Lipid production

Abstract

The present invention relates to at least one cell for producing at least one lipid having the general formula II from at least one carbon substrate, wherein R 1 and R 2 They independently contain the same or different organic residues each having 5-13 carbon atoms, wherein the cell is a non-pathogenic cell that has been genetically modified to increase the heterologous expression of an enzyme (E2) relative to a wild-type cell, wherein the enzyme (E2) is capable of converting 3-hydroxyalkanoyl-3-hydroxyalkanoyl-CoA / ACP or 3-(3-hydroxyalkanoyloxy)alkanoic acid (HAA) and NDP-glucose into β-D-pyranosyl-3-hydroxyalkanoyl-3-hydroxyalkanoate #imgabs0#.

Description

Field of the Invention

[0001] The present invention relates to recombinant cells and biotechnological methods for producing lipids. In particular, the cells are non-pathogenic cells that have been genetically modified to produce at least one rubiwettin. Background of the Invention

[0003] Today, most of the available surfactants such as sodium laureth sulfate (SLES), betaine, etc. are chemically produced on an industrial scale. These chemically produced surfactants have all the disadvantages that usually come with chemical production processes, such as the formation of harmful byproducts. For example, during the production of SLES, at least one harmful byproduct 1,4-dioxane is produced. In order to reduce the amount of toxic products produced, and considering the increasing consumer demand for environmentally friendly products, the production and use of biosurfactants is a general trend. In addition to producing fewer toxic byproducts during the production process, biosurfactants also have useful properties such as high structural diversity, beneficial surfactant properties, low environmental toxicity, antibiotic or biologically active properties, and complete biodegradability. Therefore, there is generally an incentive to produce and use biosurfactants instead of chemical surfactants.

[0004] Rubiwettin is at least one example of such a biosurfactant. Rubiwettins represent a class of surfactants that are economically interesting because they may be a substitute for chemically produced surfactants.

[0005] Rubiwettin is an exolipid composed of a β-D-glucose molecule linked to a 3-hydroxy fatty acid dimer, with a chain length of C 14 and C 10 As the main component of lipids. They have surface active properties. Currently, wild-type Serratia rubrum ( Serratia rubidae ) isolate, which is a human and animal pathogen. The fact that this production organism is able to cause disease significantly reduces consumer acceptance of these conventionally produced rubiwettins. In addition, higher safety requirements are required during the production of rubiwettin, and this increases costs due to increased capital expenditures and possible additional production steps.

[0006] The methods currently available for producing biosurfactants such as rubiwettin involve the use of these pathogenic organisms. The yield can be optimized by changing the pH, oxygen supply, medium composition, feeding strategy, nitrogen supply, temperature, choice of substrate, etc. However, if rubiwettin is used as a surfactant on a large scale, they will have to compete with the surfactants currently used. The latter are bulk chemicals that can be produced at very low cost. Therefore, rubiwettin must also be produced at the lowest possible cost, without health risks to customers, and with as certain performance as possible. It is impossible to optimize performance parameters only through process optimization.

[0007] Therefore, there is a need in the art for a cheaper and more efficient method of producing biosurfactants such as rubiwettin with high product yields. DETAILED DESCRIPTION OF THE INVENTION

[0009] The present invention attempts to solve the above problems by providing a biotechnological means of producing biosurfactants such as lipids, especially rubiwettin from carbon sources using non-pathogenic cells. Specifically, non-pathogenic cells can be genetically modified to increase the expression of at least one enzyme (E2), and the enzyme (E2) can convert 3-hydroxyalkanoyl-3-hydroxyalkanoyl-CoA / ACP and / or 3-(3-hydroxyalkanoyloxy)alkanoic acid (HAA) combined with NDP-glucose into β-D-pyranose-glucopyranose-3-hydroxyalkanoyl-3-hydroxyalkanoate. Then the genetically modified cells can be used to convert a suitable carbon source into a lipid with the following general formula II:

[0010]

[0011] Formula II

[0012] Where R 1 and R 2 are independently the same or different alkyl groups having 5 to 13 carbon atoms. Specifically, the alkyl group may be saturated or unsaturated. More specifically, R 1 and / or R 2 The alkyl group of R may be a monounsaturated alkyl residue. Even more specifically, R 1 and / or R 2 It may be selected from pentenyl, heptenyl, nonenyl, undecenyl, tridecenyl and (CH2) n -CH3, where n=4-12.

[0013] This lipid of general formula II may also be referred to as glycolipid, more particularly as rubiwettin RG1. The genetically modified cells according to any aspect of the present invention have the advantages of being non-pathogenic and easy to culture. This allows cells to be produced more safely and can reduce costs because laboratories do not require special safety requirements during the production and use of rubiwettin. The cells according to any aspect of the present invention have other advantages, namely, the ability to use a variety of carbon substrates to produce lipids according to any aspect of the present invention. For example, simple carbon such as glucose can be used as a carbon substrate. And, the lipids formed according to any aspect of the present invention have defined and flexible properties. Another advantage of any aspect of the present invention is that non-pathogenic cells can be used to produce rubiwettin. Another advantage is that rubiwettin can be produced with higher space-time yields, higher carbon yields, product concentrations, and product uniformity (fatty acid types) compared to cells without increasing these activities.

[0014] According to one aspect of the present invention, there is provided a microbial cell for producing at least one lipid having the general formula II from at least one carbon substrate,

[0015]

[0016] Formula II

[0017] Where R 1 and R 2 are independently the same or different alkyl groups having 5 to 13 carbon atoms, and

[0018] The cell is a non-pathogenic cell that has been genetically modified to increase the heterologous expression of an enzyme (E2) relative to wild-type cells, wherein the enzyme (E2) is capable of converting 3-hydroxyalkanoyl-3-hydroxyalkanoyl-CoA / ACP and / or 3-(3-hydroxyalkanoyloxy)alkanoic acid (HAA) into β-D-pyranoglucopyranosyl-3-hydroxyalkanoyl-3-hydroxyalkanoate.

[0019] In particular, glucose can be added to allow any one or both of the conversions to occur. Thus, glucose can be added to convert 3-hydroxyalkanoyl-3-hydroxyalkanoyl-CoA / ACP into β-D-glucopyranosyl-3-hydroxyalkanoyl-3-hydroxyalkanoate. In another embodiment, glucose can be added to convert HAA into β-D-glucopyranosyl-3-hydroxyalkanoyl-3-hydroxyalkanoate. In another embodiment, HAA and 3-hydroxyalkanoyl-3-hydroxyalkanoyl-CoA / ACP can be present simultaneously to produce β-D-glucopyranosyl-3-hydroxyalkanoyl-3-hydroxyalkanoate.

[0020] Enzyme E2 may be able to convert 3-hydroxyalkanoyl-3-hydroxyalkanoyl-CoA / ACP into β-D-glucopyranosyl-3-hydroxyalkanoyl-3-hydroxyalkanoate. In one embodiment, enzyme E2 may be able to convert HAA into β-D-glucopyranosyl-3-hydroxyalkanoyl-3-hydroxyalkanoate. In another embodiment, enzyme E2 may be able to convert 3-hydroxyalkanoyl-3-hydroxyalkanoyl-CoA / ACP and HAA into β-D-glucopyranosyl-3-hydroxyalkanoyl-3-hydroxyalkanoate. In all these embodiments, NDP-glucose may be particularly present to include the glucose portion in general formula II. Enzyme E2 may be a glycosyltransferase (EC 2.4). Specifically, enzyme E2 comprises SEQ ID NO: 4 or a variant thereof. The term "variant" as used herein comprises an amino acid or nucleic acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% identical to a reference amino acid or nucleic acid sequence, respectively, wherein preferably amino acids other than essential amino acids for function (e.g., catalytic activity of a protein, or folding or structure of a molecule) are deleted, substituted or replaced by insertion, or essential amino acids are replaced in a conservative manner to achieve the effect of retaining the biological activity of the reference sequence or a molecule derived therefrom. The prior art includes algorithms that can be used to compare two given nucleic acid or amino acid sequences and calculate the degree of identity, see Arthur Lesk (2008), Thompson equal 1994, with Katoh equal2005. The term "variant" is used synonymously and interchangeably with the term "homolog". Such variants can be prepared by introducing deletions, insertions or substitutions in an amino acid or nucleic acid sequence and in a fusion comprising such a macromolecule or a variant thereof. In one embodiment, with respect to an amino acid sequence, in addition to the above sequence identity, the term "variant" also comprises an amino acid sequence comprising one or more conservative amino acid changes compared to the respective reference or wild-type sequence, or comprises a nucleic acid sequence encoding an amino acid sequence comprising one or more conservative amino acid changes. In one embodiment, in addition to the above degree of sequence identity, the term "variant" of an amino acid sequence or nucleic acid sequence comprises any active portion and / or fragment of the amino acid sequence or nucleic acid sequence, respectively, or any nucleic acid sequence encoding an active portion and / or fragment of an amino acid sequence. The term "active portion" as used herein means an amino acid sequence or nucleic acid sequence that is less than the full-length amino acid sequence or encodes less than the full-length amino acid sequence, respectively, wherein the amino acid sequence or the encoded amino acid sequence retains at least some of its basic biological activity, respectively. For example, an active portion and / or fragment of a protease may be capable of hydrolyzing peptide bonds in a polypeptide. As used herein, the phrase "retains at least some of its essential biological activity" means that the target amino acid sequence has a biological activity that is above and different from the background activity and the kinetic parameters that characterize the activity, more specifically k cat and K M Preferably, the value of reference molecule about specific substrate is shown within 3,2 or 1 order of magnitude.Similarly, the "variant" of term nucleic acid comprises nucleic acid complementary strand, and it is preferably hybridized with reference or wild-type nucleic acid under stringent conditions.In one embodiment, SEQ ID: 4 variant can have 60% sequence identity with SEQ ID NO: 4.

[0021] In one embodiment, the enzyme E2 can have the polypeptide sequence SEQ ID NO: 4 or a polypeptide sequence in which up to 25%, preferably up to 20%, particularly preferably up to 15%, especially up to 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% of the amino acid residues are modified by deletion, insertion, substitution or a combination thereof compared to the reference sequence SEQ ID NO: 4, and still have at least 10%, preferably 50%, particularly preferably 80%, especially more than 90% of the enzyme activity of the enzyme with the reference sequence SEQ ID NO: 4, wherein the enzyme activity of the enzyme E2 is understood to mean preferably the ability to convert 3-hydroxyalkanoyl-3-hydroxyalkanoyl-CoA / ACP or 3-(3-hydroxyalkanoyloxy)alkanoic acid (HAA) into β-D-pyranoglucopyranosyl-3-hydroxyalkanoyl-3-hydroxyalkanoate. NDP-glucose can be present in this conversion.

[0022] The lipid of formula II may also be referred to as rubiwettin. 1 and R 2 The length of the group can be different. 1 and R 2 can be independently selected from saturated and unsaturated alkyl groups. More specifically, R 1 and / or R 2 It may be a saturated or unsaturated alkyl group having 5 to 13 carbon atoms. Even more specifically, R 1 and R 2 It may be a saturated or monounsaturated alkyl group having 5 to 13 carbon atoms. 1 and R 2 The alkyl group may contain 5-13 carbon atoms, 7-13 carbon atoms, 9-13 carbon atoms, 5-11 carbon atoms, 5-9 carbon atoms, etc. 1 and / or R 2 In embodiments where the alkyl group is a saturated alkyl group, the alkyl group may contain 5, 6, 7, 8, 9, 10, 11, 12, or 13 carbon atoms. 1 and / or R 2 In the embodiment where the alkyl group is an unsaturated alkyl group, the alkyl group may be a monounsaturated alkyl group comprising 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1 or 13:1 carbon atoms. The cell according to any aspect of the present invention may be capable of producing alkyl groups having different R 1 and R 2 In one embodiment, the lipid of formula II produced according to any aspect of the present invention may be rubiwettin RG1 (CAS-Nr. 129039-46-9). Rubiwettin RG1 may also be referred to as a glycolipid named β-D-glucopyranosyl-3-(3'-hydroxytetradecanoyloxy)decanoate or β-glucopyranosyl-3-(3'-hydroxytetradecanoyloxy)decanoate.

[0023] The cells according to any aspect of the invention may be further genetically modified to increase heterologous expression of an enzyme (E1) relative to wild-type cells, said enzyme (E1) being capable of converting 3-hydroxyalkanoyl-CoA / ACP into 3-hydroxyalkanoyl-3-hydroxyalkanoyl-CoA / ACP and further into 3-(3-hydroxyalkanoyloxy)alkanoic acid (HAA).

