Glycolipid production

Abstract

The invention relates to a microbial cell for producing at least one lipid having general formula II from at least one carbon substrate, in which R1 and R2 independently of each other comprise the same or different organic groups each having 5 to 13 carbon atoms, wherein the cell is a non-pathogenic cell genetically modified to increase the heterologous expression of the following enzymes relative to the wild-type cell: Enzyme E1, a 3-(3-hydroxyalkanoyloxy) alkanoic acid (HAA) synthase comprising SEQ ID NO: 1, SEQ ID NO: 7, SEQ ID NO: 11, SEQ ID NO: 15 or a variant thereof wherein the variant comprises a 60% sequence identity to SEQ ID NO: 1, 7, 11 or 15; and-an enzyme E2 comprising a glycosyltransferase (EC 2.4) of SEQ ID NO: 2, SEQ ID NO: 8 or SEQ ID NO: 12 or a variant thereof wherein the variant comprises a 60% sequence identity to SEQ ID NO: 2, 8 or 12.

Description

Technical Field

[0001] This invention relates to recombinant cells and biotechnological methods for producing glycolipids. In particular, the cells are non-pathogenic cells that have been genetically modified to produce at least one glycolipid. Background Technology

[0002] Currently, most available surfactants, such as sodium lauryl ether sulfate (SLES) and betaine, are produced chemically on an industrial scale. These chemically produced surfactants have all the drawbacks typically associated with chemical production processes, such as the formation of harmful byproducts. For example, at least one harmful byproduct, 1,4-dioxane, is generated during SLES production. To reduce the amount of toxic products generated and considering the growing consumer demand for environmentally friendly products, there is a general trend towards the production and use of biosurfactants. Besides generating less toxic byproducts during manufacturing, biosurfactants possess useful properties such as high structural diversity, beneficial surfactant properties, low environmental toxicity, antibiotic or bioactive properties, and complete biodegradability. Therefore, there is a general impetus for the production and use of biosurfactants to replace chemical surfactants.

[0003] Rubiwettin, a class of glycolipids, is at least one example of such biosurfactants. Rubiwettins represent a class of economically interesting surfactants because they have the potential to replace chemically produced surfactants.

[0004] Rubiwettin is composed of hydroxy fatty acid chains linked to C16, which are the main lipid components. 14 Exolipids are 3-hydroxy fatty acid dimers between C8 and C9 composed of a β-D-glucose molecule. They possess surface-active properties. Rubiwettin is currently derived from wild-type... Serratia rubidaea The synthesis of isolates (which are pathogens in both humans and animals) significantly reduces consumer acceptance of these conventionally produced rubiwettins due to the fact that the producing organisms are pathogenic. Furthermore, the production of rubiwettin requires higher safety standards, which increases costs due to increased capital expenditures and potentially additional production steps.

[0005] Current methods for producing biosurfactants such as rubiwettin involve using pathogenic organisms. Production yields can be optimized by altering pH, oxygen supply, culture medium composition, feeding strategy, nitrogen supply, temperature, and substrate selection. However, if rubiwettin is to be used on a large scale as a surfactant, it must compete with currently used surfactants, which are bulk chemicals that can be produced at very low cost. Therefore, rubiwettin must also be produced at the lowest possible cost, without posing a health risk to consumers, and with the most limited properties possible. This cannot be achieved simply by optimizing performance parameters through process optimization.

[0006] WO 2019 / 154984 discloses a non-pathogenic cell that has been genetically modified to introduce a specific enzyme capable of producing rubiwettin from a carbon substrate. However, there remains a need in the art for alternative, more efficient methods to produce rubiwettin in higher product yields. Summary of the Invention

[0007] This invention seeks to address the aforementioned problems by providing a biotechnological means for producing biosurfactants, such as lipids, particularly 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) capable of converting 3-hydroxyalkanoyl-3-hydroxyalkanoyl-CoA / ACP and / or 3-(3-hydroxyalkanoyloxy)alkanoic acid (HAA) in combination with NDP-glucose to β-D-glucopyranosyl-3-hydroxyalkanoyl-3-hydroxyalkanoate, wherein said enzyme E2 is a glycosyltransferase (EC2.4) comprising SEQ ID NO: 1, SEQ ID NO: 7, SEQ ID NO: 11, or SEQ ID NO: 15 or a variant thereof. The genetically modified cells can then be used to convert suitable carbon sources into lipids having the following general formula II: Formula II Where R 1 and R 2 Each alkyl group is independently an identical or different alkyl group having 5 to 13 carbon atoms. In particular, the alkyl group can be saturated or unsaturated. More specifically, R 1 and / or R 2 The alkyl group can be a saturated alkyl group. More specifically, R 1 and / or R 2 It can be selected from pentyl, heptyl, nonyl, undecyl, tridecyl and (CH2). n -CH3, where n=4-12.

[0008] In particular, the cell can be further genetically modified to increase the expression of at least one enzyme (E1) 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 can be a 3-(3-hydroxyalkanoyloxy)alkanoic acid (HAA) synthase comprising SEQ ID NO: 1, SEQ ID NO: 7, SEQ ID NO: 11, SEQ ID NO: 15 or variants thereof.

[0009] Cells according to any aspect of the invention can be used to produce rubiwettin from a carbon source using non-pathogenic cells.

[0010] Lipids of general formula II can also be called glycolipids, more specifically rubiwettin or glycolipids. Genetically modified cells according to any aspect of the invention have the advantages of being non-pathogenic and easy to culture. This makes the production of these cells safer and also keeps costs lower, as no special safety requirements are required in the laboratory during the production and use of rubiwettin. Cells according to any aspect of the invention have the further advantage of being able to produce lipids according to any aspect of the invention using a variety of carbon substrates. For example, simple carbon such as glucose can be used as a carbon substrate. Furthermore, lipids formed according to any aspect of the invention have defined and flexible properties. A further advantage is that rubiwettin can be produced with higher space-time yields, higher carbon yields, higher product concentrations, and higher product uniformity (fatty acid compounds) compared to cells without enhanced activities.

