for transcription of the nucleic acid sequence.
  • Such plasmids, as well as other expression vectors are well known to those of ordinary skill in the art.
  • Useful vectors may for example be conveniently obtained from commercially available yeast or bacterial vectors.
  • Successful transformants can be selected using the attributes contributed by the marker or selection gene. Screening can be performed by PCR or Southern analysis to confirm that the desired genetic modifications (e.g.
  • ANT1 activity in a cell or microorganism can be detected by any suitable method known in the art.
  • suitable detection methods include enzymatic assays (e.g. based on pH in peroxisomes described by van Roermund et al (2004, J. Cell Sci 1 17:4231 -4237)), PCR based assays (e.g., qPCR, RT-PCR), immunological detection methods (e.g., antibodies specific for ANT1 ), the like and combinations thereof.
  • the ANT1 nucleic acid sequence to be genetically modified may comprise a polynucleotide sequence selected from the group consisting of SEQ ID NO: 1 ; SEQ ID NO: 2; SEQ ID NO: 3; and a nucleic acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% nucleic acid sequence identity therewith and encoding a polypeptide having adenine nucleotide transporter activity between the cytosol and the peroxisomal lumen.
  • ANT1 polynucleotide refers to any polynucleotide, such as single or double-stranded DNA (genomic DNA or cDNA) or RNA, comprising a nucleic acid sequence encoding an ANT1 polypeptide or a conservative sequence variant thereof.
  • polynucleotide refers to any polynucleotide, such as single or double-stranded DNA (genomic DNA or cDNA) or RNA, comprising a nucleic acid sequence encoding a polypeptide in question or a conservative sequence variant thereof.
  • the term "conservative sequence variant” refers to nucleotide sequence modifications, which do not significantly alter biological properties of the encoded polypeptide.
  • Conservative nucleotide sequence variants include variants arising from the degeneration of the genetic code and from silent mutations. Nucleotide substitutions, deletions and additions are also contemplated.
  • the ANT1 polypeptide comprises an amino acid sequence that is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence depicted in SEQ ID NO: 4, 5 or 6.
  • variant refers to a sequence having minor changes in the amino acid or nucleic acid sequence as compared to a given sequence.
  • Such a variant may occur naturally e.g. as an allelic variant within the same strain, species or genus, or it may be generated by mutagenesis or other gene modification. It may comprise amino acid or nucleic acid substitutions, de- letions or insertions, but it still functions in substantially the same manner as the given enzymes, in particular it retains its function as an enzyme.
  • Identity of any sequence or fragments thereof compared to the sequence of this disclosure refers to the identity of any sequence compared to the entire sequence of the present invention.
  • the comparison of sequences and determination of identity percentage between two sequences can be accomplished using mathematical algorithms available in the art. This applies to both amino acid and nucleic acid sequences.
  • Sequence identity may be determined for example by using BLAST (Basic Local Alignment Search Tools) or FASTA (FAST-AII). In the searches, setting parameters "gap penalties" and "matrix" are typically selected as default.
  • a microorganism selected for the present invention is suitable for genetic manipulation and often can be cultured at cell densities useful for industrial production of a target product.
  • a microorganism selected may be maintained in a fermentation device.
  • the genetically modified micro-organisms used in the invention are obtained by performing specific genetic modifications to the micro-organism.
  • a "recombinant micro-organism” refers to any micro-organism that has been genetically modified to contain different genetic material compared to the micro-organism before modification (e.g. comprise a deletion, substitution, disruption or insertion of one or more nucleic acids compared to the micro-or- ganism before modification).
  • the micro-organism of the invention is genetically modified to produce diacids by modification of at least the ANT1 gene or any regulatory nucleotide sequence of the ANT1 gene.
  • the recombinant micro-organism" of the invention also refers to a host cell comprising said genetic modification.
  • Micro-organisms suitable for the production of diacids according to the present invention comprise or have omega oxidation ability.
  • an engineered microorganism is a single cell organism.
  • a micro-organism as described herein may be a prokaryotic organism (e.g., an organism of the kingdom Eubacteria) or a eukaryotic cell.
  • a prokaryotic cell lacks a mem- brane-bound nucleus, while a eukaryotic cell has a membrane-bound nucleus. Both plant and animal cells are included with the scope of a micro-organism.
  • the micro-organism may be selected from the group consisting of bacteria, algae and fungi. "Fungi" "fungus” and “fungal” as used herein refer to yeast and filamentous fungi (i.e. moulds).