[0024] Enzyme E1 can be 3-(3-hydroxyalkanoyloxy)alkanoic acid (HAA) synthase. In one embodiment, enzyme E1 comprises SEQ ID NO: 2 or a variant thereof. In another embodiment, enzyme E1 comprises a sequence selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14 and variants thereof, wherein the variants have 60% sequence identity to SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12 and SEQ ID NO: 14, respectively.

[0025] In one embodiment, the enzyme E1 can have the polypeptide sequence SEQ ID NO: 6 or a polypeptide sequence in which up to 25%, preferably up to 20%, particularly preferably up to 15%, especially up to 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% of the amino acid residues are modified by deletion, insertion, substitution or a combination thereof compared to the reference sequence SEQ ID NO: 6, and still has at least 10%, preferably 50%, particularly preferably 80%, especially more than 90% of the enzymatic activity of the enzyme having the reference sequence SEQ ID NO: 6, wherein the enzymatic activity of the enzyme E1 is understood to refer to the ability to preferably convert 3-hydroxyalkanoyl-CoA / ACP into 3-hydroxyalkanoyl-3-hydroxyalkanoyl-CoA / ACP and further into HAA.

[0026] In another embodiment, the enzyme E1 can have the polypeptide sequence SEQ ID NO: 8 or a polypeptide sequence in which up to 25%, preferably up to 20%, particularly preferably up to 15%, especially up to 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% of the amino acid residues are modified by deletion, insertion, substitution or a combination thereof compared to the reference sequence SEQ ID NO: 8, and still has at least 10%, preferably 50%, particularly preferably 80%, especially more than 90% of the enzymatic activity of the enzyme having the reference sequence SEQ ID NO: 8, wherein the enzymatic activity of the enzyme E1 is understood to refer to the ability to preferably convert 3-hydroxyalkanoyl-CoA / ACP into 3-hydroxyalkanoyl-3-hydroxyalkanoyl-CoA / ACP and further into 3-(3-hydroxyalkanoyloxy)alkanoic acid (HAA).

[0027] In another embodiment, the enzyme E1 can have the polypeptide sequence SEQ ID NO: 10 or a polypeptide sequence in which up to 25%, preferably up to 20%, particularly preferably up to 15%, especially up to 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% of the amino acid residues are modified by deletion, insertion, substitution or a combination thereof compared to the reference sequence SEQ ID NO: 10, and still has at least 10%, preferably 50%, particularly preferably 80%, especially more than 90% of the enzymatic activity of the enzyme having the reference sequence SEQ ID NO: 10, wherein the enzymatic activity of the enzyme E1 is understood to refer to the ability to preferably convert 3-hydroxyalkanoyl-CoA / ACP into 3-hydroxyalkanoyl-3-hydroxyalkanoyl-CoA / ACP and further into 3-(3-hydroxyalkanoyloxy)alkanoic acid (HAA).

[0028] In another embodiment, the enzyme E1 can have the polypeptide sequence SEQ ID NO: 12 or a polypeptide sequence in which up to 25%, preferably up to 20%, particularly preferably up to 15%, especially up to 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% of the amino acid residues are modified by deletion, insertion, substitution or a combination thereof compared to the reference sequence SEQ ID NO: 12, and still has at least 10%, preferably 50%, particularly preferably 80%, especially more than 90% of the enzymatic activity of the enzyme having the reference sequence SEQ ID NO: 12, wherein the enzymatic activity of the enzyme E1 is understood to refer to the ability to preferably convert 3-hydroxyalkanoyl-CoA / ACP into 3-hydroxyalkanoyl-3-hydroxyalkanoyl-CoA / ACP and further into 3-(3-hydroxyalkanoyloxy)alkanoic acid (HAA).

[0029] In another embodiment, the enzyme E1 can have the polypeptide sequence SEQ ID NO: 14 or a polypeptide sequence in which up to 25%, preferably up to 20%, particularly preferably up to 15%, especially up to 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% of the amino acid residues are modified by deletion, insertion, substitution or a combination thereof compared to the reference sequence SEQ ID NO: 14, and still has at least 10%, preferably 50%, particularly preferably 80%, especially more than 90% of the enzymatic activity of the enzyme having the reference sequence SEQ ID NO: 14, wherein the enzymatic activity of the enzyme E1 is understood to refer to the ability to preferably convert 3-hydroxyalkanoyl-CoA / ACP into 3-hydroxyalkanoyl-3-hydroxyalkanoyl-CoA / ACP and further into 3-(3-hydroxyalkanoyloxy)alkanoic acid (HAA).

[0030] The cell according to any aspect of the invention may produce a further lipid having the general formula I from a carbon substrate,

[0031] Formula I

[0032] Where R 1 and R 2 are independently the same or different alkyl groups having 5 to 13 carbon atoms. Specifically, the alkyl group may be saturated or unsaturated. More specifically, R 1 and / or R 2 The alkyl group of R may be a monounsaturated alkyl residue. Even more specifically, R 1 and / or R 2 It may be selected from pentenyl, heptenyl, nonenyl, undecenyl, tridecenyl and (CH2) n -CH3, where n=4-12.

[0033] The lipids of formula I may have R 1 and R 2 Specifically, R 1 and R 2 can be independently selected from saturated and unsaturated alkyl groups. More specifically, R 1 and / or R 2 It may be a saturated or unsaturated alkyl group having 5 to 13 carbon atoms. Even more specifically, R 1 and R 2 It may be a saturated or monounsaturated alkyl group having 5 to 13 carbon atoms. 1 and R 2 The alkyl group may contain 5-13 carbon atoms, 7-13 carbon atoms, 9-13 carbon atoms, 5-11 carbon atoms, 5-9 carbon atoms, etc. 1 and / or R 2 In embodiments where the alkyl group is an unsaturated alkyl group, the alkyl group may be a monounsaturated alkyl group containing 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, or 13:1 carbon atoms. 1 and / or R 2 In embodiments where the alkyl group is a saturated alkyl group, the alkyl group may contain 5, 6, 7, 8, 9, 10, 11, 12 or 13 carbon atoms. The cell according to any aspect of the invention may be capable of producing a cell having different R 1 and R 2The lipid of formula I can be referred to as rubiwettin R1 (CAS-Nr.129039-45-8). Specifically, the lipid is a mixture of 3-(3'-hydroxytetradecanoyloxy) tetradecanoate, 3-(3'-hydroxydecanoyloxy) decanoate, 3-(3'-hydroxyhexadecenoyloxy) hexadecenoate, 3-(3'-hydroxytetradecanoyloxy) decanoate, 3-(3'-hydroxyhexadecenoyloxy) decanoate, 3-(3'-hydroxyhexadecenoyloxy) tetradecanoate and minor molecular isomers.

[0034] Surprisingly, it can be demonstrated that the recombinant cells according to any aspect of the present invention with increased expression of E2 and / or E1 are capable of producing increased amounts of lipids having formula II and / or I compared to the wild type of the cell. Thus, the cells according to any aspect of the present invention can allow highly selective production of rubiwettin RG1 and reduce the production of undesirable intermediates such as dimers of β-hydroxy fatty acids.

[0035] As used herein, the phrase "heterologous expression of enzyme increases" should be understood as an increase in intracellular activity. Basically, the increase in enzyme activity can be achieved as follows: using a strong promoter or adopting a gene or allele encoding the corresponding enzyme with increased activity, and optionally combining these measures to increase the copy number of one or more gene sequences of the encoding enzyme. For example, by conversion, transduction, conjugation or these methods with a carrier containing a desired gene, an allele of the gene or its part and a combination of carriers that make the expression of the gene possible, production is used for the genetically modified cell in the method according to the present invention. Heterologous expression is specifically achieved by the integration of the gene or the allele in the chromosome of the cell or in the extrachromosomal replication of the carrier. Specifically, the enzyme can be 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% greater than the wild-type cell in terms of the activity increase of the wild-type cell.

[0036] The technician will be able to use any method known in the art to genetically modify cells. Whether the nucleic acid molecules, polypeptides, and more specifically enzymes used according to any aspect of the present invention are recombinant or not, it is not necessarily affected by their expression levels. However, in one embodiment, one or more recombinant nucleic acid molecules, polypeptides, or enzymes used according to any aspect of the present invention can be overexpressed. The term "overexpression" used herein refers to that the various polypeptides encoded or expressed are expressed at a higher level or with a higher activity than those usually found in cells (e.g., in various wild-type cells) under the same conditions without the presence of genetic modification for increasing expression. Those skilled in the art are familiar with the various ways of causing overexpression. For example, the nucleic acid molecules to be overexpressed or the nucleic acid molecules encoding the polypeptides or enzymes to be overexpressed can be placed under the control of a strong inducible promoter such as the lac promoter. The prior art describes standard plasmids that can be used for this purpose, such as the pET system of the carrier of pET-3a (commercially available from Novagen). It can be determined whether a nucleic acid or polypeptide is overexpressed, in the case of nucleic acid molecules, by quantitative PCR reaction, in the case of polypeptides, by SDS polyacrylamide electrophoresis, protein blotting, or comparative activity assays. Genetic modification can be directed to transcription, translation and / or post-translational modification, which causes enzymatic activity and / or selective change under the culture condition selected and / or identified.Therefore, in different embodiments of the present invention, in order to work more effectively, microorganism can comprise one or more gene deletions.By the mutant gene deletion scheme and / or from having one or more reductions in these enzymes expressed or no mutant strain expressed and / or additive methods well known by persons skilled in the art, gene deletion can be realized.

[0037] A general survey of the possibilities of increasing the activity of intracellular enzymes, such as pyruvate carboxylase, is given in DE-A-100 31 999, which is hereby incorporated by reference and whose disclosure content on the possibilities of increasing the activity of intracellular enzymes forms part of the disclosure content of the present invention.

[0038] The above and all enzymes mentioned subsequently or the expression of genes can be detected as follows: separate by means of one-dimensional and two-dimensional protein gels, and then use appropriate analytical software to optically identify the protein concentration in the gel. If the increase of enzymatic activity is only based on the increase of corresponding gene expression, then the contrast of one-dimensional or two-dimensional protein separation between wild-type and genetically modified cells can determine the quantitative increase of enzymatic activity in a simple manner. The conventional method for preparing protein gel (in the case of rod-shaped bacteria) and identifying protein is the method described by people such as Hermann (Electrophoresis, 22: 1712.23 (2001)). Can analyze protein concentration as follows equally: use the protein to be detected has specific antibody to carry out western blot hybridization (Sambrook equalMolecular Cloning: a laboratory manual, 2nd edition Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY USA, 1989), and then optically analyzed using appropriate software to determine the concentration (Lohaus and Meyer (1989) Biospektrum, 5: 32-39; Lottspeich (1999) Angewandte Chemie 111: 2630-2647). The activity of DNA binding proteins can be measured by DNA band shift assays (also known as gel retardation) (Wilson et al. (2001) Journal of Bacteriology, 183: 2151-2155). The effects of DNA binding proteins on the expression of other genes can be detected by various well-described reporter gene assays (Sambrook et al. equal Molecular Cloning: a laboratory manual, 2nd edition Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY USA, 1989). Intracellular enzyme activity can be determined according to various described methods (Donahue et al. (2000) Journal of Bacteriology 182 (19): 5624-5627; Ray et al. (2000) Journal of Bacteriology 182 (8): 2277-2284; Freedberg et al. (1973) Journal of Bacteriology 115 (3): 816-823). If no practical method for determining the activity of an enzyme is indicated in the following embodiments, it is preferably determined by the Hermann equal Electrophoresis, 22: 1712-23 (2001), Lohaus equal Biospektrum 532-39 (1998), Lottspeich, Angewandte Chemie 111: 2630-2647 (1999) and Wilson etc peopleThe method described in Journal of Bacteriology 183: 2151-2155 (2001) is used to determine the increase of enzyme activity and the reduction of enzyme activity. If the increase of enzyme activity is achieved by the sudden change of endogenous genes, this sudden change can be randomly generated as follows: by conventional methods, for example, by ultraviolet irradiation or by mutagenic chemicals, or by genetic engineering methods such as deletion, insertion and / or nucleotide exchange selectively. Modified cells are obtained by these sudden changes. Particularly preferred enzyme mutants are also particularly those enzymes that can no longer be inhibited by feedback, product or substrate, or at least those enzymes that are inhibited to a reduced degree compared to the wild-type enzyme.