[0011] According to one aspect of the invention, a microbial cell is provided for producing at least one lipid having general formula II from at least one carbon substrate. Formula II Where R 1 and R 2 Each contains, independently, the same or different organic groups, each having 5 to 13 carbon atoms. The cells mentioned above are non-pathogenic cells that have been genetically modified to increase the heterologous expression of the following enzymes relative to wild-type cells: - Enzyme E1, comprising a 3-(3-hydroxyalkanoyloxy)alkyl acid (HAA) synthase of SEQ ID NO: 1, SEQ ID NO: 7, SEQ ID NO: 11, SEQ ID NO: 15 or variants thereof, wherein said variant comprises 60% sequence identical to SEQ ID NO: 1, 7, 11 or 15; and - Enzyme E2, comprising a glycosyltransferase (EC 2.4) of SEQ ID NO: 2, SEQ ID NO: 8 or SEQ ID NO: 12 or a variant thereof, wherein the variant comprises 60% sequence identical to SEQ ID NO: 2, 8 or 12.

[0012] Specifically, glucose can be added to allow either or both of these conversions to occur. Therefore, glucose can be added to convert 3-hydroxyalkyl-3-hydroxyalkyl-CoA / ACP to β-D-glucopyranosyl-3-hydroxyalkyl-3-hydroxyalkylate. In another example, glucose can be added to convert HAA to β-D-glucopyranosyl-3-hydroxyalkyl-3-hydroxyalkylate. In yet another example, for the production of β-D-glucopyranosyl-3-hydroxyalkyl-3-hydroxyalkylate, HAA and 3-hydroxyalkyl-3-hydroxyalkyl-CoA / ACP can coexist.

[0013] Cells according to any aspect of the invention can be genetically modified to increase heterologous expression relative to wild-type cells of an enzyme (E1) capable of converting 3-hydroxyalkanoyl-CoA / ACP to 3-hydroxyalkanoyl-3-hydroxyalkanoyl-CoA / ACP and further to 3-(3-hydroxyalkanoyloxy)alkyl acid (HAA). Specifically, enzyme E1 can be a 3-(3-hydroxyalkanoyloxy)alkyl acid (HAA) synthase. In one example, enzyme E1 comprises a sequence selected from SEQ ID NO: 1, 7, 11, 15 or variants thereof. In one example, variants of SEQ ID NO: 1, 7, 11, or 15 each contain 60% sequence identity with SEQ ID NO: 1, 7, 11, or 15.

[0014] In one example, enzyme E1 may have a polypeptide sequence SEQ ID NO: 1 or wherein, compared with the reference sequence SEQ ID NO: 1, it is modified by deletion, insertion, substitution or a combination thereof by 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 amino acid groups and still has at least 10%, particularly 50%, more particularly 80%, and even more particularly greater than 90% of the polypeptide sequence containing the enzyme activity of the reference sequence SEQ ID NO: 1, wherein the enzyme activity of enzyme E1 is understood to mean, preferably, the ability to convert 3-hydroxyalkyl-CoA / ACP to 3-hydroxyalkyl-3-hydroxyalkyl-CoA / ACP and further to HAA.

[0015] In another example, enzyme E1 may have a polypeptide sequence SEQ ID NO: 7 or wherein, compared with the reference sequence SEQ ID NO: 7, it is modified by deletion, insertion, substitution or a combination thereof by 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 amino acid groups and still has at least 10%, particularly 50%, more particularly 80%, and even more particularly greater than 90% of the polypeptide sequence containing the enzyme activity of the reference sequence SEQ ID NO: 7, wherein the enzyme activity of enzyme E1 is understood to mean, preferably, the ability to convert 3-hydroxyalkyl-CoA / ACP to 3-hydroxyalkyl-3-hydroxyalkyl-CoA / ACP and further to HAA.

[0016] In a further example, enzyme E1 may have the polypeptide sequence SEQ ID NO: 11 or wherein, compared with the reference sequence SEQ ID NO: 11, it is modified by deletion, insertion, substitution or a combination thereof by 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 amino acid groups and still has at least 10%, particularly 50%, more particularly 80%, and even more particularly greater than 90% of the polypeptide sequence containing the enzyme activity of the reference sequence SEQ ID NO: 11, wherein the enzyme activity of enzyme E1 is understood to mean, preferably, the ability to convert 3-hydroxyalkyl-CoA / ACP to 3-hydroxyalkyl-3-hydroxyalkyl-CoA / ACP and further to HAA.

[0017] In a further example, enzyme E1 may have a polypeptide sequence SEQ ID NO: 15 or wherein, compared with the reference sequence SEQ ID NO: 15, it is modified by deletion, insertion, substitution or a combination thereof by 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 amino acid groups and still has at least 10%, particularly 50%, more particularly 80%, and even more particularly greater than 90% of the polypeptide sequence containing the enzyme activity of the reference sequence SEQ ID NO: 15, wherein the enzyme activity of enzyme E1 is understood to mean, preferably, the ability to convert 3-hydroxyalkyl-CoA / ACP to 3-hydroxyalkyl-3-hydroxyalkyl-CoA / ACP and further to HAA.

[0018] Enzyme E2 is capable of converting 3-hydroxyalkyl-3-hydroxyalkyl-CoA / ACP to β-D-glucopyranosyl-3-hydroxyalkyl-3-hydroxyalkyl ester. In one example, enzyme E2 is capable of converting HAA to β-D-glucopyranosyl-3-hydroxyalkyl-3-hydroxyalkyl ester. In yet another example, enzyme E2 is capable of converting both 3-hydroxyalkyl-3-hydroxyalkyl-CoA / ACP and HAA to β-D-glucopyranosyl-3-hydroxyalkyl-3-hydroxyalkyl ester. In all these examples, NDP-glucose may be specifically present to include the glucose moiety in general formula II. Enzyme E2 may be a glycosyltransferase (EC 2.4). In particular, enzyme E2 comprises SEQ ID NO: 2 or a variant thereof. As used herein, the term "variant" 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, wherein preferably, amino acids other than those essential for function (e.g., catalytic activity of a protein) or folding or structure of the molecule are deleted, substituted, or replaced by insertion, or essential amino acids are replaced in a conserved manner to preserve the biological activity of the reference sequence or molecules derived therefrom. Prior art includes algorithms that can be used to compare two given nucleic acid or amino acid sequences and calculate their similarity; see Arthur Lesk (2008), Thompson et al., 1994, and Katoh et al., 2005. The term "variant" is synonymous with and interchangeable with the term "homolog." Such variants can be prepared by introducing deletions, insertions, or substitutions into amino acid or nucleic acid sequences and fusions comprising such macromolecules or variants thereof. In one instance, the term "variant" regarding an amino acid sequence, in addition to the sequence consistency described above, also includes an amino acid sequence containing one or more conserved amino acid variations relative to its respective reference or wild-type sequence, or includes a nucleic acid sequence encoding an amino acid sequence containing one or more conserved amino acid variations. In one instance, the term "variant" regarding an amino acid sequence or nucleic acid sequence, in addition to the sequence consistency described above, also includes any active portion and / or fragment of an amino acid sequence or nucleic acid sequence, or any nucleic acid sequence encoding an active portion and / or fragment of an amino acid sequence. As used herein, the term "active portion" refers to an amino acid sequence or nucleic acid sequence, respectively smaller than the full-length amino acid sequence or encoding an amino acid sequence smaller than the full-length amino acid sequence, wherein the amino acid sequence or the encoded amino acid sequence retains at least a portion of its essential biological activity. For example, the 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 a portion of its essential biological activity" means that the amino acid sequence in question has biological activity exceeding and different from background activity, and the kinetic parameter characterizing said activity, more specifically k cat and KM Preferably, the value exhibited by the reference molecule for a particular substrate is within the order of 3, 2, or 1. Similarly, the term "variant" for nucleic acids includes nucleic acids whose complementary strands preferably hybridize with a reference or wild-type nucleic acid under stringent conditions. In one instance, a variant of SEQ ID: 4 may have 60% sequence identity with SEQ ID NO: 4.