  • the micro-organism is an oleaginous micro-organism i.e. a micro-organism (e.g. yeast, filamentous fungus, bacteria or algae) capable of producing lipids.
  • a micro-organism e.g. yeast, filamentous fungus, bacteria or algae
  • the term "oleaginous micro-organism” refers to micro-organisms, which accumulate at least 10%, 12.5%, 15%, 17.5%, preferably at least 20% or even at least 25% (w/w) of their biomass as lipid. They may even accumulate at least 30%, 40%, 50%, 60%, 70%, 80% (w/w) or more of their biomass as lipids.
  • the biomass is usually measured as cell dry weight (CDW).
  • the micro-organism is an alkane utilising micro-organism. These micro-organisms are known to comprise or have effective omega oxidation.
  • the micro-organism may be either non-pathogenic (i.e. not causing infections in plants, animals or people) or pathogenic, in a specific embodiment non-pathogenic.
  • the micro-organism is a yeast or filamentous fungus.
  • the micro-organism is a yeast se- lected from the genera Arxula, Cryptococcus, Candida, Debaryomyces, Galac- tomyces, Hansenula, Lipomyces, Lodderomyces, Metschnikowia, Millerozyma, Priceomyces, Rhodosporidium, Rhodotorula, Sugiyamaella, Trichosporon, Pichia and Yarrowia, specifically from the group consisting of Arxula adeninivo- rans, Candida sp., Candida catenulata, Candida haemulonii, Candida maltosa, Candida parapsilopsis, Candida rhagii, Candida rugosa, Candida sake, Candida tenuis, Cryptococcus curvatus, Cryptococcus albidus, Debaryomyces hansenii, Debaryomyces robert
  • the present invention provides a micro-organism which is not capable of degrading specific diacids.
  • a micro- organism is not capable of degrading diacids having short or medium chain length.
  • a micro-organism is not capable of degrading diacids having long chain length. When diacids are not degraded, diacids are produced in higher amounts compared to wild type micro-organisms.
  • the produced diacid is a C2-C15-di- acid, C2-C5-diacid, C6-C15-diacid, C4-C15-diacids, C6-C14-diacids, C6-C12- diacids, C6-C10-diacid, C6-C9-diacid, C7-C9-diacid, C8-C12 diacid, C8-C10-di- acid, C9-C12-diacid, C6-diacid, C7-diacid, C8-diacid, C9-diacid, C10-diacid, C1 1 -diacid, C12-diacid, C13-diacid, C14-diacid, C15-diacid or a diacid having at least C16 chain length.
  • the diacid is a medium chain diacid.
  • short and medium chain fatty acids or diacids refers to fatty acids or diacids having chain length of C2-C15.
  • Short chain fatty acids or diacids refers to fatty acids or diacids having chain length of C2-C5.
  • “Medium chain fatty acids or diacids” refers to fatty acids or diacids having chain length of C6-C15.
  • Long chain fatty acids and diacids have chain length of at least C16 e.g. C16-C21 .
  • the produced diacid is a long chain diacid (e.g. C18).
  • methods for producing diacid comprise cul- turing an engineered micro-organism under culture conditions in which the cultured micro-organism produces diacids.
  • the genetically modified fungi of the present invention are capable of producing increased levels of specific diacids.
  • the increase may be at least a 1 .5, 3, 5, 10, 15 or 20 fold increase in diacid concentration in transformants compared to the unmodified strain or any strain with other modifications during cultivation.
  • it may be at least a 1 .5, 3, 5, 10, 15 or 20 fold increase in diacid yield per used carbon source in transformants compared to the unmodified strain or any strain with other modifica- tions.
  • diacid production rate mg/l/h
  • This increase of diacid production can be detected either intracellularly or in the amount of diacids in culture medium.
  • Diacids are produced from carbon substrates.
  • carbon substrates are lipid containing substrates.
  • the carbon substrate is selected from the group consisting of vegetable oils, fats, oils, fatty acids, phospholipids, glycolipids, triglycerides, alkanes, glucose, pentoses, hexoses and sugars derived from lignocellulose.
  • lipid refers to a group of organic compounds that are relatively or completely insoluble in water but soluble in nonpolar organic solvents. These properties are a result of long hydrocarbon tails, which are hydrophobic in nature.