[0039] If the increase of enzyme activity is achieved by the increase of enzyme synthesis, the copy number of the corresponding gene will increase, or the promoter and regulatory region or ribosome binding site located upstream of the structural gene will mutate. The expression cassette integrated into the upstream of the structural gene works in the same way. It is also possible to increase expression at any desired time point by an inducible promoter. However, in addition, "enhancers" can also be assigned to enzyme genes as regulatory sequences, which also lead to increased gene expression through improved interactions between RNA polymerase and DNA. As a result of measures to extend the life of mRNA, expression is also improved. In addition, by preventing the degradation of enzyme proteins, enzyme activity is also increased. Here, genes or gene constructs are present in plasmids with different copy numbers, or are integrated in chromosomes and amplified therein. Alternatively, overexpression of related genes can also be achieved by changing the composition of the culture medium and culture management. The person skilled in the art will find guidance in this respect in particular in the following literature: Martin et al. (Bio / Technology 5, 137-146 (1987)), Guerrero et al. (Genes 138, 35-41 (1994)), Tsuchiya and Morinaga (Bio / Technology 6, 428-430 (1988)), Eikmanns et al. (Genes 102, 93-98 (1991)), EP-A-0 472 869, US 4,601,893, Schwarzer and Pühler (Bio / Technology 9, 84-87 (1991)), Reinscheid et al. (Applied and Environmental Microbiology 60, 126-132 (1994)), LaBarre et al. (Journal of Bacteriology 175, 1001-1007). (1993)), WO-A-96 / 15246, Malumbres et al. (Genes 134, 15-24 (1993)), JP-A-10-229891, Jensen and Hammer (Biotechnology and Bioengineering 58, 191-195 (1998)), and known textbooks of genetics and molecular biology. Like mutations, the above measures also lead to genetically modified cells.

[0040] For example, use an additional plasmid to increase the expression of each gene. Suitable plasmids or carriers are all embodiments that those skilled in the art can use for this purpose in principle. Such plasmids and carriers can for example derive from the publicity materials of Novagen, Promega, New England Biolabs, Clontech or Gibco BRL. Other preferred plasmids and vectors can be found in the following literature: Glover, DM (1985) DNA cloning: a practical approach, Volumes I-III, IRL Press Ltd., Oxford; Rodriguez, RL and Denhardt, DT (eds.) (1988) Vectors: a survey of molecular cloning vectors and their uses, 179-204, Butterworth, Stoneham; Goeddel, DV (1990) Systems for heterologous gene expression, Methods Enzymol. 185, 3-7; Sambrook, J.; Fritsch, EF and Maniatis, T. (1989), Molecular cloning: a laboratory manual, 2nd edition, Cold Spring Harbor Laboratory Press, New York.

[0041] The plasmid vector containing the gene to be amplified is then transferred into the desired strain by conjugation or transformation. equal The conjugation method is described in Applied and Environmental Microbiology 60: 756-759 (1994). equal Applied Microbiology and Biotechnology 29: 356-362 (1988), Dunican and Shivnan, Bio / Technology 7: 1067-1070 (1989) and Tauch equal The transformation method is described in FEMS Microbiology Letters 123: 343-347 (1994). After homologous recombination by a "crossover" event, the resulting strain contains at least two copies of the gene of interest.

[0042] According to any aspect of the present invention, the cell can be genetically modified so that within a defined time interval, within 2 hours, particularly within 8 hours or 24 hours, it forms at least 2 times, particularly at least 10 times, at least 100 times, at least 1000 times or at least 10000 times more lipid of general formula I or II than wild-type cells. The increase in product formation can be determined as follows: for example, by separately culturing cells according to any aspect of the present invention and wild-type cells in a suitable nutrient medium under the same conditions (same cell density, same nutrient medium, same culture conditions) for a specified time interval, and then determining the amount of the target product (lipid with general formula II or I) in the nutrient medium.

[0043] Changes in the amino acid residues of a given polypeptide sequence that do not result in significant changes in the properties and functions of a given polypeptide are known to those skilled in the art. Thus, for example, "conservative amino acids" can be exchanged for one another; examples of such suitable amino acid substitutions are: Ala for Ser; Arg for Lys; Asn for Gln or His; Asp for Glu; Cys for Ser; Gln for Asn; Glu for Asp; Gly for Pro; His for Asn or Gln; Ile for Leu or Val; Leu for Met or Val; Lys for Arg or Gln or Glu; Met for Leu or Ile; Phe for Met or Leu or Tyr; Ser for Thr; Thr for Ser; Trp for Tyr; Tyr for Trp or Phe; Val for Ile or Leu. It is also known that changes, especially at the N- or C-terminus of a polypeptide, for example in the form of amino acid insertions or deletions, often have no significant effect on the function of the polypeptide.

[0044] By destroying the cells containing enzyme activity in a manner known to those skilled in the art, for example by means of a ball mill, a French press or an ultrasonic disintegrator, then by separating cells, cell debris and a rupture aid (e.g., glass beads) at 13,000 rpm and 4 ° C for 10 minutes by centrifugation, this activity can be determined. Using the resulting cell-free crude extract, the enzyme assay and subsequent LC-ESI-MS detection of the product can then be carried out. Alternatively, by chromatographic methods (such as nickel-nitrilotriacetic acid affinity chromatography, streptavidin affinity chromatography, gel filtration chromatography or ion exchange chromatography), the enzyme can be enriched in a manner known to those skilled in the art, or purified to homogeneity.

[0045] In one embodiment, the method for determining the activity of enzyme E2 comprises first destroying the cells containing the activity (i.e., cells according to any aspect of the present invention) in a manner known to those skilled in the art, for example, by means of a ball mill, a French press, or an ultrasonic disintegrator, followed by separation of cells, cell fragments, and a rupture aid (e.g., glass beads) by centrifugation at 16,100 g for 10 minutes at 4 ° C. Using the resulting cell-free crude extract, an enzyme assay of the product and subsequent LC-ESI-MS detection can be performed. As an alternative, the enzyme can be enriched in a manner known to those skilled in the art, or purified to homogeneity, by a chromatographic method (such as nickel / nitrilotriacetic acid affinity chromatography, streptavidin affinity chromatography, gel filtration chromatography, or ion exchange chromatography). The sample can then be used to measure the activity of enzyme E2. Specifically, the activity of the enzyme E2 can be determined using a standard assay, which can consist of 185µl 10 mM Tris-HCl (pH 7.5), 10µl mM NDP-glucose and 50µl crude protein extract (approximately 1 mg total protein) or purified protein in solution (5µg purified protein). The reaction can be started by adding 10µl of a mM ethanolic solution of 3-hydroxytetradecanoyl-3-hydroxydecanoic acid or 3-hydroxyhexadecanoyl-3-hydroxydecanoic acid and incubated at 30°C for 1 h with shaking (600 rpm). Subsequently, the reaction can be treated with 1 ml acetone. Undissolved components can be precipitated by centrifugation (16,100 g, 5 min room temperature), and the sample can then be analyzed by means of LC-ESI-MS. The identification of the product can then be performed by analyzing the corresponding mass trace and MS2 spectrum. This method can be used to measure the activity of E2.

[0046] In another embodiment, the method for determining the activity of enzyme E1 includes first destroying cells containing the activity (i.e., cells according to any aspect of the present invention) in a manner known to those skilled in the art, and then separating cells, cell fragments, and rupture aids (e.g., glass beads) by centrifugation at 16,100 g for 10 min at 4 ° C. Using the resulting cell-free crude extract, enzyme assays and subsequent LC-ESI-MS detection of the product can be performed. As an alternative, the enzyme can be enriched in a manner known to those skilled in the art, or purified to homogeneity, by chromatographic methods (such as nickel / nitrilotriacetic acid affinity chromatography, streptavidin affinity chromatography, gel filtration chromatography, or ion exchange chromatography). The sample can then be used to measure the activity of enzyme E1. Specifically, the activity of enzyme E1 can be determined using a standard assay, which can contain 100µM E. coli ACP, 1 mM β-mercaptoethanol, 200µM malonyl-CoA, 40µM octanoyl-CoA and 40µM dodecanoyl-CoA or 40µM octanoyl-CoA and 40 mM tetradecanoyl-CoA, 100µM NADPH, 2µg E. coli FabD, 2µg Mycobacterium tuberculosis FabH, 1µg E. coli FabG, 0.1 M sodium phosphate buffer (pH 7.0) and 5µg enzyme E1 in a final volume of 120µl. ACP, β-mercaptoethanol and sodium phosphate buffer can be pre-incubated at 37°C for 30 min to completely reduce ACP. The reaction can be started by adding enzyme E1. The reaction can be terminated with 2 ml of water, which has been acidified to pH 2.0 with HCl, and subsequently extracted twice with 2 ml of chloroform / methanol (2:1 (v:v)). Phase separation can be performed by centrifugation (16,100 g, 5 min, room temperature). The lower organic phase can be removed, completely evaporated in a vacuum centrifuge, and the precipitate can then be dissolved in 50 µl of methanol. Undissolved components can be precipitated by centrifugation (16,100 g, 5 min room temperature), and the sample can be analyzed by means of LC-ESI-MS. Identification of the product can be performed by analyzing the corresponding mass trace and MS2 spectrum.

[0047] The enzyme used according to any aspect of the present invention may be recombinant. The term "recombinant" as used herein refers to a molecule or a molecule encoded by such a molecule, particularly a polypeptide or nucleic acid, which thus does not occur naturally but is the result of genetic engineering, or refers to a cell comprising a recombinant molecule. For example, a nucleic acid molecule is recombinant if it comprises a promoter that is functionally linked to a sequence encoding a catalytically active polypeptide and that has been engineered so that the catalytically active polypeptide is overexpressed compared to the polypeptide level in a corresponding wild-type cell comprising the original, unaltered nucleic acid molecule.

[0048] The cells used according to any aspect of the present invention may also be non-pathogenic cells. Non-pathogenic cells refer to cells that do not cause disease, injury or death to another organism. The cells according to any aspect of the present invention may be any non-pathogenic prokaryotic or eukaryotic organism. These may be mammalian cells (e.g., cells from humans), plant cells or microorganisms such as yeast, fungi or bacteria, wherein microorganisms, especially bacteria and yeast are preferred.

[0049] Suitable bacteria, yeasts or fungi are especially those bacteria, yeasts or fungi which are deposited as bacteria, yeasts or fungal strains at Deutsche Sammlung von Mikroorganismen und Zellkulturen (German Collection of Microorganisms and Cell Cultures) GmbH (DSMZ), Brunswick, Germany. Suitable bacteria according to the invention belong to the genera listed below:

[0050] http: / / www.dsmz.de / species / bacteria.htm,

[0051] Suitable yeasts according to the invention belong to the genera listed below:

[0052] http: / / www.dsmz.de / species / yeasts.htm

[0053] And suitable fungi according to the invention are those listed below:

[0054] http: / / www.dsmz.de / species / fungi.htm.