[0019] In one example, enzyme E2 may have the polypeptide sequence SEQ ID NO: 2, or wherein at most 25%, particularly at most 20%, more particularly preferably at most 15%, particularly at most 10, 9, 8, 7, 6, 5, 4, 3, 2, 1% of amino acid groups compared to the reference sequence SEQ ID NO: 2, and still have at least 10%, particularly 50%, more particularly 80%, and even more particularly greater than 90% of the polypeptide sequence containing the enzyme activity of the reference sequence SEQ ID NO: 2, wherein the enzyme activity of enzyme E2 is understood to specifically refer to the ability to convert 3-hydroxyalkyl-3-hydroxyalkyl-CoA / ACP or 3-(3-hydroxyalkyloxy)alkyl acid (HAA) to β-D-glucopyranosyl-3-hydroxyalkyl-3-hydroxyalkyl ester. NDP-glucose may be present in this conversion.

[0020] In another example, enzyme E2 may have the polypeptide sequence SEQ ID NO: 8, or wherein the polypeptide sequence is modified by deletion, insertion, substitution, or a combination thereof by up to 25%, particularly up to 20%, more particularly preferably up to 15%, particularly up to 10, 9, 8, 7, 6, 5, 4, 3, 2, 1% of amino acid groups compared with the reference sequence SEQ ID NO: 8, and still has at least 10%, particularly 50%, more particularly 80%, and even more particularly greater than 90% of the polypeptide sequence containing the enzyme activity of the reference sequence SEQ ID NO: 8, wherein the enzyme activity of enzyme E2 is understood to specifically refer to the ability to convert 3-hydroxyalkyl-3-hydroxyalkyl-CoA / ACP or 3-(3-hydroxyalkyloxy)alkyl acid (HAA) to β-D-glucopyranosyl-3-hydroxyalkyl-3-hydroxyalkyl ester. NDP-glucose may be present in this conversion.

[0021] In one example, enzyme E2 may have the polypeptide sequence SEQ ID NO: 12 or wherein, compared with the reference sequence SEQ ID NO: 12, it is modified by deletion, insertion, substitution, or a combination thereof by up to 25%, particularly up to 20%, more particularly preferably up to 15%, particularly up to 10, 9, 8, 7, 6, 5, 4, 3, 2, 1% of amino acid groups and still has at least 10%, particularly 50%, more particularly 80%, and even more particularly greater than 90% of the polypeptide sequence containing the enzyme activity of the reference sequence SEQ ID NO: 12, wherein the enzyme activity of enzyme E2 is understood to refer specifically to the ability to convert 3-hydroxyalkyl-3-hydroxyalkyl-CoA / ACP or 3-(3-hydroxyalkyloxy)alkyl acid (HAA) to β-D-glucopyranosyl-3-hydroxyalkyl-3-hydroxyalkyl ester. NDP-glucose may be present in this conversion.

[0022] In particular, compared with the use of other organisms, such as Serratia rubidaea Compared to E2, use SEQ ID NO: 2, SEQ ID NO: 8, or SEQ ID NO: 12 (i.e., from...). Pantoea spec. E2 can lead to the production of higher amounts of rubiwettin.

[0023] In one instance, cells according to any aspect of the invention are genetically modified to increase heterologous expression of the following enzymes relative to wild-type cells: - Enzyme E1 comprises a 3-(3-hydroxyalkanoyloxy)alkanoic acid (HAA) synthase of SEQ ID NO: 1 or a variant thereof, wherein said variant contains 60% sequence identity with SEQ ID NO: 1; and - Enzyme E2, comprising a glycosyltransferase (EC 2.4) of SEQ ID NO: 2 or a variant thereof, wherein the variant comprises 60% sequence identical to SEQ ID NO: 2.

[0024] In another example, cells according to any aspect of the invention are genetically modified to increase heterologous expression of the following enzymes relative to wild-type cells: - Enzyme E1, comprising a 3-(3-hydroxyalkanoyloxy)alkanoic acid (HAA) synthase of SEQ ID NO: 7 or a variant thereof, wherein said variant comprises 60% sequence identical to SEQ ID NO: 7; and - Enzyme E2, comprising a glycosyltransferase (EC 2.4) of SEQ ID NO: 8 or a variant thereof, wherein the variant comprises 60% sequence identical to SEQ ID NO: 8.

[0025] In a further example, cells according to any aspect of the invention are genetically modified to increase heterologous expression of the following enzymes relative to wild-type cells: - Enzyme E1, comprising a 3-(3-hydroxyalkanoyloxy)alkanoic acid (HAA) synthase of SEQ ID NO: 11 or a variant thereof, wherein said variant comprises 60% sequence identical to SEQ ID NO: 11; and - Enzyme E2, comprising a glycosyltransferase (EC 2.4) of SEQ ID NO: 12 or a variant thereof, wherein the variant comprises 60% sequence identical to SEQ ID NO: 12.