  • the term thus encompasses fats, oils (vegetable or animal), waxes, fatty acids, fatty acid derivatives, like phospholipids, glycolipids, acylglycerids such as monoglycer- ides, diglycerides, and triglycerides and terpenoids such as carotenoids and steroids.
  • phospholipid refers to any lipid containing a diglycer- ide combined with a phosphate group and a simple organic molecule such as choline or ethanolamine.
  • glycolipid refers to a lipid attached with a carbohydrate.
  • triglyceride refers to an ester derived from glycerol and three fatty acids.
  • Triglycerides may be unsaturated or saturated.
  • “Sugars derived from lignocellulose” refer to sugar monomers of lignocellulose including glucose, xylose, fructose, mannose, galactose, rhamnose, and arabi- nose.
  • “derived from” refers to products obtained from or isolated from a starting product, as well as modifications thereof.
  • the genetically modified fungi are cultivated in a medium containing appropriate carbon sources together with other optional ingredients selected from the group consisting of nitrogen or a source of nitrogen (such as amino acids, proteins, inorganic nitrogen sources such as ammonia or ammonium salts), yeast extract, peptone, minerals and vitamins, such as KH2PO 4 , Na 2 HPO , MgSO , CaCI 2 , FeCIs, ZnSO , citric acid, MnSO , C0CI2, CuSO , Na2MoO 4 , FeSO 4 , HsBO 4 , D-biotin, Ca-Pantothenate, nicotinic acid, myoinositol, thiamine, pyridoxine, p-amino benzoic acid.
  • nitrogen or a source of nitrogen such as amino acids, proteins, inorganic nitrogen sources such as ammonia or ammonium salts
  • yeast extract such as amino acids, proteins, inorganic nitrogen sources such as ammonia or ammonium salt
  • Suitable cultivation conditions such as temperature, cell density, selection of nutrients, and the like are within the knowledge of a skilled person and can be selected to provide an economical process with the micro-organism in question. Temperatures during each of the growth phase and the production phase may range from above the freezing temperature of the medium to about 50°C, although the optimal temperature will depend somewhat on the particular micro-organism. In a specific embodiment the temperature, particularly during the production phase, is from about 25 to 30°C.
  • the pH of the cultivation process may or may not be controlled to remain at a constant pH, but is usually between 5 and 9, depending on the production organism. Optimally the pH is controlled to a constant pH of 7 and 8.
  • Suitable buffering agents include, for example, calcium hydroxide, calcium carbonate, sodium hydroxide, potassium hydroxide, potassium carbonate, sodium carbonate, ammonium carbonate, ammonia, ammonium hydroxide and the like. In general, those buffering agents that have been used in conventional cultivation methods are also suitable here.
  • the cultivation is conveniently conducted aerobically or microaerobi- cally. If desired, specific oxygen uptake rate can be used as a process control.
  • the process of the invention can be conducted continuously, batch-wise, or some combination thereof.
  • the resulting diacids can be recovered.
  • the micro-organism cells e.g. yeast or filamentous fungal cells
  • the micro-organism cells can be normally separated from the culture medium.
  • the cells can be disrupted (e.g. by rapid compression), where after the pH of the centrifuged supernatant may be lowered. Soluble diacids precipitate in the supernatant having the pH 3 or less.
  • the diacids can be recovered directly from the culture medium without disrupting the cells e.g. by dropping the pH. In some embodiments both intracellular and extracellular diacids are recovered.
  • a method of producing diacids comprises an optional step e) of isolating and/or purifying the diacids.
  • Diacids may be isolated and purified (for example from the medium) by using any conventional methods known in the art such as ion exchange, two phase extraction, molecular distillation, melt crystallization, hexane extraction, CO2 extraction or distillation.
  • the diacids obtained by the method of the present invention can be used for preparing any products utilizing the properties of diacids such as poly- mers, hot-melt adhesives, surfactants, lubricants, additives such as grease and corrosion inhibitors, food additives, solvents and cleaning additives, plasticizers, fragrances, insecticides, fungicides, cosmetics, pharmaceuticals, biocides, herbicides or pesticides.
  • diacids such as poly- mers, hot-melt adhesives, surfactants, lubricants, additives such as grease and corrosion inhibitors, food additives, solvents and cleaning additives, plasticizers, fragrances, insecticides, fungicides, cosmetics, pharmaceuticals, biocides, herbicides or pesticides.