[0055] In particular, the cell may be selected from the following genera: Aspergillus ( Aspergillus ), Corynebacterium ( Corynebacterium ), Brevibacterium ( Brevibacterium ), Bacillus ( Bacillus ), Acinetobacter spp. Acinetobacter )、Alcaligenes spp. Alkaligenes ), Lactobacillus spp. Lactobacillus ), Paracoccus ( Paracoccus ), Lactococcus ( Lactococcus ), Candida spp. White ), Pichia Peach ), Hansenula ( Little Hansen ), Kluyveromyces ( Kluyveromyces )、Saccharomyces ( Saccharomyces ), Escherichia coli ( Escherichia ), Zymomonas spp. Zymomonas), Ascomycetes ( Yarrow ), Methylobacterium ( Methylobacterium ), Ralstonia ( Ralstonia ), Pseudomonas ( Pseudomonas )、Rhodospirilla spp. Rhodospirillum ), Rhodobacterium ( Rhodobacter ), Burkholderia spp. Burkholderia ), Clostridium ( Clostridium ) and Cupriavidus ( Copper-loving More specifically, the cell may be selected from Aspergillus nidulans ( Aspergillus nidulans ), Aspergillus niger ( Aspergillus niger ), widespread alkali-producing bacteria ( Alkaligenes side )、Bacillus megaterium( Bacillus megaterium ), Bacillus subtilis ( Bacillus subtilis )、Breibacterium flavum( Brevibacterium flavum ), Brevibacterium lactofermentum ( Brevibacterium lactofermentum ), Burkholderia androstemonis ( Burkholderia andropogoni ), B. brasiliensis , B. caledonica , B. caribbean , B. caryophylli , B. fungi , B. gladioli , B. glathei , B. glumes , Blumeria graminis B. graminis ), B. hostess , B. kururiensis , B. phenazine , Burkholderia tumouri B. phymatum ), B. phytofirmans , B. plantarii , B. sacchari , B. singaporeensis , B. sordidicola , B. terricola , B. tropical , B. tuber , B. ubonensis , B. unamae , B. xenophobic , B. anthina , B. pyrrhocinia , Burkholderia thailandensis ( B. thailandensis ), Candida blankii , Candida rugosa ( White wrinkled ), Corynebacterium glutamicum ( Corynebacterium glutamic ), Corynebacterium efficiens , Escherichia coli ( Escherichia coli ), Hansenula polymorpha ( Hansenula polymorpha )、Kluyveromyces lactis( Kluveromyces lactis ), Methylobacterium extorquens ( Methylobacterium extorquens ), The clever Paracoccus ,Pseudomonas argentinensis , P. borbori , P. citronellolis , P. flavescens , P. mendocina , P. nitroreducens , P. oil-eating , P. pseudoalcaligenes , P. resinovorans , P. straminea , P. aurantiaca , P. gold-making , P. chlororaphis , P. fragi , P. lundensis , P. taetrolens , P. Antarctica , P. azotoformans , 'P. blatchfordae' , P. brassicacearum , P. brenneri , P. cedar , P. corrugata , P. fluorescens , P. gessardii , P. libanensis , P. mandelii , P. marginal , P. mediterranea , P. meridiana , P. migulae , P. mucidolens , P. oriental , P. panacis , P. proteolytica , P. rhodesiae , P. synxantha , P. Thiverval , P. tolaasii , P. veronii , P. denitrificans , P. pertucinogena , P. cream-colored , P. fulva , P. monteilii , P. mosselii , P. parafulva , Pseudomonas putida ( P. putida ), P. balearica , P. stutzeri , P. amygdali , P. avellanae , P. carica papaya , P. chicory , P. coronafaciens , P. ficus erectae , 'P. helianthi' , P. honey , P. savastanoi , Pseudomonas syringae ( P. syringae ), P. tomato , P. viridiflava , P. abietaniphila , P. acidophila , P. agaricus , P. alcaliphila , P. alkanolytica , P. amyloidosis , P. asplenii , P. azotifigens , P. cannabina , P. coenobios , P. freezing , P. constantinii , P. cruciviae , P. delhiensis , P. excibis , P. Far Eastern , P. frederiksbergensis 、Pseudomonas sphaeroides P. fuscovaginae ), P. icy , P. grimontii , P. indica , P. jessenii , P. jinjuensis , P. kilonensis , Pseudomonas kluyveri ( P. knackmussii ), P. koreensis , P. linii , P. lutea , P. moraviensis , P. restlessness , P. pachastrellae , P. palleroniana , Poppy seeds , P. pelí , P. perolens , P. poa , P. pohangensis , P. psychrophila , P. psychrotolerans , P. rathonis , P. reptile-eating , P. resiniphila , P. rhizosphaerae , P. rubescens , P. salomonii , P. crops , P. septica , P. simiae , Grams of his , P. thermotolerans 、Pseudomonas aeruginosa( P. aeruginosa ), P. tremae , P. trivialis , P. turbinellae , P. tuticorinensis , P. from Umsong , P. vancouverensis , P. vranovensis , P. xanthomarina , Ralstonia eutropha ( Ralstonia eutropha ), Rhodospirilla rubrum ( Rhodospirillum rubrum ), Rhodobacter sphericalus ( Rhodobacter spherical )、Saccharomyces cerevisiae( Saccharomyces cerevisiae ), Yarrowia lipolytica ( Yarrow lipolytic ) and Zymomonas mobileum ( Zymomonas mobile More specifically, the cell may be a bacterial cell selected from the group consisting of: Acinetobacter species ( Acinetobacter sp. ), Bacillus species ( Bacillus sp. ), Brevibacterium species ( Brevibacterium sp. ), Burkholderia species ( Burkholderia sp. ), Chlorella species ( Chlorella sp. ), Clostridium species ( Clostridium sp. ), Corynebacterium species ( Corynebacterium sp. ), cyanobacteria (Cyanobacteria) , Escherichia species ( Escherichia sp. ), Pseudomonas species ( Pseudomonas sp. ), Klebsiella species ( Klebsiella sp. ), Salmonella species ( Salmonella sp. ), Rhizobium species ( Rhizobium sp. ), Saccharomyces species ( Saccharomyces sp. ), Pichia species ( Pichia sp. ) and Nostoc species ( Nostoc sp. ). Even more specifically, the cell may be selected from Bacillus subtilis ( Bacillus subtle ), Burkholderia thailandensis ( Burkholderia thailandensis ), Corynebacterium glutamicum ( Corynebacterium glutamicum ), Escherichia coli ( E. coli ), Klebsiella oxytoca ( Klebsiella oxytoca ), Pseudomonas fluorescens ( Pseudomonas fluorescens ), Pseudomonas putida ( Pseudomonas putida )、Pseudomonas stutzeri( Pseudomonas stutzeri ), Rhizobium meliloti ( Rhizobium meliloti )、Saccharomyces cerevisiae( Saccharomyces cerevisiae ) and Pichia pastoris ( Pichia pastoris ).

[0056] In one embodiment, the cell according to any aspect of the present invention may be a cell that has been genetically modified to increase the expression of enzyme E2 comprising SEQ ID NO: 4 or a variant thereof and the expression of enzyme E1 comprising SEQ ID NO: 2 or a variant thereof.

[0057] The cell according to any aspect of the present invention may be a non-pathogenic cell which has been genetically modified to increase heterologous expression of the enzyme (E2) and the enzyme (E1) relative to a wild-type cell,

[0058] The enzyme (E2) is capable of converting 3-hydroxyalkanoyl-3-hydroxyalkanoyl-CoA / ACP and / or 3-(3-hydroxyalkanoyloxy)alkanoic acid (HAA) into β-D-glucopyranosyl-3-hydroxyalkanoyl-3-hydroxyalkanoate, wherein E2 is a glycosyltransferase (EC 2.4);

[0059] The enzyme (E1) is capable of converting 3-hydroxyalkanoyl-CoA / ACP into 3-hydroxyalkanoyl-3-hydroxyalkanoyl-CoA / ACP and further into 3-(3-hydroxyalkanoyloxy)alkanoic acid (HAA), wherein E1 is a 3-(3-hydroxyalkanoyloxy)alkanoic acid (HAA) synthase.

[0060] The cell according to another aspect of the present invention may be a non-pathogenic cell that has been genetically modified to increase heterologous expression of enzyme (E2) and enzyme (E1) relative to wild-type cells,

[0061] The enzyme (E2) is capable of converting 3-hydroxyalkanoyl-3-hydroxyalkanoyl-CoA / ACP or HAA into β-D-glucopyranosyl-3-hydroxyalkanoyl-3-hydroxyalkanoate, wherein E2 is a glycosyltransferase (EC2.4) comprising SEQ ID NO: 4;

[0062] The enzyme (E1) is capable of converting 3-hydroxyalkanoyl-CoA / ACP into 3-hydroxyalkanoyl-3-hydroxyalkanoyl-CoA / ACP and further into 3-(3-hydroxyalkanoyloxy)alkanoic acid (HAA), wherein E1 is a 3-(3-hydroxyalkanoyloxy)alkanoic acid (HAA) synthase comprising SEQ ID NO: 2.

[0063] The cell according to any aspect of the present invention may be a non-pathogenic cell which has been genetically modified to increase heterologous expression of the enzyme (E2) and the enzyme (E1) relative to a wild-type cell,

[0064] The enzyme (E2) is capable of converting 3-hydroxyalkanoyl-3-hydroxyalkanoyl-CoA / ACP or 3-(3-hydroxyalkanoyloxy)alkanoic acid (HAA) into β-D-glucopyranosyl-3-hydroxyalkanoyl-3-hydroxyalkanoate, wherein E2 is a glycosyltransferase (EC 2.4);

[0065] The enzyme (E1) is capable of converting 3-hydroxyalkanoyl-CoA / ACP into 3-hydroxyalkanoyl-3-hydroxyalkanoyl-CoA / ACP and further into HAA, wherein E1 is a 3-(3-hydroxyalkanoyloxy)alkanoic acid (HAA) synthase comprising SEQ ID NO:6.

[0066] The cell according to one further aspect of the invention may be a non-pathogenic cell which has been genetically modified to increase heterologous expression of the enzyme (E2) and the enzyme (E1) relative to a wild-type cell,

[0067] The enzyme (E2) is capable of converting 3-hydroxyalkanoyl-3-hydroxyalkanoyl-CoA / ACP or HAA into β-D-glucopyranosyl-3-hydroxyalkanoyl-3-hydroxyalkanoate, wherein E2 is a glycosyltransferase (EC 2.4);

[0068] The enzyme (E1) is capable of converting 3-hydroxyalkanoyl-CoA / ACP into 3-hydroxyalkanoyl-3-hydroxyalkanoyl-CoA / ACP and further into 3-(3-hydroxyalkanoyloxy)alkanoic acid (HAA), wherein E1 is a 3-(3-hydroxyalkanoyloxy)alkanoic acid (HAA) synthase comprising SEQ ID NO: 8.

[0069] The cell according to another aspect of the present invention may be a non-pathogenic cell that has been genetically modified to increase heterologous expression of enzyme (E2) and enzyme (E1) relative to wild-type cells,

[0070] The enzyme (E2) is capable of converting 3-hydroxyalkanoyl-3-hydroxyalkanoyl-CoA / ACP or HAA into β-D-glucopyranosyl-3-hydroxyalkanoyl-3-hydroxyalkanoate, wherein E2 is a glycosyltransferase (EC 2.4);

[0071] The enzyme (E1) is capable of converting 3-hydroxyalkanoyl-CoA / ACP into 3-hydroxyalkanoyl-3-hydroxyalkanoyl-CoA / ACP and further into 3-(3-hydroxyalkanoyloxy)alkanoic acid (HAA), wherein E1 is a 3-(3-hydroxyalkanoyloxy)alkanoic acid (HAA) synthase comprising SEQ ID NO: 10.

[0072] The cell according to another aspect of the present invention may be a non-pathogenic cell that has been genetically modified to increase heterologous expression of enzyme (E2) and enzyme (E1) relative to wild-type cells,

[0073] The enzyme (E2) is capable of converting 3-hydroxyalkanoyl-3-hydroxyalkanoyl-CoA / ACP or HAA into β-D-glucopyranosyl-3-hydroxyalkanoyl-3-hydroxyalkanoate, wherein E2 is a glycosyltransferase (EC2.4) comprising SEQ ID NO: 4;

[0074] The enzyme (E1) is capable of converting 3-hydroxyalkanoyl-CoA / ACP into 3-hydroxyalkanoyl-3-hydroxyalkanoyl-CoA / ACP and further into 3-(3-hydroxyalkanoyloxy)alkanoic acid (HAA), wherein E1 is a 3-(3-hydroxyalkanoyloxy)alkanoic acid (HAA) synthase comprising SEQ ID NO: 12.

[0075] The cell according to another aspect of the present invention may be a non-pathogenic cell that has been genetically modified to increase heterologous expression of enzyme (E2) and enzyme (E1) relative to wild-type cells,

[0076] The enzyme (E2) is capable of converting 3-hydroxyalkanoyl-3-hydroxyalkanoyl-CoA / ACP or HAA into β-D-glucopyranosyl-3-hydroxyalkanoyl-3-hydroxyalkanoate, wherein E2 is a glycosyltransferase (EC2.4) comprising SEQ ID NO: 4;

[0077] The enzyme (E1) is capable of converting 3-hydroxyalkanoyl-CoA / ACP into 3-hydroxyalkanoyl-3-hydroxyalkanoyl-CoA / ACP and further into 3-(3-hydroxyalkanoyloxy)alkanoic acid (HAA), wherein E1 is a 3-(3-hydroxyalkanoyloxy)alkanoic acid (HAA) synthase comprising SEQ ID NO: 14.