[0026] In one instance, cells according to any aspect of the invention are genetically modified to increase heterologous expression of the following enzymes relative to wild-type cells: - Enzyme E1, comprising a 3-(3-hydroxyalkanoyloxy)alkanoic acid (HAA) synthase of SEQ ID NO: 15 or a variant thereof, wherein said variant comprises 60% sequence identical to SEQ ID NO: 15; and - Enzyme E2, comprising a glycosyltransferase (EC 2.4) of SEQ ID NO: 2 or a variant thereof, wherein the variant comprises 60% sequence identical to SEQ ID NO: 2.

[0027] In another example, cells according to any aspect of the invention are genetically modified to increase heterologous expression of the following enzymes relative to wild-type cells: - Enzyme E1, comprising a 3-(3-hydroxyalkanoyloxy)alkanoic acid (HAA) synthase of SEQ ID NO: 15 or a variant thereof, wherein said variant comprises 60% sequence identical to SEQ ID NO: 15; and - Enzyme E2, comprising a glycosyltransferase (EC 2.4) of SEQ ID NO: 8 or a variant thereof, wherein the variant comprises 60% sequence identical to SEQ ID NO: 8.

[0028] In another example, cells according to any aspect of the invention are genetically modified to increase heterologous expression of the following enzymes relative to wild-type cells: - Enzyme E1, comprising a 3-(3-hydroxyalkanoyloxy)alkanoic acid (HAA) synthase of SEQ ID NO: 15 or a variant thereof, wherein said variant comprises 60% sequence identical to SEQ ID NO: 15; and - Enzyme E2, comprising a glycosyltransferase (EC 2.4) of SEQ ID NO: 12 or a variant thereof, wherein the variant comprises 60% sequence identical to SEQ ID NO: 12.

[0029] Lipids of general formula II can also be called glycolipids. 1 and R 2 The length of the group can vary. Specifically, R... 1 and R 2 It can be independently selected from saturated and unsaturated alkyl groups. More specifically, R 1 and / or R 2 It can be a saturated or unsaturated alkyl group having 5 to 13 carbon atoms. More specifically, R 1 and R 2 It can be a saturated or monounsaturated alkyl group having 5 to 13 carbon atoms. R 1 and R 2 Alkyl groups can contain 5 to 13 carbon atoms, 7 to 13 carbon atoms, 9 to 13 carbon atoms, 5 to 11 carbon atoms, 5 to 9 carbon atoms, and so on. In R 1 and / or R 2 In the case of an alkyl group, which is a saturated alkyl group, the alkyl group may contain 5, 6, 7, 8, 9, 10, 11, 12, or 13 carbon atoms. In R 1 and / or R 2 In instances 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. More specifically, R 1 and / or R 2 The alkyl group is a saturated alkyl group having 5 to 13 carbon atoms. Cells according to any aspect of the invention can produce cells with different R... 1 and R 2 A mixture of rubiwettin groups. In one example, the lipid of general formula II produced according to any aspect of the invention may be rubiwettin RG1 (CAS-Nr. 129039-46-9). Rubiwettin RG1 may also be referred to as a glycolipid, and is named β-D-glucopyranosyl-3-(3'-hydroxytetradecanoyloxy)decanoate or β-glucopyranosyl-3-(3'-hydroxytetradecanoyloxy)decanoate.

[0030] Cells according to any aspect of the invention can produce additional lipids having general formula I from carbon substrates. General Formula I Where R 1 and R 2 Each alkyl group is independently an identical or different alkyl group having 5 to 13 carbon atoms. In particular, the alkyl group can be saturated or unsaturated. More specifically, R 1 and / or R2 The alkyl group can be a saturated alkyl group. More specifically, R 1 and / or R 2 It can be selected from pentyl, heptyl, nonyl, undecyl, tridecyl and (CH2). n -CH3, where n=4-12.

[0031] Lipids of general formula I can have varying lengths of R 1 and R 2 Group. Specifically, R 1 and R 2 It can be independently selected from saturated and unsaturated alkyl groups. More specifically, R 1 and / or R 2 It can be a saturated or unsaturated alkyl group having 5 to 13 carbon atoms. More specifically, R 1 and R 2 It can be a saturated or monounsaturated alkyl group having 5 to 13 carbon atoms. R 1 and R 2 Alkyl groups can contain 5 to 13 carbon atoms, 7 to 13 carbon atoms, 9 to 13 carbon atoms, 5 to 11 carbon atoms, 5 to 9 carbon atoms, and so on. In R 1 and / or R 2 In instances 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. In R 1 and / or R 2 In examples 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. Cells according to any aspect of the invention can be able to produce cells with different R... 1 and R 2 A mixture of lipids with a group. In particular, lipids having formula I can also be called rubiwettin R1 (CAS-Nr. 129039-45-8). Specifically, the lipid is a mixture of 3-(3'-hydroxytetradecanoyloxy)tetradecanoate, 3-(3'-hydroxydecanoyloxy)decanoate, 3-(3'-hydroxyhexadecanoyloxy)hexadecanoate, 3-(3'-hydroxytetradecanoyloxy)decanoate, 3-(3'-hydroxyhexadecanoyloxy)tetradecanoate and minor molecular isomers.

[0032] Surprisingly, it can be shown that recombinant cells according to any aspect of the invention, with increased E2 and / or E1 expression compared to wild-type cells, are able to produce increased amounts of lipids having formula II and / or I. Therefore, cells according to any aspect of the invention can achieve highly selective production of rubiwettin RG1 while reducing the generation of undesirable intermediates, such as dimers of β-hydroxy fatty acids.

[0033] Specifically, using from Pantoea spec. The substitution of SEQ-ID 1, 7, and 11 for the E1 sequence from *Pseudomonas aeruginosa* as E1 causes rubiwettin derivatives to shift from C10:0-C12:1 and C10:0-C12:0 to C8:0-C10:0. Rubiwettin derivatives with shorter 3-hydroxy fatty acid chain lengths may have different applications than those with longer 3-hydroxy fatty acid chain lengths.

[0034] As used herein, the phrase "increased heterologous expression of the enzyme" should be understood as increased intracellular activity. Essentially, increased enzyme activity can be achieved by increasing the copy number of the gene sequence encoding the enzyme, using a strong promoter, or employing a gene or allele encoding the corresponding enzyme with increased activity, and optionally by combining these measures. Genetically modified cells used in the method according to the invention are produced, for example, by transformation, transduction, conjugation, or a combination of these methods, using a vector containing the desired gene, an allele of such gene, or a portion thereof, and a vector enabling the expression of that gene. Heterologous expression is achieved, in particular, by integrating the gene or allele into the cell chromosome or an extrachromosomal replication vector. Specifically, the increase in enzyme activity relative to wild-type cells can be 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% higher than that of wild-type cells.