  • the plasmid YI_lox-PGK-TEF-lox_pMA (Geneart AG, Germany) contains Yarrowia lipolytica PGK1 promoter and TEF1 terminator with flanking loxP sites.
  • the plasmid pRLMEX30 (Mach et al. 1994 Curr Genet 25:567-570) was digested with Nsi ⁇ and Xba ⁇ and a 1035 bp fragment containing Escherichia coli hygromycin ⁇ hph) gene was gel isolated and ligated to a 3818 fragment obtained by digesting a plasmid YI_lox-PGK-TEF-lox_pMA with Nsi ⁇ and Xba ⁇ . The resulting plasmid was designated as lox-PGK-Hph-TEF-lox.
  • the plasmid Yl_ant1_5+3_flanks contains 5' and 3' UTR regions of Yarrowia lipolytica ANT1 gene (YALI0E03058g, SEQ ID NO:1 ) with flanking Not ⁇ restriction sites.
  • the plasmid Yl_ant1_5+3_flanks was digested with C/al and Xma ⁇ . A 3677 bp fragment was gel isolated.
  • the plasmid lox-PGK-Hph-TEF-lox was digested with C/al and Xmal and a 2472 bp fragment was gel isolated.
  • the 2472 fragment originating from lox-PGK-Hph-TEF-lox plasmid was ligated to the 3677 bp fragment from Yl_ant1_5+3_flanks plasmid.
  • the resulting plasmid was designated as antl Cassette 1 .
  • the plasmid pPGK-Nat-tTEF (Geneart AG, Germany) contains a Natl encoding gene which has been codon optimised according to Yarrowia lipolytica yeast codon usage. Natl gene encodes nourseothricin resistance gene.
  • the plasmid pPGK-Nat-tTEF was digested with Nsi ⁇ and Xba ⁇ and a 582 bp fragment was gel isolated and ligated to a 51 17 bp fragment obtained by digesting a plasmid as antl Cassette 1 with Nsi ⁇ and Xba ⁇ .
  • the resulting plasmid was designated as pBC10 ( Figure 2).
  • the plasmid pBC10 contains flanks to delete the whole open reading frame of ANT1.
  • Example 2. Construction of PXA2 deletion cassette (pBC31 , Figure 3)
  • pRS426 plasmid was linearised with Not ⁇ digestion and gel purified.
  • the PGK promoter, Natl gene and TEF terminator cassette was PCR amplified with oligos AKE36 (SEQ ID NO: 9) and AKE37 (SEQ ID NO: 10) by using plasmid pBC10 (see e.g. Figure 2 or Example 1 ) as a template.
  • a 2026 bp PCR fragment was amplified and gel purified.
  • Yarrowia lipolytica PXA2 gene (YALI0D04246g) was PCR amplified with oligos BC149 (SEQ ID NO: 15) and BC150 (SEQ ID NO: 16) by using genomic DNA of Yarrowia lipolytica C- 12909 strain as a template.
  • the BC149 oligonucleotide contains flank to pRS426 plasmid and the BC150 oligonucleotide contains flank to the PGK pro- moter, Natl gene and TEF terminator cassette.
  • a 1000 bp PCR fragment was amplified and gel purified. 3' UTR of Y.
  • oligos BC151 SEQ ID NO: 17
  • BC152 SEQ ID NO: 18
  • the BC151 oligonucleotide contains flank to the PGK promoter, Natl gene and TEF terminator cassette and the BC152 oligonucleotide contains flank to pRS426 plasmid.
  • a 999 bp PCR fragment was amplified and gel purified.
  • PGK-Nat1 -TEF cassette, PXA2 5' UTR fragment and PXA2 3' UTR fragment were ligated to Not ⁇ linearised pRS426 plasmid with yeast recombination system.
  • the resulting plasmid was designated as pBC31 ( Figure 3).
  • pRS426 plasmid was linearised with Sma ⁇ digestion and gel purified.
  • the PGK promoter, Natl gene and TEF terminator cassette was PCR amplified with oligos AKE36 (SEQ ID NO: 9) and AKE37 (SEQ ID NO: 10) by using plasmid pBC10 (see e.g. Example 1 or Figure 2) as a template.
  • a 2026 bp PCR fragment was amplified and gel purified. 5'UTR of Y.