[0078] The cells according to any aspect of the invention may be used to produce lipids according to formula I and / or II from carbon substrates:

[0079] Formula I

[0080] Formula II

[0081] Wherein R in the general formula I or II 1 and R 2 are independently the same or different alkyl groups having 5 to 13 carbon atoms. Specifically, the alkyl group may be saturated or unsaturated. More specifically, R 1 and / or R 2 The alkyl group of R may be a monounsaturated alkyl residue. Even more specifically, R 1 and / or R 2 It may be selected from pentenyl, heptenyl, nonenyl, undecenyl, tridecenyl and (CH2) n -CH3, where n=4-12.

[0082] The lipid formed may be a combination of lipids of formula I and II with different R groups that can be produced in a single reaction process.

[0083] For the purpose of producing the above-mentioned product, can make the cell according to genetic modification of the present invention contact with nutrient medium continuously or discontinuously in batch process (batch culture) or in fed-batch process (fed-batch process) or repeated fed-batch process (repeated fed-batch process), and cultivate like this.As described in GB-A-1009370, also semi-continuous process can be considered.In the textbook of Chmiel (" Bioprozesstechnik 1. Einführung in die Bioverfahrenstechnik " [introduction of bioprocess technology 1. bioprocess technology] (Gustav FischerVerlag, Stuttgart, 1991)) or in the textbook of Storhas (" Bioreaktoren und periphere Einrichtungen " [bioreactor and surrounding device], Vieweg Verlag, Brunswick / Wiesbaden, 1994), the summary of known culture method has been described.

[0084] The culture medium to be used must be adapted to the requirements of the respective strain. For example, a description of culture media for different yeast strains is contained in "Nonconventional yeast in biotechnology" (Klaus Wolf, ed., Springer-Verlag Berlin, 1996).

[0085] The carbon source used as a substrate according to any aspect of the present invention can be selected from carbohydrates, for example, glucose, sucrose, arabinose, xylose, lactose, fructose, maltose, molasses, starch, cellulose and hemicellulose, plant and animal oils and fats, for example, soybean oil, safflower oil, peanut oil, hemp seed oil, jatropha oil, coconut fat, gourd oil, linseed oil, corn oil, poppy seed oil, evening primrose oil, olive oil, palm kernel oil, palm oil, rapeseed oil, sesame oil, sunflower oil, grape seed oil, walnut oil, wheat germ oil and coconut oil, fatty acids, For example, sad, capric acid, lauric acid, myristic acid, palmitic acid, palmitoleic acid, stearic acid, arachidonic acid, behenic acid, oleic acid, linoleic acid, linolenic acid, gamma-linolenic acid and its methyl or ethyl ester and fatty acid mixture, monoglyceride, diglyceride and triglyceride containing the fatty acid just mentioned, alcohol, for example, glycerine, ethanol and methanol, hydrocarbon such as methane, ethane, propane or butane carbonaceous gas and gas mixture, such as CO, CO , synthesis gas or flue gas, amino acid such as L-glutamate or L-valine, or organic acid for example acetic acid.These materials can be used alone or as mixture.As described in US 6,136,576, use carbohydrate, especially monosaccharide, oligosaccharide or polysaccharide as carbon source, and use hydrocarbon, especially alkane, alkene and alkyne. Specifically, the carbon source can be selected from glucose, dextrose, sucrose, polysaccharides such as cellulose or hemicellulose, vegetable oils, animal fats, fatty acids, fatty acid esters, carbon-containing gases, alkanes, glycerol, acetate, ethanol and methanol. More specifically, the carbon source can be selected from glucose, sucrose, glycerol, vegetable oils, methane, ethane and butane. A great advantage of the present invention is that the cells according to the present invention are able to form lipids with general formula I and / or II from the simplest carbon sources such as glucose, sucrose or glycerol, thereby making it unnecessary to provide a long-chain C source in the culture medium during the method according to any aspect of the present invention.

[0086] According to one aspect of the present invention there is provided a method for producing at least one lipid having the general formula II:

[0087]

[0088] Formula II

[0089] Where R 1 and R 2 are independently the same or different alkyl groups having 5 to 13 carbon atoms, and

[0090] Wherein the method comprises the step of contacting at least one cell according to any aspect of the present invention with at least one carbon source.

[0091] Specifically, R 1 and / or R 2The alkyl group of R may be saturated or unsaturated. 1 and / or R 2 The alkyl group of R may be a monounsaturated alkyl residue. Even more specifically, R 1 and / or R 2 It may be selected from pentenyl, heptenyl, nonenyl, undecenyl, tridecenyl and (CH2) n -CH3, where n=4-12.

[0092] The method according to any aspect of the invention may also be used to produce additional lipids having the general formula I from carbon substrates,

[0093]

[0094] Formula I

[0095] Where R 1 and R 2 are independently the same or different alkyl groups having 5 to 13 carbon atoms. 1 and / or R 2 The alkyl group of R may be saturated or unsaturated. 1 and / or R 2 The alkyl group of R may be a monounsaturated alkyl residue. Even more specifically, R 1 and / or R 2 It may be selected from pentenyl, heptenyl, nonenyl, undecenyl, tridecenyl and (CH2) n -CH3, where n=4-12.

[0096] The method according to any aspect of the present invention can be used to produce a mixture of lipids, which comprises the lipids in general formula I and II. Specifically, the lipids of general formula I and II are produced in a ratio of 1:100, 1:90, 1:80, 1:70, 1:60, 1:50, 1:40, 1:30, 1:20, 1:10, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 10:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1 or 100:1. More particularly, the lipids of formula I and II can have alkyl groups of different lengths that exist simultaneously in the R subunits.

[0097] According to another aspect of the present invention, there is provided the use of a cell according to any aspect of the present invention for producing at least one lipid having the general formula I and / or II:

[0098] Formula I

[0099] Formula II

[0100] Where R 1 and R 2 are independently the same or different alkyl groups having 5 to 13 carbon atoms. 1 and / or R 2 The alkyl group of R may be saturated or unsaturated. 1 and / or R 2 The alkyl group of R may be a monounsaturated alkyl residue. Even more specifically, R 1 and / or R 2 It may be selected from pentenyl, heptenyl, nonenyl, undecenyl, tridecenyl and (CH2) n -CH3, where n=4-12. Example

[0101] The foregoing describes a preferred embodiment, which, as will be appreciated by those skilled in the art, may be subject to changes or modifications in design, construction or operation without departing from the scope of the claims. For example, these changes are intended to be covered by the scope of the claims.

[0102] Example 1

[0103] Construction of the Expression Vector of the Gene rbwAB from Serratia rubrum

[0104] To heterologously express genes from Serratia rubrum wxya (SEQ ID NO. 1) as enzyme E1 and RBW (SEQ ID NO. 3) as enzyme E2, and the plasmid pACYC_rbwAB_Srub was constructed. The plasmids encoding 3-(3'-hydroxyalkanoyloxy)alkanoic acid (HAA) synthase (RbwA, SEQ ID NO: 2) and glucosyltransferase (RbwB, SEQ ID NO: 4) were respectively inserted into the plasmid pACYC_rbwAB_Srub. rbwAB_Srub The synthetic operon consisting of (SEQ ID NO: 15) is under the control of the rhamnose-inducible promoter P rha The operon was cloned into the vector pACYCATh-5, which is based on pAYCY184 (New England Biolabs, Frankfurt / Main, Germany) under the control of . The terminator sequence is located downstream of the synthetic operon. S. rubidaea ) genomic DNA amplification of the gene. P RhaPromoter cassette (SEQ ID NO: 16) and terminator sequence (SEQ ID NO: 17). Plasmid pACYCATh-5 carries the p15A replication origin for E. coli and the pVS1 replication origin for replication in Pseudomonas putida KT2440. pVS1 origin is derived from Pseudomonas plasmid pVS1 (Itoh Y, et al. Plasmid 1984, 11(3), 206-20). wxya and RBW The optimized operon was generated by cross PCR fusion. For amplification, Phusion from New England Biolabs (Frankfurt / Main, Germany) was used according to the manufacturer's instructions. TM High-Fidelity Master Mix. In the next step, restriction sites are used Apa I / Psp XI The fusion construct is cloned into the vector pACYCATh-5. The connected product is transformed into chemically competent Escherichia coli DH5α cells (New England Biolabs, Frankfurt / Main, Germany). The procedures for PCR purification, cloning and transformation are carried out according to the manufacturer's manual. The correct insertion of the target gene is checked by restriction analysis, and the authenticity of the introduced DNA fragment is verified by DNA sequencing. The resulting plasmid is named pACYC_rbwAB_Srub (SEQ ID NO: 18).

[0105] By means of electroporation (Iwasaki K, et al., Biosci. Biotech. Biochem . 1994. 58(5):851-854)) Pseudomonas putida strain KT2440 was transformed with plasmid pACYC_rbwAB_Srub and plated on LB-agar plates supplemented with kanamycin (50µg / mL). The transformants were checked for the presence of the correct plasmid by plasmid preparation and restriction analysis. The resulting strain was named BS-PP-360 (Pseudomonas putida KT2440 pACYC_rbwAB_Srub).

[0106] Example 2

[0107] Construction of expression vector of gene rbwA from Serratia rubrum

[0108] To heterologously express genes from Serratia rubrum wxya (SEQ ID NO: 1), the plasmid pACYC_rbwA_Srub was constructed. N I and XOtI cut the plasmid pACYC_rbwAB_Srub (see Example 1) to eliminate RBW To reconnect the modified vector, the plasmid was treated with T4 DNA polymerase (New England Biolabs, Frankfurt / Main, Germany) to remove the 3' overhang and fill the 5' overhang to form a blunt end. The reconnected product was transformed into chemically competent Escherichia coli DH5α cells (New England Biolabs, Frankfurt / Main, Germany). The PCR purification, cloning and transformation procedures were performed according to the manufacturer's manual. The correct insertion of the target gene was checked by restriction analysis, and the authenticity of the introduced DNA fragment was verified by DNA sequencing. The resulting plasmid was named pACYC_rbwA_Srub (SEQ ID NO: 19).

[0109] By means of electroporation (Iwasaki K, et al. Biosci. Biotech. Biochem. 1994. 58(5):851-854) Pseudomonas putida strain KT2440 was transformed with plasmid pACYC_rbwA_Srub and plated on LB-agar plates supplemented with kanamycin (50 μg / mL). The transformants were checked for the presence of the correct plasmid by plasmid preparation and restriction analysis. The resulting strain was named BS-PP-433 (Pseudomonas putida KT2440 pACYC_rbwA_Srub).

[0110] Example 3

[0111] Pseudomonas aeruginosa Construction of expression vectors for gene rhlA and gene rbwB of Serratia rubrum

[0112] For genes from Pseudomonas aeruginosa rhA (SEQ ID NO: 5) and from Serratia rubrum RBW The plasmid pACYC_rhlA_ParbwB_Srub was constructed by heterologous expression of 3-(3-hydroxyalkanoyloxy)alkanoic acid (HAA) synthase (RhlA, SEQ ID NO: 6) and glucosyltransferase (RbwB, SEQ ID NO: 4). rhlA_Pa (SEQ ID NO: 20) is a synthetic operon composed of a rhamnose-inducible promoter P rha The terminator sequence was located downstream of the synthetic operon. The gene was amplified by PCR from genomic DNA of Pseudomonas aeruginosa and Serratia rubrum, respectively. RhaThe promoter cassette (SEQ ID NO: 16) and the terminator sequence (SEQ ID NO: 17). The vector is based on pACYC184 (New England Biolabs, Frankfurt / Main, Germany) and carries the p15A replication origin for Escherichia coli and the pVS1 replication origin for replication in Pseudomonas putida KT2440. The pVS1 origin is derived from the Pseudomonas plasmid pVS1 (Itoh Y, Watson JM, Haas D, Leisinger T, Plasmid 1984, 11 (3), 206-20). rhA and RBW The optimized operon was generated by cross PCR fusion. For amplification, Phusion from NewEngland Biolabs (Frankfurt / Main, Germany) was used according to the manufacturer's instructions. TM High-fidelity master mix. In the next step, restriction sites are used Apa I / Psp XI The fusion construct is cloned into the vector pACYCATh-5. The connected product is transformed into chemically competent Escherichia coli DH5α cells (New England Biolabs, Frankfurt / Main, Germany). The procedures for PCR purification, cloning and transformation are carried out according to the manufacturer's manual. The correct insertion of the target gene is checked by restriction analysis, and the authenticity of the introduced DNA fragment is verified by DNA sequencing. The resulting plasmid is named pACYC_rhlA_ParbwB_Srub (SEQID NO: 21).