[0035] Those skilled in the art can genetically modify cells using any method known in the art. Whether the nucleic acid molecule, polypeptide, and more specifically the enzyme used according to any aspect of the invention is recombinant does not necessarily affect its expression level. However, in one instance, one or more recombinant nucleic acid molecules, polypeptides, or enzymes used according to any aspect of the invention may be overexpressed. As used herein, the term "overexpression" means that the corresponding polypeptide encoded or expressed is expressed at a higher level or with higher activity than it is normally found in cells, for example, in their respective wild-type cells, under the same conditions without genetic modifications to increase expression. Those skilled in the art are familiar with many ways to induce overexpression. For example, the nucleic acid molecule to be overexpressed or the nucleic acid molecule encoding the polypeptide or enzyme to be overexpressed can be placed under the control of a strongly inducible promoter such as a lac promoter. Standard plasmids that can be used for this purpose are described in the prior art, such as vectors of the pET system, for example, pET-3a (available from Novagen). Whether a nucleic acid or polypeptide is overexpressed can be determined by quantitative PCR (in the case of nucleic acid molecules), SDS-PAGE, Western blotting, or comparative activity assays (in the case of polypeptides). Genetic modification can involve transcriptional, translational, and / or post-translational modifications that result in changes in enzyme activity and / or selectivity under selected and / or defined culture conditions. Therefore, in various embodiments of the invention, microorganisms may contain one or more gene deletions for more efficient operation. Gene deletions can be achieved by a mutant gene deletion method, and / or by starting with mutant strains that reduce or eliminate the expression of one or more of these enzymes, and / or by other methods known to those skilled in the art.

[0036] DE-A-100 31 999 provides a general survey of the possibility of increasing enzyme activity in cells, taking pyruvate carboxylase as an example. It is incorporated herein by reference, and its disclosure regarding the possibility of increasing enzyme activity in cells forms part of the disclosure of this invention.

[0037] The expression of the enzymes or genes mentioned above and subsequently all mentioned below can be detected by means of 1D and 2D protein gel separation and subsequent optical identification of the protein concentration in the gel using appropriate analytical software. If the increase in enzyme activity is entirely based on the increase in the expression of the corresponding gene, the quantification of the increase in enzyme activity can be determined in a simple manner by comparing 1D or 2D protein separations between wild-type cells and genetically modified cells. In the case of Corynebacterium, the routine method used for preparing protein gels and identifying proteins is the procedure described by Hermann et al. (Electrophoresis, 22: 1712.23 (2001)). Protein concentration can also be analyzed by Western blot hybridization using specific antibodies against the protein being tested (Sambrook et al., Molecular Cloning: a laboratory manual, 2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY USA, 1989) and subsequent optical analysis using appropriate concentration assay software (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 analysis (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 using various well-described reporter gene assays (Sambrook et al., Molecular Cloning: a laboratory manual, 2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY USA, 1989). Intracellular enzyme activity can be measured using 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 the actual method for determining the activity of an enzyme is not specified in the following implementation scheme, the determination of an increase in enzyme activity and the determination of a decrease in enzyme activity are preferably performed by means of the methods described in Hermann et al., Electrophoresis, 22: 1712-23 (2001), Lohaus et al., Biospektrum 5 32-39 (1998), Lottspeich, Angewandte Chemie 111: 2630-2647 (1999), and Wilson et al., Journal of Bacteriology 183: 2151-2155 (2001).

[0038] If the increase in enzyme activity is achieved through mutations in an endogenous gene, such mutations can be generated randomly by conventional methods (e.g., by UV irradiation or by mutagenic chemicals) or selectively by genetic engineering methods (e.g., deletions, insertions, and / or nucleotide exchanges). Modified cells are obtained through these mutations. Particularly preferred enzyme mutants are those that are no longer repressible for feedback, product, or substrate, or at least to a reduced extent compared to the wild-type enzyme.

[0039] If increased enzyme activity is achieved by increasing enzyme synthesis, this can be done by increasing the copy number of the corresponding gene, or by mutating the promoter and regulatory regions or ribosome binding sites upstream of the structural gene. Expression cassettes incorporated upstream of the structural gene function in the same way. Alternatively, expression can be increased at any desired time point using inducible promoters. However, "enhancers" can also be assigned as regulatory sequences to enzyme genes, which similarly increases gene expression by improving the interaction between RNA polymerase and DNA. Expression is also improved as a result of mRNA lifetime extension measures. Furthermore, enzyme activity is also increased by preventing enzyme protein degradation. The gene or gene construct can be integrated and amplified in plasmids with different copy numbers or in chromosomes. Alternatively, overexpression of the relevant gene can be achieved by altering the culture medium composition and culture management. Those skilled in the art, particularly those familiar with the publications, have noted 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 472869, US 4,601,893, Schwarzer and Pühler (Bio / Technology 9, 84-87 (1991)), Reinscheid et al. (Applied and Environmental Microbiology 60, 126-132 (1994)), and LaBarre et al. (Journal of Bacteriology 175, 1001-1007). Descriptions of this can be found in (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 on genetics and molecular biology. Similar to mutations, the above measures also result in genetically modified cells.

[0040] Plasmids are used, for example, to increase the expression of their respective genes. Suitable plasmids or vectors are, in principle, all embodiments available to those skilled in the art for this purpose. Such plasmids and vectors can be obtained, for example, from brochures of Novagen, Promega, New England Biolabs, Clontech, or GibcoBRL. Further optimized plasmids and vectors can be found in: Glover, DM (1985) DNA cloning: a practical approach, Vol. I-III, IRL Press Ltd., Oxford; Rodriguez, RL and Denhardt, D. T (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 transformed into the desired strain through conjugation or transformation. Conjugation methods are described, for example, in Schäfer et al., Applied and Environmental Microbiology 60: 756-759 (1994). Transformation methods are described, for example, in Thierbach et al., Applied Microbiology and Biotechnology 29: 356-362 (1988), Dunican and Shivnan, Bio / Technology 7: 1067-1070 (1989), and Tauch et al., FEMS Microbiology Letters 123: 343-347 (1994). After homologous recombination via a "cross-over" event, the resulting strain contains at least two copies of the relevant gene.