  • lipolytica MFE1 gene (YALI0E15378g) was PCR amplified with oligos AKE34 (SEQ ID NO: 7) and AKE35 (SEQ ID NO: 8) by using genomic DNA of Yarrowia lipolytica C-12909 strain as a template.
  • AKE34 oligonucleotide contains flank to pRS426 plasmid and the AKE35 oligonucleotide contains flank to the PGK promoter, Natl gene and TEF terminator cassette.
  • An 1 136 bp PCR fragment was amplified and gel purified. 3' UTR of Y.
  • lipolytica MFE1 gene was PCR amplified with oligos AKE38 (SEQ ID NO: 1 1 ) and AKE39 (SEQ ID NO: 12) by using genomic DNA of Yarrowia Iipolytica C-12909 strain as a template.
  • AKE38 oligonucleotide contains flank to the PGK promoter, Natl gene and TEF terminator cassette and the AKE39 contains flank to pRS426 plasmid.
  • An 1 145 bp PCR fragment was amplified and gel purified.
  • PGK-Nat1 -TEF cassette, MFE1 5' UTR fragment and MFE1 3' UTR fragment were ligated to Sma ⁇ linearised pRS426 plasmid with yeast recombination system.
  • the resulting plasmid was designated as pBC14 ( Figure 4) and pBC22.
  • Example 4 The construction of recombinase plasmid (pBC27, Figure 5)
  • Autonomously replicating sequence of Y. Iipolytica was PCR amplified with oligos BC1 (SEQ ID NO: 13) and BC2 (SEQ ID NO: 14) by using genomic DNA of Y. Iipolytica C-00365 strain as a template.
  • the oligonucleotides BC1 and BC2 contain ARS18 sequence of Y. Iipolytica with flanking Kpnl re- striction sites.
  • PCR amplified ARS18 was digested with Kpn ⁇ and gel purified. A 1338 bp fragment was ligated to a Kpn ⁇ digested pBluescript plasmid. The resulting plasmid was designated as pBC7.
  • the plasmid YI_Tef1 p_Cre_Tdh3t contains Yarrowia Iipolytica TEF1 gene promoter and TDH3 gene terminator and a 1031 Cre encoding gene which has been codon optimised according to Yarrowia Iipolytica yeast codon usage.
  • the plasmid YI_Tef1 p_Cre_Tdh3t was digested with EcoR ⁇ and Sac ⁇ and Pvul and a 2354 bp fragment was gel isolated and ligated to a 2950 bp fragment obtained by digesting a plasmid pBluescript with EcoR ⁇ and Sad. The resulting plasmid was designated as pBC5.
  • the plasmid YI_Eno2p_Hph_Pgk1t (Geneart AG, Germany) contains
  • the plasmid YI_Eno2p_Hph_Pgk1 t was digested with Xho ⁇ and Pme ⁇ and Xmn ⁇ and a 2347 bp fragment was gel isolated and ligated to a 5314 bp fragment obtained by digesting a plasmid pBC5 with Xho ⁇ and EcoRV. The resulting plasmid was designated as pBC20.
  • the plasmid pBC7 was digested with Kpn ⁇ and a 1338 bp fragment was gel isolated and ligated to a 7571 bp fragment obtained by digesting a plasmid pBC20 with Kpn ⁇ .
  • the resulting plasmid was designated as pBC27 ( Figure 5).
  • Example 5 The construction of MFE deletion cassette with expression of C. tropicalis NCP1, CYP52A17 and FAOT (pBC94, Figure 6)
  • the plasmid pBC39 (GenScript USA) contains Y. lipolytica TDH1 promoter and TPI1 terminator and a Candida tropicalis NCP1 encoding gene which has been codon optimised according to Y. lipolytica yeast codon usage and flanking Xma ⁇ and Nhe ⁇ restriction sites.
  • the plasmid pBC37 (GenScript, USA) contains Y. lipolytica TPI1 promoter and PGK1 terminator and a C. tropicalis CYP52A17 encoding gene which has been codon optimised according to Y. lipolytica yeast codon usage and flanking Nhe ⁇ and Xma ⁇ restriction sites.
  • the plasmid pBC44 (GenScirpt, USA) contains Y. lipolytica EN01 promoter and TDH1 terminator and a C. tropicalis FAOT encoding gene has been codon optimised according to Y. lipolytica yeast codon usage and flanking Nhe ⁇ and Xma ⁇ restriction sites.