[0113] By means of electroporation (Iwasaki K, et al., Biosci. Biotech. Biochem. 1994. 58(5):851-854)) Pseudomonas putida strain KT2440 was transformed with plasmid pACYC_rhlA_ParbwB_Srub and plated on LB-agar plates supplemented with kanamycin (50 μg / mL). The transformants were checked for the presence of the correct plasmid by plasmid preparation and restriction analysis. The resulting strain was named BS-PP-368 (Pseudomonas putida KT2440 pACYC_rhlA_ParbwB_Srub).

[0114] Example 4

[0115] strain BS-PP-433 ( Pseudomonas putida KT2440 pACYC_rbwA_Srub) produces lipid R1

[0116] For the production of lipid R1, a DASGIP® parallel bioreactor system from Eppendorf (Hamburg, Germany) was used. Fermentation was performed using a 1 L reactor. pH and pO2 were measured online for process monitoring. OTR / CTR measurements were used, among other things, to estimate metabolic activity and cell fitness.

[0117] The pH electrode was calibrated by two-point calibration using standard solutions of pH 4.0 and pH 7.0 as specified in the technical description of DASGIP. The reactor was equipped with the necessary sensors and connections specified in the technical description and fitted with a stirrer shaft. The reactor was then filled with 300 ml of water and autoclaved at 121 °C for 20 min to ensure sterility. The pO2 electrode was connected to the measuring amplifier and polarized overnight (at least 6 h). Afterwards, the water was removed under a clean bench and the culture medium was replaced with fermentation medium (2.2 g / L (NH4)2SO4, 0.02 g / L NaCl, 0.4 g / L MgSO4 x 7H2O, 0.04 g / L CaCl2 x 2H2O, separately sterilized: 2 g / L KH2PO4, 8.51 g / L KH2PO4, 20 g / L glucose, 10 mL / L trace element solution M12 (sterile filtered: 0.2 g / L ZnSO4 x 7 H2O, 0.1 g / L MnCl2 x 4H2O, 1.5 g / L Na3-Citrat x 2 H2O,0.1 g / L CuSO4 x 5 H2O, 0.002 g / L NiCl2 x 6 H2O, 0.003 g / L Na2MoO4 x 2 H2O, 0.03 g / L H3BO3, 1 g / L FeSO4 x 7 H2O). Thereafter, the pO2 electrode was calibrated to 100% by single-point calibration (stirrer: 600 rpm / aeration, 10 sl / h air) and the feed, calibrant and inducer lines were cleaned by "cleaning in place" as specified in the technical instructions. For this purpose, the lines were first flushed with 70% ethanol, then with 1 M NaOH, then with sterile, completely demineralized water and finally filled with the corresponding medium.

[0118] Using BS-PP-433 (Pseudomonas putida strain KT2440 pACYC_rbwA_Srub), 25 ml of LB1 medium (10 g / L tryptone, 5 g / L yeast extract, 1 g / L NaCl, pH 7.0) supplemented with kanamycin (50 µg / mL) in a baffled shake flask was inoculated with 100 µl of the glycerol stock solution and incubated overnight at 30°C and 200 rpm for approximately 18 h. The first preculture was used to inoculate 50 ml seed medium in a 500 ml baffled shake flask (autoclaved: 4.4 g / L Na2HPO4 * 2 H2O, 1.5 g / L KH2PO4, 1 g / L NH4Cl, 10 g / L yeast extract, separately sterilized: 20 g / L glucose, 0.2 g / L MgSO4 * 7 H2O, 0.006 g / L FeCl3, 0.015 g / L CaCl2, 1 ml / L trace element solution SL6 (sterile filtered: 0.3 g / L H3BO3, 0.2 g / L CoCl2 x 6 H2O, 0.1 g / L ZnSO4 x 7H2O, 0.03 g / L MnCl2 x 4H2O, 0.01 g / L CuCl2 x 2 H2O, 0.03 g / L Na2MoO4 x 2 H2O, 0.02 g / L NiCl2 x 6 H2O) (starting OD 600 0.2).

[0119] The culture was incubated at 200 rpm and 30°C for about 7 h. To inoculate the reactor at an optical density of 0.7, the OD of the second pre-culture phase was measured. 600 And calculate the amount of culture required for inoculation.

[0120] With the help of a 30 ml syringe, the required amount of culture was added through the septum into the heat-treated and aerated reactor.

[0121] Use the standard procedure shown in Table 1:

[0122] a)

[0123]

[0124] b)

[0125]

[0126] c)

[0127] script Trigger Start 31% DO (1 / 60h) temperature 37℃ Induced rhamnose 3 h after feeding started Feed trigger 50%DO Feed rate 1.5 [mL / h]

[0128] Table 1. Standard procedures used for heating and venting reactors.

[0129] The pH was adjusted unidirectionally to pH 7.0 with a 12.5% ​​ammonia solution. During the growth phase and bioconversion, the dissolved oxygen (pO2 or DO) in the culture was adjusted to at least 30% by the agitator speed and aeration rate. After inoculation, the DO was reduced from 100% to these 30%, and during the rest of the fermentation, the DO was kept constant.

[0130] Fermentation was carried out in fed batch. Feeding started with a 2.5 g / L*h glucose feed, which consisted of 500 g / L glucose and was triggered by a DO peak indicating the end of the batch phase. 3 h after the start of feeding, the expression of lipid R1 production was induced with 0.2% (w / v) rhamnose. The inducer concentration represents the volume at the start of fermentation. The production of lipid R1 started with induction. At the specified time points, samples were taken from the fermentor to determine the concentration of lipid R1 produced.

[0131] Strain BS-PP-433 produced more 3-(3-hydroxyalkanoyloxy)alkanoic acid (HAA) than a reference strain with an empty plasmid.

[0132] Example 5

[0133] Production of rubiwettin using strain BS-PP-360 (Pseudomonas putida KT2440 pACYC_rbwAB_Srub) RG1

[0134] For the production of rubiwettin RG1, a DASGIP® parallel bioreactor system from Eppendorf (Hamburg, Germany) was used. The fermentation was carried out using a 1 L reactor. pH and pO2 were measured online for process monitoring. OTR / CTR measurements were used, among other things, to estimate metabolic activity and cell fitness.

[0135] The pH electrode was calibrated by two-point calibration using standard solutions of pH 4.0 and pH 7.0 as specified in the technical description of DASGIP. The reactor was equipped with the necessary sensors and connections specified in the technical description and fitted with a stirrer shaft. The reactor was then filled with 300 ml of water and autoclaved at 121 °C for 20 min to ensure sterility. The pO2 electrode was connected to the measuring amplifier and polarized overnight (at least 6 h). Afterwards, the water was removed under a clean bench and the culture medium was replaced with fermentation medium (2.2 g / L (NH4)2SO4, 0.02 g / L NaCl, 0.4 g / L MgSO4 x 7H2O, 0.04 g / L CaCl2 x 2H2O, separately sterilized: 2 g / L KH2PO4, 8.51 g / L KH2PO4, 20 g / L glucose, 10 mL / L trace element solution M12 (sterile filtered: 0.2 g / L ZnSO4 x 7 H2O, 0.1 g / L MnCl2 x 4H2O, 1.5 g / L Na3-Citrat x 2 H2O,0.1 g / L CuSO4 x 5 H2O, 0.002 g / L NiCl2 x 6 H2O, 0.003 g / L Na2MoO4 x 2 H2O, 0.03 g / L H3BO3, 1 g / L FeSO4 x 7 H2O). Thereafter, the pO2 electrode was calibrated to 100% by single-point calibration (stirrer: 600 rpm / aeration, 10 sl / h air) and the feed, calibrant and inducer lines were cleaned by "cleaning in place" as specified in the technical instructions. For this purpose, the lines were first flushed with 70% ethanol, then with 1 M NaOH, then with sterile, completely demineralized water and finally filled with the corresponding medium.

[0136] Using P. putida strain BS-PP-360, 25 ml of LB1 medium (10 g / L tryptone, 5 g / L yeast extract, 1 g / L NaCl, pH 7.0) supplemented with kanamycin (50 µg / mL) in a baffled shake flask was inoculated with 100 µl of the glycerol stock solution and incubated overnight at 30°C and 200 rpm for approximately 18 h. The first preculture was used to inoculate 50 ml seed medium in a 500 ml baffled shake flask (autoclaved: 4.4 g / L Na2HPO4 * 2 H2O, 1.5 g / L KH2PO4, 1 g / L NH4Cl, 10 g / L yeast extract, separately sterilized: 20 g / L glucose, 0.2 g / L MgSO4 *7 H2O, 0.006 g / L FeCl3, 0.015 g / L CaCl2, 1 ml / L trace element solution SL6 (sterile filtered: 0.3 g / L H3BO3, 0.2 g / L CoCl2 x 6 H2O, 0.1 g / L ZnSO4 x 7 H2O, 0.03 g / L MnCl2 x 4H2O, 0.01 g / L CuCl2 x 2 H2O, 0.03 g / L Na2MoO4 x 2 H2O, 0.02 g / L NiCl2 x 6 H2O)(starting OD 600 0.2). The culture was incubated at 200 rpm and 30°C for about 7 h. To inoculate the reactor at an optical density of 0.7, the OD of the second pre-culture stage was measured. 600 And calculate the amount of culture required for inoculation.

[0137] With the help of a 30 ml syringe, the required amount of culture was added through the septum to the heat treated and aerated reactor.The standard procedure shown in Table 1 of Example 4 was used.

[0138] The pH was adjusted unidirectionally to pH 7.0 with a 12.5% ​​concentration ammonia solution. During the growth phase and bioconversion, the dissolved oxygen (pO2 or DO) in the culture was adjusted to at least 30% by the agitator speed and aeration rate. After inoculation, the DO dropped from 100% to these 30%, and the DO remained stable during the rest of the fermentation.

[0139] Fermentation was performed in fed-batch. Feeding started with a 2.5 g / L*h glucose feed, which consisted of 500 g / L glucose and was triggered by a DO peak indicating the end of the batch phase. 3 h after the start of feeding, the expression of rubiwettin production was induced with 0.2% (w / v) rhamnose. The inducer concentrations represent the volume at the start of the fermentation. For both sugars, a stock solution of 220 g / L was used. The production of rubiwettin RG1 started with induction. At the indicated time points, samples were taken from the fermentor to determine the concentration of rubiwettin produced. After 65 h of fermentation, 0.53 g / L rubiwettin RG1 was produced.

[0140] Example 6

[0141] strain BS-PP-368 ( Pseudomonas putida KT2440 pACYC_rhlA_ParbwB_Srub) production rubiwettin RG1

[0142] For the production of rubiwettin RG1, a DASGIP® parallel bioreactor system from Eppendorf (Hamburg, Germany) was used. The fermentation was carried out using a 1 L reactor. pH and pO2 were measured online for process monitoring. OTR / CTR measurements were used in particular to estimate metabolic activity and cell fitness.

[0143] The pH electrode was calibrated by two-point calibration using standard solutions of pH 4.0 and pH 7.0 as specified in the technical description of DASGIP. The reactor was equipped with the necessary sensors and connections specified in the technical description and fitted with a stirrer shaft. The reactor was then filled with 300 ml of water and autoclaved at 121 °C for 20 min to ensure sterility. The pO2 electrode was connected to the measuring amplifier and polarized overnight (at least 6 h). Afterwards, the water was removed under a clean bench and the culture medium was replaced with fermentation medium (2.2 g / L (NH4)2SO4, 0.02 g / L NaCl, 0.4 g / L MgSO4 x 7H2O, 0.04 g / L CaCl2 x 2H2O, separately sterilized: 2 g / L KH2PO4, 8.51 g / L KH2PO4, 20 g / L glucose, 10 mL / L trace element solution M12 (sterile filtered: 0.2 g / L ZnSO4 x 7 H2O, 0.1 g / L MnCl2 x 4H2O, 1.5 g / L Na3-Citrat x 2 H2O,0.1 g / L CuSO4 x 5 H2O, 0.002 g / L NiCl2 x 6 H2O, 0.003 g / L Na2MoO4 x 2 H2O, 0.03 g / L H3BO3, 1 g / L FeSO4 x 7 H2O). Thereafter, the pO2 electrode was calibrated to 100% by single-point calibration (stirrer: 600 rpm / aeration 10 sl / h air) and the feed, calibrant and inducer lines were cleaned by "cleaning in place" as specified in the technical instructions. For this purpose, the lines were first flushed with 70% ethanol, then with 1 M NaOH, then with sterile, completely demineralized water and finally filled with the corresponding medium.