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

[0043] Variations of amino acid residues in a given polypeptide sequence that do not cause significant changes to the properties and function of the given polypeptide are known to those skilled in the art. Thus, for example, “conserved amino acids” can be interchanged; examples of such suitable amino acid substitutions are: Ala replacing Ser; Arg replacing Lys; Asn replacing Gln or His; Asp replacing Glu; Cys replacing Ser; Gln replacing Asn; Glu replacing Asp; Gly replacing Pro; His replacing Asn or Gln; Ile replacing Leu or Val; Leu replacing Met or Val; Lys replacing Arg or Gln or Glu; Met replacing Leu or Ile; Phe replacing Met or Leu or Tyr; Ser replacing Thr; Thr replacing Ser; Trp replacing Tyr; Tyr replacing Trp or Phe; Val replacing Ile or Leu. It is also known that variations in the form of amino acid insertions or deletions, particularly at the N- or C-terminus of the polypeptide, generally do not have a significant effect on the function of the polypeptide.

[0044] Enzyme activity can be determined by disrupting cells containing this activity using methods known to those skilled in the art, such as a ball mill, Freund's crusher, or an ultrasonic grinder, followed by centrifugation at 13,000 rpm and 4°C for 10 minutes to separate cells, cell debris, and disrupting agents, such as glass beads. The resulting cell-free crude extract can then be used for enzyme assays, as well as subsequent LC-ESI-MS detection of the product. Alternatively, the enzyme can be enriched or purified to homogeneity using chromatographic methods (such as nickel-nitrotriacetic acid affinity chromatography, streptavidin affinity chromatography, gel filtration chromatography, or ion exchange chromatography) in a manner known to those skilled in the art.

[0045] In one example, a method for determining the activity of enzyme E2 involves first disrupting cells containing this activity (i.e., cells according to any aspect of the invention) in a manner known to those skilled in the art, such as by means of a ball mill, Freund's crusher, or an ultrasonic grinder, followed by separation of the cells, cell debris, and disruption aids, such as glass beads, by centrifugation at 16,100 g for 10 minutes at 4°C. The resulting cell-free crude extract can be used for enzyme assays, as well as subsequent LC-ESI-MS detection of the product. Alternatively, the enzyme can be enriched or purified to homogeneity by chromatographic methods (such as nickel / triacetic acid affinity chromatography, streptavidin affinity chromatography, gel filtration chromatography, or ion exchange chromatography) in a manner known to those skilled in the art. Such a sample can then be used to measure the activity of enzyme E2. Specifically, the activity of enzyme E2 can be determined using a standard assay consisting of 185 µl of 10 mM Tris-HCl (pH 7.5), 10 µl of mM NDP-glucose, and 50 µl of crude protein extract (approximately 1 mg of total protein) or purified protein in solution (5 µg of purified protein). The reaction can be initiated by adding 10 µl of an ethanolic solution of 3-hydroxytetradecanoyl-3-hydroxydecanoic acid or 3-hydroxyhexadecanoyl-3-hydroxydecanoic acid and incubating at 30 °C with shaking (600 rpm) for 1 hour. Subsequently, the reaction can be treated with 1 ml of acetone. Undissolved components can be precipitated by centrifugation (16, 100 g, 5 min RT) and the sample can be analyzed using LC-ESI-MS. The product can then be identified by analyzing the corresponding mass trace and MS2 spectra. This method can be used to measure the activity of E2.

[0046] In another example, a method for determining the activity of enzyme E1 involves first disrupting cells containing this activity (i.e., cells according to any aspect of the invention) in a manner known to those skilled in the art, followed by separation of the cells, cell debris, and disruption aids, such as glass beads, by centrifugation at 16,100 g for 10 minutes at 4°C. The resulting cell-free crude extract can be used for enzyme assays, as well as subsequent LC-ESI-MS detection of the product. Alternatively, the enzyme can be enriched or purified to homogeneity by chromatographic methods (such as nickel / triacetic acid affinity chromatography, streptavidin affinity chromatography, gel filtration chromatography, or ion exchange chromatography) in a manner known to those skilled in the art. This sample can then be used to measure the activity of enzyme E1. In particular, the activity of enzyme E1 can be determined using a standard assay, which may contain 100 µM... Escherichia coli bacteriaACP, 1 mM β-mercaptoethanol, 200 µM malonyl-CoA, 40 µM octanoyl-CoA and 40 µM dodecanoyl-CoA or 40 µM octanoyl-CoA and 40 mM tetradecanoyl-CoA, 100 µM NADPH, 2 µg Escherichia coli FabD, 2 µg tuberculosis Mycobacterium FabH, 1 µg E. coli FabG, 0.1 M sodium phosphate buffer (pH 7.0), and 5 µg of enzyme E1 were used to make a final volume of 120 µl. ACP, β-mercaptoethanol, and sodium phosphate buffer were pre-incubated at 37 °C for 30 min to completely reduce ACP. The reaction could be initiated by adding enzyme E1. The reaction could be terminated with 2 ml of water acidified to pH 2.0 with HCl, followed by two extractions with 2 ml of chloroform / methanol (2:1 (v:v)). Phase separation could be performed by centrifugation (16,100 g, 5 min, room temperature). The organic phase below could be removed by complete evaporation in a vacuum centrifuge, and the precipitate could be placed in 50 µl of methanol. Undissolved components could be precipitated by centrifugation (16,100 g, 5 min RT), and the sample could be analyzed using LC-ESI-MS. The products could then be identified by analyzing the corresponding mass traces and MS2 spectra.

[0047] The enzymes used according to any aspect of the invention can be recombinant. As used herein, the term "recombinant" refers to a molecule that is not naturally occurring but is the result of genetic engineering, or is encoded by such a molecule, particularly a polypeptide or nucleic acid, or to a cell containing the recombinant molecule. For example, a nucleic acid molecule is recombinant if it contains a promoter functionally linked to a sequence encoding a catalytically active polypeptide and the promoter has been engineered such that the catalytically active polypeptide is overexpressed at polypeptide levels relative to the corresponding wild-type cells containing the original, unaltered nucleic acid molecule.

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

[0049] Suitable bacteria, yeasts, or fungi, particularly those strains of bacteria, yeasts, or fungi preserved at Deutsche Sammlung von Mikroorganismen und Zellkulturen (German Collection of Microorganisms and CellCultures) GmbH (DSMZ), Brunswick, Germany. According to the invention, suitable bacteria belong to the genera listed at http: / / www.dsmz.de / species / bacteria.htm, suitable yeasts belong to the genera listed at http: / / www.dsmz.de / species / yeasts.htm, and suitable fungi belong to the genera listed at http: / / www.dsmz.de / species / fungi.htm.