  • TDH1 promoter-C. tropicalis NCP1-TPI1 terminator cassette was
  • TPI1 promoter-C. tropicalis CYP52A17-PGK1 terminator cassette was PCR amplified with oligos BC217 (SEQ ID NO: 21 ) and BC218 (SEQ ID NO: 22) by using plasmid pBNC37 as a template.
  • a 2892 bp PCR fragment was amplified and gel purified.
  • the TDH1 promoter-C. tropicalis NCP1-TPI1 terminator cassette and TPI1 promoter-C. tropicalis CYP52A17-PGK1 terminator cassette were ligated into Xma ⁇ digested pBC14 plasmid with yeast recombination. The resulting plasmid was designated as pBC48.
  • the plasmid pBC44 was digested with Nhe ⁇ and a 3416 bp fragment was ligated to a 16244 bp obtained by digesting the plasmid pBC48 with Nhe ⁇ .
  • the resulting plasmid was designated as pBC94 ( Figure 6).
  • Yarrowia lipolytica transformations were carried out as follows.
  • Yarrowia lipolytica C-00365 was inoculated in 5 ml of YPD medium in 100 ml flask and cultivated 6 to 7 hours at +30°C.
  • the culture was diluted into 15 ml of YPD medium in a 250 ml flask and cultivated over night with 250 rpm shaking at +30°C.
  • the cell density was between 9 x 10 7 - 1 .5 x 10 8 cells/ml
  • the cells were harvested with 4000 rpm centrifugation for 4 minutes. The harvested cells were washed with sterile water.
  • Washed cells were resuspended to 5 x 10 7 cells/ml cell density with fresh 0.1 M lithium acetate, pH 6.0 and incubated 1 hour at +30°C with gentle shaking.
  • the cells were harvested with 4000 rpm centrifu- gation for 4 minutes and resuspended to 5 x 10 8 cells/ml cell density with fresh 0.1 M lithium acetate, pH 6.0.
  • One hundred microliter of suspended cells were transferred into new Eppendorf tube. 240 ⁇ of 50% PEG 4000, 36 ⁇ of 1 M lithium acetate, pH 6.0, 5 ⁇ of herring sperm (10 ⁇ 9/ ⁇ ) and 10-15 ⁇ g of transformed DNA (as described in Examples 7-13) were added.
  • the volume was adjusted to 460 ⁇ with sterile water.
  • the mixture was incubated with agitation for 30 min at +30°C. After incubation transformation sample was incubated at +42°C for 5 minutes.
  • the cells were spin down and resuspended in 1 ml of YPD and incubated at +30°C with 250 rpm shaking 3 hours. After incubation the cells were plated on YPD + 400 ⁇ /ml nourseothricin + 0.1 % Triton X-100 plates.
  • Example 7 Generation of genetically modified Y. lipolytica (yBC3) with an integrated nourseothricin resistance gene in ANT1 locus by transforming wild-type Y. lipolytica with digested plasmid pBC10 (example 1)
  • Plasmid pBC10 (see e.g. example 1 or Figure 2) was restricted with Not ⁇ .
  • a 4098 bp fragment was gel isolated and used to transform the wild-type Y. lipolytica strain (C-00365, VTT Culture Collection) using the transformation method described in Example 6.
  • the transformed cells were screened for nourseothricin resistance.
  • Several nourseothricin resistant colonies were analysed at DNA level by PCR.
  • the transformant originating from the transformation of the wild-type Y. lipolytica strain with Not ⁇ cut pBC10 and containing ANT1 gene deletion was designated as yBC3.
  • Example 8 Generation of genetically modified Y. lipolytica (yBC6) with an integrated nourseothricin resistance gene in PXA2 locus by transforming wild-type Y. lipolytica with digested plasmid pBC31 (example 2)
  • Plasmid pBC31 (see e.g. example 2 or Figure 3) was restricted with Not ⁇ .
  • a 4036 bp fragment was gel isolated and used to transform the wild-type Y. lipolytica strain (C-00365, VTT Culture Collection) using the transformation method described in Example 6.
  • the transformed cells were screened for nourseothricin resistance.
  • Several nourseothricin resistant colonies were analysed at DNA level by PCR.
  • the transformants originating from the transfor- mation of the wild-type Y. lipolytica strain with Not ⁇ cut pBC31 and containing PXA2 gene deletion was designated as yBC6 and yBC7.