[0144] Using P. putida strain BS-PP-368, 25 ml of LB1 medium (10 g / L tryptone, 5 g / L yeast extract, 1 g / L NaCl, pH 7.0) supplemented with kanamycin (50 µg / mL) in a baffled shake flask was inoculated with 100 µl of the glycerol stock solution and incubated overnight at 30°C and 200 rpm for approximately 18 h. The first preculture was used to inoculate 50 ml seed medium in a 500 ml baffled shake flask (autoclaved: 4.4 g / L Na2HPO4 * 2 H2O, 1.5 g / L KH2PO4, 1 g / L NH4Cl, 10 g / L yeast extract, separately sterilized: 20 g / L glucose, 0.2 g / L MgSO4 *7 H2O, 0.006 g / L FeCl3, 0.015 g / L CaCl2, 1 ml / L trace element solution SL6 (sterile filtered: 0.3 g / L H3BO3, 0.2 g / L CoCl2 x 6 H2O, 0.1 g / L ZnSO4 x 7 H2O, 0.03 g / L MnCl2 x 4H2O, 0.01 g / L CuCl2 x 2 H2O, 0.03 g / L Na2MoO4 x 2 H2O, 0.02 g / L NiCl2 x 6 H2O)(starting OD 600 0.2). The culture was incubated at 200 rpm and 30°C for about 7 h. To inoculate the reactor at an optical density of 0.7, the OD of the second pre-culture stage was measured. 600 And calculate the amount of culture required for inoculation.

[0145] With the help of a 30 ml syringe, the required amount of culture was added to the heat treated and aerated reactor through the septum.The standard procedure shown in Table 1 of Example 4 was used for the heated and aerated reactor.

[0146] The pH was adjusted unidirectionally to pH 7.0 with a 12.5% ​​concentration ammonia solution. During the growth phase and bioconversion, the dissolved oxygen (pO2 or DO) in the culture was adjusted to at least 30% by the agitator speed and aeration rate. After inoculation, the DO dropped from 100% to these 30%, and the DO remained stable during the rest of the fermentation.

[0147] Fermentation was performed in fed-batch. Feeding started with a 2.5 g / L*h glucose feed, which consisted of 500 g / L glucose and was triggered by a DO peak indicating the end of the batch phase. 3 h after the start of feeding, the expression of rubiwettin production was induced with 0.2% (w / v) rhamnose. The inducer concentrations represent the volume at the start of the fermentation. For both sugars, a stock solution of 220 g / L was used. The production of rubiwettin RG1 started with induction. At the indicated time points, samples were taken from the fermentor to determine the concentration of rubiwettin produced. After 65 h of fermentation, 11.1 g / L rubiwettin RG1 was produced.

[0148] Example 7

[0149] HPLC-based quantification of rubiwettin

[0150] Quantification of lipids R1 and RG1 was performed by HPLC. Using a displacement pipette (Combitip), 900 µl of 70% (v / v) n-propanol were introduced into a 2 ml reaction vessel and the reaction vessel was immediately closed to reduce evaporation. 100 µl of fermentation broth was subsequently added. After shaking at 30 Hz for 1 min in a Retsch grinder, the resulting crude extract mixture was centrifuged at 13,000 rpm for 5 min and 800 µl of the clear supernatant was transferred into an HPLC vial. Further dilutions of the cell broth were performed in 55% (v / v) propanol. The samples were stored at -20 °C before measurement.

[0151] For the detection and quantification of lipids, an evaporative light scattering detector (Sedex LT-ELSD Model 85LT) was used. The measurements were performed with the aid of an Agilent Technologies 1200 Series (Santa Clara, Calif.) and a Zorbax SB-C8 Rapid Resolution column (4,6 x 150 mm, 3,5µm, Agilent). The injection volume was 5.0µl and the run time was 20 min. Mobile phase A: 0.1% TFA in water (trifluoroacetic acid, solution); mobile phase B: methanol. The column temperature was 40°C. ELSD (detector temperature 60°C) and DAD (diode array, 210 nm) were used as detectors.

[0152] gradient:

[0153] t [min] Flow rate [1ml / min] 0.00 70% 1.00 15.00 100% 1.00 15.01 70% 1.00 20.00 70% 1.00

[0154] Table 2. Mobile phase gradients of A and B over time.

[0155] The gradient used was from 70% B in A to 100% B in 15 min at a flow rate of 1 mL / min, followed by re-equilibration with 70% B in A for 5 min (see Table 2). Reference materials were used and their identity and purity were checked by HPLC-MS / MS and NMR.

[0156] Example 8

[0157] For production Construction of Agrobacterium tumefaciens strains expressing rubiwettin R1 and rubiwettin RG1

[0158] To demonstrate the production of rubiwettin by another microbial species, Agrobacterium tumefaciens, we prepared electrocompetent cells of A. tumefaciens LBA 4404 and expressed the plasmid pACYC_rbwA_Srub (SEQ ID NO: 19) , pACYC_ rbwAB_Srub (SEQ ID NO: 18) and pACYC_rhlA_ParbwB_Srub (SEQ ID NO: 21) were used to transform it.

[0159] For this purpose, freshly grown cells of Agrobacterium tumefaciens LBA 4404 (1-2 days old) were plated on LB agar plates (90 mm diameter, 10 g / L tryptone, 5 g / L yeast extract, 5 g / L NaCl, and 15 g / L agar supplemented with 50 μg / mL rifampicin) and incubated overnight (about 16 h) at 27°C to produce a bacterial lawn that completely covered the plate surface.

[0160] Carefully wash the bacterial cells off the plate with 4 mL of ice-cold 10% (v / v) sterile glycerol. Use an inoculation loop to scrape off the cells growing on the surface of the plate, avoiding damaging the agar medium, and suspend them in the glycerol solution. Then transfer the bacterial suspension into two sterile 2 mL centrifuge tubes.

[0161] The suspensions in both tubes were centrifuged at 14,000 rpm (18,000 g) for 1 min at 4°C; the supernatant was discarded.

[0162] 1 mL of ice-cold 10% (v / v) sterile glycerol was added to each tube containing the bacterial pellet. The tubes were then vortexed thoroughly to resuspend the cells, and this wash step was repeated one more time.

[0163] After two centrifugation steps, the supernatant was removed and discarded again, and the bacterial pellets from both tubes were resuspended in 200 μl of ice-cold 10% (v / v) sterile glycerol each and combined in one tube (yielding a total of 400 μl).

[0164] Keep the tube containing the Agrobacterium cell suspension on ice until electroporation.

[0165] For electroporation, mix 70-80 μl of an ice-cold suspension of electrocompetent bacterial cells with 1-3 μl of plasmid DNA (1-100 ng) in a sterile centrifuge tube.

[0166] The mixture was loaded into a cooled electroporation vessel (gap = 2 mm) and placed in the vessel holder. An electroporator (Gene Pulser Xcell™ Microbial Electroporation Systems; Bio-Rad) was used with the following parameters: 2.5 kV, 25 μF capacitance, and 400 Ohm resistance.

[0167] Immediately add 1 mL of SOC medium (20 mM glucose, 20 g / L tryptone, 5 g / L yeast extract, 10 mM NaCl, 2.5 mM MgCl2, and 10 mM MgSO4) to the electroporation vessel, transfer the resulting bacterial suspension into a 15 mL centrifuge tube, and incubate the tube at 27°C with rotation for 1 h.

[0168] After incubation, 100 μl of each electroporated cell suspension was spread on LB plates supplemented with kanamycin (50 μg / mL). The plates were incubated at 27°C for 2 days, and successfully transformed colonies were confirmed by plasmid isolation and analytical restriction digestion.

[0169] The following strains were produced:

[0170] Agrobacterium tumefaciens LBA 4404 pACYC_rbwA_Srub for production of rubiwettin R1

[0171] Agrobacterium tumefaciens LBA 4404 pACYC_rbwAB_Srub for production of rubiwettin RG1

[0172] Agrobacterium tumefaciens LBA 4404 pACYC_rhlA_ParbwB_Srub for production of rubiwettin RG1.

[0173] Example 9

[0174] Production of rubiwettin R1 by Agrobacterium tumefaciens LBA 4404 pACYC_rbwA_Srub and LBA 4404 pACYC_rbwAB_Srub and Agrobacterium tumefaciens LBA 4404 pACYC_rhlA_ParbwB_Srub Rubiwettin RG1

[0175] For the production of rubiwettin R1 and RG1, a DASGIP® parallel bioreactor system from Eppendorf (Hamburg, Germany) was used. Fermentation was performed using a 1 L reactor. pH and pO2 were measured online for process monitoring. OTR / CTR measurements were used in particular to estimate metabolic activity and cell fitness.

[0176] The pH probe was calibrated by two-point calibration using measuring solutions of pH 4.0 and pH 7.0 according to the technical reference of DASGIP. The reactor was provided with the required sensors and connections according to the technical reference and the stirrer shaft was installed. The reactor was then filled with 300 ml of water and autoclaved at 121 °C for 20 min to ensure sterility. The pO2 probe was connected to the measuring amplifier and polarized overnight (at least 6 hours). The water was then removed under a clean bench and replaced with a high cell density medium consisting of (NH4)2SO4 1.76 g / l, K2HPO4 19.08 g / l, KH2PO4 12.5 g / l, yeast extract 6.66 g / l, trisodium citrate dihydrate 11.2 g / l, 17 ml / l of filter-sterilized 1% concentration ammonium ferric citrate solution and 5 ml / l of filter-sterilized trace element stock solution consisting of HCl (37%) 36.50 g / l, MnCl2*4H2O 1.91 g / l, ZnSO4*7H2O 1.87 g / l, EDTA dihydrate 0.84 g / l, H3BO3 0.30 g / l, Na2MoO4*2H2O 0.25 g / l, CaCl2*2H2O 4.70 g / l, FeSO4*7H2O 17.80 g / l, CuCl2*2H2O 0.15 g / l), which contained 15 g / l glucose as carbon source (added by metering 30 ml / l of a sterile feed solution consisting of 500 g / l glucose, 1% (w / v) MgSO4*7H2O and 2.2% (w / v) NH4Cl) and 50 mg / l kanamycin.

[0177] Subsequently, the pO2 probe was calibrated to 100% using a single-point calibration (stirrer: 600 rpm / aeration: 10 sL / h air) and the feed, calibrator and inducer lines were cleaned by cleaning in place according to the technical reference. To this end, the lines were first flushed with 70% ethanol, then with 1 M NaOH, then with sterile demineralized water and finally filled with the corresponding medium.

[0178] To produce rubiwettin R1 with Agrobacterium tumefaciens LBA 4404 pACYC_rbwA_Srub and rubiwettin RG1 with Agrobacterium tumefaciens LBA 4404 pACYC_rbwAB_Srub and Agrobacterium tumefaciens LBA 4404 pACYC_rhlA_ParbwB_Srub, the three strains were first grown overnight from frozen cultures in LB medium (25 ml in a 100 ml baffled shake flask) containing 50 mg / l kanamycin at 28°C and 200 rpm for about 18 h. Then, 2 ml of this culture was transferred to the second pre-culture stage into 25 ml of high cell density medium in a 100 ml shake flask, which consisted of (NH4)2SO4 1.76 g / L, K2HPO4 19.08 g / l, KH2PO4 12.5 g / l, yeast extract 6.66 g / l, trisodium citrate dihydrate 11.2 g / l, 17 ml / l of filter-sterilized 1% strength ammonium ferric citrate solution and 5 ml / l of filter-sterilized trace element stock solution (consisting of HCl (37%) 36.50 g / l, MnCl2*4H2O 1.91 g / l, ZnSO4*7H2O 1.87 g / l, EDTA dihydrate 0.84 g / l, H3BO3 0.30 g / l. Na2MoO4*2H2O0.25 g / l, CaCl2*2H2O 4.70 g / l, FeSO4*7H2O 17.80 g / l, CuCl2*2H2O 0.15 g / l), contained 15 g / l glucose as carbon source (added by metering 30 ml / l of a sterile feed solution consisting of 500 g / l glucose, 1% (w / v) MgSO4*7H2O and 2.2% (w / v) NH4Cl), contained the antibiotics already described, and incubated at 28°C / 200 rpm for a further 6 h.