[0050] Specifically, the cells can be selected from Aspergillus, Corynebacterium, Breobranch, Bacillus, Acinetobacter, Alcaligenes, Lactobacillus, Paracoccus, Lactococcus, Candida, Pichia pastoris, Hansenula, Kluyveromyces, Yeast, Escherichia, Fermentomonas, and Yersinia. Yarrowia ), Methylobacterium, Rawlstonella, Pseudomonas, Rhodospirillum, Rhodospirillum, Burkholderia ( Burkholderia Clostridium and copper-loving bacteria. More specifically, cells can be selected from... Aspergillus nidus, Aspergillus niger, and Alcaligenes (lactus), Bacillus megaterium, Bacillus subtilis, Bacillus flavus, and Brevibacterium lactose-fermenting bacteria. lactofermentum), Burkholderia andropogonis, Burkholderia brasiliensis Bacillus brasilensis , B. caledonica , Burkholderia caribensis , Burkholderia caryophylli , B. fungorum , Burkholderia gladioli (B.) gladioli) , B. glathei , Burkholderia glumae , B. graminis , B. hospita , B. kururiensis , Burkholderia phenazinium , B. phymatum , B. phytofirmans , plant Burkholderia plantarii , B. sacchari , Burkholderia sacchariformis (B. sacchariformis) singaporensis) , B. sordidicola , B. terricola , Burkholderia tropicala , B. tuberum , B. ubonensis , Burkholderia unamae , B. xenovorans , B. Anthina , Burkholderia pyrrocinia , Burkholderia tsiniana (B.) Thailandensis , Candida blankii , Candida folds , Corynebacterium glutamicum , Corynebacterium efficiens , E. coli , Hansenula polymorpha , Kluveromyces lactis lactis) , Methylobacterium tauren , paracoccus variegata , Pseudomonas argentinensis , Mud Fake P. borbori , Citronellolis and Pseudomonas flavum. *Pseudomonas flavescens*, *P. mendocina*, and *P. nitroreducens*. *Pseudomonas oleovorans*, *Pseudomonas pseudoalcaligenes*, and *Pseudomonas affinis* Pseudomonas resinovorans, Pseudomonas straminea, Pseudomonas aurantiaca, and others. *Pseudomonas aureofaciens*, *Pseudomonas chlororaphis*, and *Pseudomonas berryensis*. *Pseudomonas fragi*, *P. lundensis*, *P. taetrolens*, and *P. antarcticus*. *Pseudomonas antarctica*, *P. azotoformans*, *P. blatchfordae*, and *P. brassicae*. *Pseudomonas brassicacearum*, *P. brenneri*, *P. cedrina*, *P. wrinkle* *Pseudomonas corrugata*, *Pseudomonas fluorescens*, *Pseudomonas gessardii*, *Pseudomonas leptospira* *Pseudomonas libanensis*, *Pseudomonas mandelii*, *Pseudomonas marginalis*. marginalis), Pseudomonas mediterranea, Pseudomonas meridiana, and Miscellanea. Pseudomonas migulae, Pseudomonas mucidolens, Pseudomonas orientalis *Pseudomonas panacis*, *Pseudomonas proteolytica*, and *Pseudomonas hominis*. *Pseudomonas rhodesiae*, *P. synxantha*, *P. thivervalensis ... Pseudomonas tolaasii, Pseudomonas veronii, Pseudomonas denitrificans (P. *Pseudomonas denitrificans*, *P. pertucinogena*, and *P. miltiorrhizos*. *Pseudomonas cremoricolorata*, *P. fulva*, *P. monteilii*, *P. mossioides* *Pseudomonas mosselii*, *Pseudomonas parafulva*, *Pseudomonas putida*, *Pseudomonas bariensis* *Pseudomonas balearica*, *Pseudomonas stutzeri*, *Pseudomonas amygdali*, and hazelnut *Pseudomonas avellanae*, *Pseudomonas caricapapayae*, *Pseudomonas chicoryensis*. *Pseudomonas cichorii*, *P. coronafaciens*, *P. ficuserectae*, *P. spp.* *Pseudomonas helianthi*, *Pseudomonas meliae*, and *Pseudomonas sassini*. *Pseudomonas savastanoi*, *P. syringae*, *P. tomato*, and *P. aeruginosa*. *Pseudomonas viridiflava*, *P. abietaniphila*, *P. acidophila*, *P. umbellifera* Pseudomonas agarici, Pseudomonas alcaliphila, Pseudomonas alkalophila. *Pseudomonas alkanolytica*, *P. amyloderamosa*, *P. asplenii*, and *P.* azotifigens, Pseudomonas cannabina, Pseudomonas coenobios, Pseudomonas icariae. *Pseudomonas congelans*, *P. costantinii*, *P. cruciviae*, *P. derry* Pseudomonas delhiensis, Pseudomonas excibis, Pseudomonas far-easternis. *Pseudomonas extremorientalis*, *P. frederiksbergensis*, and *Pseudomonas brownis* (P. fuscovaginae), P. gelidicola, P. grimontii, and others *Pseudomonas indica*, *Pseudomonas jessenii*, *Pseudomonas jinjuensis* *Pseudomonas kilonensis*, *Pseudomonas knackmussii*, *Pseudomonas koreensis*, *Pseudomonas linteus* *Pseudomonas lini*, *Pseudomonas lutea*, *Pseudomonas moraviensis*, otitis media Pseudomonas otitidis, Pseudomonas pachastrellae, Pseudomonas pallensii (P. otitidis). *Pseudomonas palleroniana*, *P. papaveris*, *P. peli*, and *P. rotting egg* are all species of *Pseudomonas palleroniana*. (P. perolens), P. poae, P. pohangensis, P. psychrophilic *Pseudomonas psychrophila*, *Pseudomonas psychrotolerans*, *Pseudomonas lasalis*. *Pseudomonas rathonis*, *P. reptilivora*, *P. resiniphila*, rhizosphere Pseudomonas rhizosphaerae, Pseudomonas rubescens, Pseudomonas sassiniformes. *Pseudomonas salomonii*, *P. segitis*, *P. septica*, *P. simiae*, *P. suis* *Pseudomonas suis*, *Pseudomonas thermotolerans*, *Pseudomonas aeruginosa*, *Pseudomonas spp.* *Pseudomonas tremae*, *P. trivialis*, *P. turbinellae*, *P. dutigolinii* *Pseudomonas tuticorinensis*, *Pseudomonas umsongensis*, *Pseudomonas wansongensis*. *Pseudomonas vancouverensis*, *P. vranovensis*, and *P. xanthomarina*. , Ralstonia eutropha , Deep red rhodospirel , Rhodopsyllosis , brewing yeast , Yarrowia lipolytica Yarrowia lipolytica and Zymomonas mobile. More specifically, the cells can be selected from... Acinetobacter, Bacillus, Brevibacterium, Burkholderia sp., Chlorella Clostridium, Corynebacterium, Cyanobacteria, Escherichia, Pseudomonas, Klebsiella, Salmonella, Rhizobium Genus, Yeast, Pichia pastoris and CandidaBacterial cells. More specifically, the cells can be selected from... Bacillus subtilis, Burkholderia thailandensis, Corynebacterium glutamicum, Escherichia coli, and Klebsiella pneumoniae. Burke's bacteria, *Pseudomonas fluorescens*, *Pseudomonas putida*, *Pseudomonas stearothermia*, *Rhizobium alfalfa*, *Saccharomyces cerevisiae* and Pichia pastoris .