  • Example 9 Generation of genetically modified Y. Iipolytica (yBC1) with an integrated nourseothricin resistance gene in MFE2 locus by transforming wild-type Y. Iipolytica with digested plasmid pBC22 (example 3)
  • Plasmid pBC22 was restricted with Not ⁇ .
  • a 4300 bp fragment was gel isolated and used to transform the wild-type Y.
  • Iipolytica strain C-00365, VTT Culture Collection
  • the transformed cells were screened for nourseothricin resistance.
  • Several nourseothricin resistant colonies were analysed at DNA level by PCR.
  • the transit) formants originating from the transformation of the wild-type Y. Iipolytica strain with Not ⁇ cut pBC22 and containing MFE2 gene deletion was designated as yBC1 and yBC2.
  • Example 10 Generation of genetically modified Y. Iipolytica (yBC28 and
  • Plasmid pBC94 was restricted with Not ⁇ .
  • a 13940 bp fragment was
  • Plasmid pBC48 was restricted with Not ⁇ .
  • a 10539 bp fragment was gel isolated and used to transform the wild type Y. Iipolytica using the transformation method described in Example 6.
  • the transformed cells were screened for nourseothricin resistance.
  • Several nourseothricin resistant colonies were analysed at DNA level by PCR.
  • Example 11 Generation of antibiotic marker free Y. Iipolytica with ANT1 deletion by transforming genetically modified strain yBC3 with plasmid pBC27 (Example 4)
  • Plasmid pBC27 was transformed to the genetically modified strain yBC3, using the transformation method described in Example 6. The transformed cells were screened for hygromycin resistance. Several hygromycin resistant colonies were analysed at DNA level with PCR. Couple of colonies having pBC27 plasmid were cultivated in YPD+400 g/ ⁇ hygromycin with daily re- inoculation into new YPD+400 g/ ⁇ hygromycin. After one week cultivation di- lution series from the cultivations were prepared and plated on YPD plates. Individual colonies were streaked on YPD, YPD+400 g/ ⁇ hygromycin and YPD+400 g/ ⁇ nourseothricin plates.
  • Example 12 Generation of genetically modified Y. Iipolytica (yBC44) with an ANT1 deletion and an integrated NCP1 , CYP52A17 and FAOT encoding genes and nourseothricin resistance gene in MFE2 locus by transforming genetically modified strain yBC24 with digested plasmid pBC94 (Example 5)
  • Plasmid pBC94 was restricted with Not ⁇ .
  • a 13940 bp fragment was gel isolated and used to transform the genetically modified strain yBC24 using the transformation method described in Example 6.
  • the transformed cells were screened for nourseothricin resistance.
  • Several nourseothricin resistant colonies were analysed at DNA level by PCR.
  • Example 13 Generation of genetically modified Y. lipolytica (yBC19) with an ANT1 deletion and an integrated nourseothricin resistance gene in PXA2 locus by transforming genetically modified strain yBC24 with digested plasmid pBC31 (Example 5)
  • Plasmid pBC31 was restricted with Not ⁇ .
  • a 4036 bp fragment was gel isolated and used to transform the genetically modified strain yBC24 using the transformation method described in Example 6.
  • the transformed cells were screened for nourseothricin resistance.
  • Several nourseothricin resistant colonies were analysed at DNA level by PCR.
  • the transformants originating from the transformation of the genetically modified yBC24 strain with Not ⁇ cut pBC31 and containing ANT1 and PXA2 gene deletions were designated as yBC19 and yBC20.
  • MTBE extracts were evaporated into dryness in 2 ml GC-vials and dissolved into 50 ⁇ of dichloromethane. Silylation was performed by adding 25 ⁇ of N-Methyl-N-(trimethylsilyl) trifluoroacetamide (MSTFA) and by heating the samples at 80°C for 20 minutes.
  • MSTFA N-Methyl-N-(trimethylsilyl) trifluoroacetamide
  • Helium has been used as the carrier gas and the split ratio of20:1 or 25:1 has been used.
  • the injector temperature of 250°C, MSD transfer line heater temperature of 280°C, MS source temperature of 230°C and quadrupole temperature of 150°C were used.
  • the data was collected at a mass range of 40- 600 or 40-700. Samples were run with Agilent GC-MS (GC6890 + MS5973N or GC7890A + MS5975C mass selective detector).