[0179] To inoculate the reactor at an optical density of 0.1, measure the OD of the second pre-culture stage. 600 , and calculate the amount of culture required for inoculation. With the help of a 5 ml syringe, add the required amount of culture through the septum to the heat-treated and aerated reactor.

[0180] The standard procedure used is shown in Table 3:

[0181]

[0182]

[0183] script Trigger Start 31% DO (1 / 60h) Induced rhamnose 3 h after feeding started Feed trigger 50%DO Feed rate 1 [ml / h]

[0184] Table 3. Standard procedure for the production of rubiwettin using Agrobacterium strains.

[0185] The pH was adjusted to 6.8 in one direction with a 12.5% ​​ammonia solution. During the cultivation and bioconversion, the dissolved oxygen (pO2 or DO) in the culture was adjusted to at least 30% by the agitator speed and the aeration rate. After inoculation, the DO dropped from 100% to this 30% and remained stable during the rest of the fermentation. The temperature was kept stable at 28°C.

[0186] Fermentation was carried out in fed-batch, where the feed start was triggered by 1.5 g / l*h glucose feed consisting of 500 g / l glucose, 1% (w / v) MgSO4*7H2O and 2.2% (w / v) NH4Cl as delivery to the feed phase, and the end of the batch phase was induced by a DO peak. Rubiwettin production was induced with 0.2% (w / v) rhamnose 3 h after the start of the feed. The inducer concentrations represent the volume at the start of the fermentation. A 220 g / L rhamnose stock solution was used.

[0187] Quantification of the formation of rubiwettin R1 and RG1 was performed as described in Example 7.

[0188] It was confirmed that Agrobacterium tumefaciens LBA 4404 pACYC_rbwA_Srub produces rubiwettin R1.

[0189] It was also confirmed that both Agrobacterium tumefaciens LBA 4404 pACYC_rbwAB_Srub and Agrobacterium tumefaciens LBA 4404 pACYC_rhlA_ParbwB_Srub produced rubiwettin RG1.

[0190] Example 10

[0191] Construction of Escherichia coli strains for producing rubiwettin R1 and rubiwettin RG1

[0192] Plasmid was electroporated pACYC_rbwA_Srub (SEQ ID NO: 19) 、pACYC_rbwAB_Srub (SEQ ID NO: 18) and pACYC_rhlA_ParbwB_Srub (SEQ ID NO: 21) were transformed into E. coli W3110 and plated on LB agar plates containing kanamycin (50 μg / ml). The presence and authenticity of the plasmids in the transformants were screened by plasmid preparation and restriction enzyme digestion analysis. The following strains were generated:

[0193] E. coli W3110 pACYC_rbwA_Srub for production of rubiwettin R1

[0194] E. coli W3110 pACYC_rbwAB_Srub for production of rubiwettin RG1

[0195] E. coli W3110 pACYC_rhlA_ParbwB_Srub was used to produce rubiwettin RG1.

[0196] Embodiment 11

[0197] Production of rubiwettin R1 using E. coli W3110 pACYC_rbwA_Srub and production of rubiwettin R1 using E. coli W3110 pACYC_rbwAB_Srub and E. coli W3110 pACYC_rhlA_ParbwB_Srub to produce rubiwettin RG1

[0198] For the production of rubiwettin R1 and RG1, a DASGIP® parallel bioreactor system from Eppendorf (Hamburg, Germany) was used. Fermentation was performed using a 1 L reactor. pH and pO2 were measured online for process monitoring. OTR / CTR measurements were used in particular to estimate metabolic activity and cell fitness.

[0199] The pH probe was calibrated by two-point calibration using measuring solutions of pH 4.0 and pH 7.0 according to the technical reference of DASGIP. The reactor was provided with the required sensors and connections according to the technical reference and the stirrer shaft was installed. The reactor was then filled with 300 ml of water and autoclaved at 121 °C for 20 min to ensure sterility. The pO2 probe was connected to the measuring amplifier and polarized overnight (at least 6 hours). The water was then removed under a clean bench and replaced with a high cell density medium consisting of (NH4)2SO4 1.76 g / l, K2HPO4 19.08 g / l, KH2PO4 12.5 g / l, yeast extract 6.66 g / l, trisodium citrate dihydrate 11.2 g / l, 17 ml / l of filter-sterilized 1% concentration ammonium ferric citrate solution and 5 ml / l of filter-sterilized trace element stock solution consisting of HCl (37%) 36.50 g / l, MnCl2*4H2O 1.91 g / l, ZnSO4*7H2O 1.87 g / l, EDTA dihydrate 0.84 g / l, H3BO3 0.30 g / l, Na2MoO4*2H2O0.25 g / l, CaCl2*2H2O 4.70 g / l, FeSO4*7H2O 17.80 g / l, CuCl2*2H2O 0.15 g / l), contained 15 g / l glucose as carbon source (added by metering 30 ml / l of a sterile feed solution consisting of 500 g / l glucose, 1% (w / v) MgSO4*7H2O and 2.2% (w / v) NH4Cl) and 50 mg / l kanamycin.

[0200] Subsequently, the pO2 probe was calibrated to 100% using a single-point calibration (stirrer: 600 rpm / aeration: 10 sL / h air) and the feed, calibrator and inducer lines were cleaned by cleaning in place according to the technical reference. To this end, the lines were first flushed with 70% ethanol, then with 1 M NaOH, then with sterile demineralized water and finally filled with the corresponding medium.

[0201] To produce rubiwettin R1 with E. coli W3110 pACYC_rbwA_Srub and rubiwettinRG1 with E. coli W3110 pACYC_rbwAB_Srub and E. coli W3110 pACYC_rhlA_ParbwB_Srub, the three strains were first grown overnight from frozen cultures in LB medium (25 ml in a 100 ml baffled shake flask) containing 50 mg / l kanamycin at 37°C and 200 rpm for approximately 18 h. Then, 2 ml of this culture was transferred to the second pre-culture stage into 25 ml of high cell density medium in a 100 ml shake flask, which consisted of (NH4)2SO41.76 g / L, K2HPO4 19.08 g / l, KH2PO4 12.5 g / l, yeast extract 6.66 g / l, trisodium citrate dihydrate 11.2 g / l, 17 ml / l of filter-sterilized 1% strength ammonium ferric citrate solution and 5 ml / l of filter-sterilized trace element stock solution (consisting of HCl (37%) 36.50 g / l, MnCl2*4H2O 1.91 g / l, ZnSO4*7H2O 1.87 g / l, EDTA dihydrate 0.84 g / l, H3BO3 0.30 g / l. Na2MoO4*2H2O 0.25 g / l, CaCl2*2H2O4.70 g / l, FeSO4*7H2O 17.80 g / l, CuCl2*2H2O 0.15 g / l), contained 15 g / l glucose as carbon source (added by metering 30 ml / l of a sterile feed solution consisting of 500 g / l glucose, 1% (w / v) MgSO4*7H2O and 2.2% (w / v) NH4Cl), contained the antibiotics already described, and was incubated at 37°C / 200 rpm for a further 6 h.

[0202] To inoculate the reactor at an optical density of 0.1, measure the OD of the second pre-culture stage. 600 , and calculate the amount of culture required for inoculation. With the help of a 5 ml syringe, add the required amount of culture through the septum to the heat-treated and aerated reactor.

[0203] The standard procedure used is shown in Table 4:

[0204]

[0205]

[0206] script Trigger Start 31% DO (1 / 60h) Induced rhamnose 3 h after feeding started Feed trigger 50%DO Feed rate 3 [ml / h]

[0207] Table 4. Standard procedure for the production of rubiwettin using E. coli strains.

[0208] The pH was adjusted to pH 6.8 using a 12.5% ​​ammonia solution. During the culture and bioconversion, the dissolved oxygen (pO2 or DO) in the culture was adjusted to at least 30% by the agitator speed and the aeration rate. After inoculation, the DO dropped from 100% to this 30%, and the DO remained stable during the rest of the fermentation. The temperature was kept stable at 37°C.

[0209] Fermentation was carried out in fed-batch, with feed start triggered by 5 g / l*h glucose feed consisting of 500 g / l glucose, 1% (w / v) MgSO4*7H2O and 2.2% (w / v) NH4Cl as delivery to the feed phase, and the end of the batch phase was induced by a DO peak. Rubiwettin production was induced with 0.2% (w / v) rhamnose 3 h after feed start. Inducer concentrations represent the volume at the start of the fermentation. A 220 g / L rhamnose stock solution was used.

[0210] Quantification of the formation of rubiwettin R1 and RG1 was performed as described in Example 7.

[0211] It was confirmed that E. coli W3110 pACYC_rbwA_Srub produced rubiwettin R1.

[0212] It was also confirmed that both E. coli W3110 pACYC_rbwAB_Srub and E. coli W3110 pACYC_rhlA_ParbwB_Srub produced rubiwettin RG1.

Claims

1. A microbial cell that produces at least one lipid of formula II from at least one carbon substrate, Where R 1 and R 2 independently of one another contain identical or different organic residues each having 5 to 13 carbon atoms, wherein the cell is a non-pathogenic cell that has been genetically modified to increase heterologous expression of an enzyme E2 relative to a wild-type cell, the enzyme E2 being capable of converting 3-hydroxyalkanoyl-3-hydroxyalkanoyl-CoA / ACP and / or 3-(3-hydroxyalkanoyloxy)alkanoic acid (HAA) in combination with NDP-glucose into β-D-glucopyranosyl-3-hydroxyalkanoyl-3-hydroxyalkanoate, wherein the enzyme E2 is a glycosyltransferase (EC 2.4) consisting of SEQ ID NO: 4; The cell is further genetically modified to increase heterologous expression of an enzyme E1 relative to a wild-type cell, wherein the enzyme E1 is capable of converting 3-hydroxyalkanoyl-CoA / ACP into 3-hydroxyalkanoyl-3-hydroxyalkanoyl-CoA / ACP and further into 3-(3-hydroxyalkanoyloxy)alkanoic acid (HAA), wherein the enzyme E1 consists of a sequence selected from SEQ ID NO: 2 or SEQ ID NO:

6.

2. The cell according to claim 1, wherein R in the lipid having the general formula II is a monounsaturated alkyl residue.

3. The cell of claim 2, wherein the alkyl residue is selected from the group consisting of nonenyl, undecenyl and tridecenyl. The cell according to claim 1 , wherein the enzyme E1 is 3-(3-hydroxyalkanoyloxy)alkanoic acid (HAA) synthase.

5. The cell according to any one of claims 1 to 4, wherein the cell is genetically modified to increase the expression of enzyme E2 and enzyme E1, The enzyme E2 consists of SEQ ID NO: 4; and The enzyme E1 consists of SEQ ID NO:

2.

6. The cell of any one of claims 1-4, wherein the cell produces an additional lipid having the general formula I from a carbon substrate, Where R 1 and R 2 They independently contain identical or different organic residues each having 5 to 13 carbon atoms.

7. The cell of claim 6, wherein R in the lipid having the general formula I is a monounsaturated alkyl residue.

8. The cell of any one of claims 1-4 and 7, wherein the carbon substrate is selected from the group consisting of glucose, dextrose, sucrose, polysaccharides, vegetable oils, animal fats, fatty acids, fatty acid esters, carbon-containing gases, alkanes, glycerol, acetate, ethanol and methanol.

9. The cell of any one of claims 1-4 and 7, wherein the cell is selected from the group consisting of: Acinetobacter sp., Bacillus sp., Brevibacterium sp., Burkholderia sp., Chlorella sp., Clostridium sp., Corynebacterium sp., Cyanobacteria, Escherichia sp., Pseudomonas sp., Klebsiella sp., Salmonella sp., Rhizobium sp., Saccharomyces sp., Pichia sp., and Nostoc sp.

10. A method for producing at least one lipid having Formula II and / or Formula I: Where R 1 and R 2 independently of one another contain identical or different organic residues each having 5 to 13 carbon atoms, and wherein the method comprises the step of contacting at least one cell according to any one of claims 1 to 9 with at least one carbon source.

11. Use of a cell according to any one of claims 1 to 9 for producing at least one lipid having the general formula I and / or II: Where R 1 and R 2 They independently contain identical or different organic residues each having 5 to 13 carbon atoms.

Images