  • Example 15 Diacid production from pelargonic acid with the strains hav- ing MFE2 gene deleted (yBC1 , Example 9), ANT1 gene deleted (yBC3, example 7) and MFE2 gene deleted and NCP1 and CYP52A17 genes expressed (yBC28, Example 10)
  • Transformants yBC1 , yBC3 and yBC28 were separately cultivated in 50 ml of YPD medium in 250 ml shake flask with 250 rpm shaking at 30°C for 3 days to produce biomass. After cultivation the cells were harvested and washed with 0.9% NaCI. Washed cells were resuspended into 10 ml of SC medium (Y- min+ SC-stock). Pelargonic acid (C-9 fatty acid) was added (1 g/l final concentration). pH was adjusted to pH 7.0 with 4 N NaOH. Pelargonic acid was added (1 g/l final concentration) and pH adjusted to 7.0 every day. Samples for lipid extraction were withdrawn periodically during cultivation. Lipid extraction and diacid measurement was carried out as described in Example 14.
  • Example 16 Production of diacids by strains of Y. lipolytica modified by addition of genes NCP1, CYP52A17 and FAOT and deletion of genes ANT1 and MFE (yBC44, example 12) in high cell density cultures having pelargonic acid feed Transformant was cultivated in bioreactors at pH 6.0 with glucose to produce biomass. After biomass production (26 g/L biomass produced) pelar- gonic acid was fed in 0.5 g/L pulses in addition to glucose feed. After 73 hours cultivation from 3.7 g/L pelargonic acid 0.15 g/L azelaic acid was produced.
  • Example 17 Production of diacids from lauric acid by strains of Y. Iipolytica having ANT1 gene deleted (yBC3, Example 7)
  • Y. Iipolytica strain having ANT1 deletion (3 transformants) were cultivated with wild type Y. Iipolytica overnight in 50 ml of synthetic complete (SC) medium containing 2% glucose in 250 ml shaken flask. Next day (OD600 about 16) 5 ml of SC+ 1 % lauric acid + 0.5 ml of Tween 20 and 5 ml of sodium phosphate buffer, pH 8.0 was added. After 3 days cultivations diacids were extracted and analysed as described in Example 14.
  • SC synthetic complete
  • Example 18 Production of diacids from lauric acid by strains of Y. Iipolytica having ANT1 gene deleted (yBC3), MFE2 gene deleted (yBC1) or ANT1 and PXA2 genes deleted (yBC19)
  • Y. Iipolytica strain having ANT1 gene deletion (yBC3), MFE2 gene deletion (yBC1 ) or ANT1 and PXA2 gene deletions (yBC19) were cultivated with wild type Y. Iipolytica as follows: Yeast colony was inoculated in 100 ml of Smit- YPD medium (10 g Yeast extract, 10 g Bacto peptone and 20 g glucose in 1 liter, pH 5.5) and cultivated in 500 ml shaken flask two days at +30°C with 250 rpm shaking. After two days 3 ml of pelargonic acid (3% final concentration) and glucose 0.88 g/ 1 g biomass was added. pH was adjusted to 8.0.
  • Example 19 Production of diacids from oleic acid by strains of Y. lipolytica having ANT1 gene deleted (yBC3) or ANT1 and PXA2 genes deleted (yBC19)
  • PXA2 gene deletions were cultivated with wild type Y. lipolytica as follows: Yeast colony was inoculated in 50 ml of YP+ 4% glucose medium (10 g Yeast extract, 10 g Bacto peptone and 40 g glucose in 1 liter, pH 5.5) and cultivated in 250 ml shaken flask two days at +30°C with 250 rpm shaking. After two days cells were harvested and washed with 0.9% NaCI. Cell pellet was resus- pended into 10 ml of synthetic complete medium and 100 ⁇ of oleic acid (1 % final concentration) was added. pH was adjusted to 8.0. In the following days pH was adjusted to 8.0 and 100 ⁇ of oleic acid was added. Samples for diacid analysis were collected after 26 and 46 hours cultivation. Diacids (C18 diacid) were extracted and analysed as described in Example 14.

Abstract

La présente invention concerne le domaine des diacides, et plus précisément un procédé de production de diacides. L'invention concerne également des micro-organismes de recombinaison comprenant une oméga-oxydation et une production de diacide accrue, et des utilisations et procédés associés.
PCT/FI2016/050228 2015-04-09 2016-04-08 Amélioration de production de diacide avec des micro-organismes génétiquement modifiés WO2016162605A1 (fr)

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