[0051] In one instance, the cell according to any aspect of the invention can be a genetically modified cell to increase the expression of the following enzymes: - Contains enzyme E2, SEQ ID NO: 2, and - Enzyme E1 containing SEQ ID NO: 1 or a variant thereof.

[0052] In another example, the cell according to any aspect of the invention can be a genetically modified cell to increase the expression of the following enzymes: - Enzyme E1 containing SEQ ID NO: 1 or a variant thereof and enzyme E2 containing SEQ ID NO: 8 or a variant thereof, or - Enzyme E1 containing SEQ ID NO: 1 or a variant thereof and enzyme E2 containing SEQ ID NO: 12 or a variant thereof, or - Enzyme E1 or a variant thereof containing SEQ ID NO: 7 or a variant thereof and enzyme E2 or a variant thereof containing SEQ ID NO: 2 or a variant thereof, or - Enzyme E1 containing SEQ ID NO: 7 or a variant thereof and enzyme E2 containing SEQ ID NO: 8 or a variant thereof, or - Enzyme E1 containing SEQ ID NO: 7 or a variant thereof and enzyme E2 containing SEQ ID NO: 12 or a variant thereof, or - Enzyme E1 containing SEQ ID NO: 11 or a variant thereof and enzyme E2 containing SEQ ID NO: 2 or a variant thereof, or - Enzyme E1 containing SEQ ID NO: 11 or a variant thereof and enzyme E2 containing SEQ ID NO: 8 or a variant thereof, or - Enzyme E1 containing SEQ ID NO: 11 or a variant thereof and enzyme E2 containing SEQ ID NO: 12 or a variant thereof, or - Enzyme E1 containing SEQ ID NO: 15 or a variant thereof and enzyme E2 containing SEQ ID NO: 2 or a variant thereof, or - Enzyme E1 containing SEQ ID NO: 15 or a variant thereof and enzyme E2 containing SEQ ID NO: 8 or a variant thereof, or - Enzyme E1 containing SEQ ID NO: 15 or a variant thereof and enzyme E2 containing SEQ ID NO: 12 or a variant thereof.

[0053] Cells according to any aspect of the invention can be used to produce lipids according to general formulas I and / or II from carbon substrates: General Formula I Formula II Where R 1 and R 2 In general formula I or II, each group is an identical or different alkyl group having 5 to 13 carbon atoms. In particular, the alkyl group can be saturated or unsaturated. More specifically, R... 1 and / or R 2 The alkyl group can be a saturated alkyl group. More specifically, R 1 and / or R 2 It can be selected from pentyl, heptyl, nonyl, undecyl, tridecyl and (CH2). n -CH3, where n=4-12.

[0054] The lipids formed can be combinations of lipids of general formulas I and II with different R groups, which can be generated in a single reaction process.

[0055] Genetically modified cells according to the invention can be continuously or discontinuously contacted with nutrient medium in a batch process (batch culture), a fed-batch process (feeding process), or a repeated fed-batch process (repeated feeding process) to produce the aforementioned products, and thus be cultured. A semi-continuous method is also possible, as described in GB-A-1009370. An overview of known culture methods is described in Chmiel's textbook (“Bioprozesstechnik 1. Einführung in die Bioverfahrenstechnik” [BioprocessTechnology 1. Introduction to the BioprocessTechnique] (Gustav FischerVerlag, Stuttgart, 1991)) or Storhas's textbook (“Bioreaktoren undperiphereEinrichtungen” [Bioreactors and Peripheral Devices], Vieweg Verlag, Brunswick / Wiesbaden, 1994).

[0056] The culture medium to be used must be suitably formulated to meet the needs of the respective yeast strains. Descriptions of culture media for different yeast strains are found, for example, in "Nonconventional yeast in biotechnology" (edited by Klaus Wolf, Springer-Verlag Berlin, 1996).

[0057] According to any aspect of the invention, the carbon source used as the substrate may be selected from carbohydrates, such as glucose, sucrose, arabinose, xylose, lactose, fructose, maltose, molasses, starch, cellulose and hemicellulose, and vegetable and animal oils and fats, such as soybean oil, safflower oil, peanut oil, hemp seed oil, jatropha oil, coconut oil, and calabash oil. Oils, flaxseed oil, corn oil, evening primrose oil, olive oil, palm kernel oil, palm oil, rapeseed oil, sesame oil, sunflower oil, grapeseed oil, walnut oil, wheat germ oil, and coconut oil; fatty acids, such as caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, palmitoleic acid, stearic acid, arachidonic acid, benzyl acid, oleic acid, linoleic acid, linolenic acid, gamma-linolenic acid, and their methyl or ethyl esters, as well as mixtures of fatty acids; monoglycerides, diglycerides, and triglycerides containing the aforementioned fatty acids; alcohols, such as glycerol, ethanol, and methanol; hydrocarbons, such as methane, ethane, propane, or butane; carbon-containing gases and gas mixtures, such as CO, CO2, syngas, or flue gas; amino acids, such as L-glutamic acid or L-valine; or organic acids, such as acetic acid. These substances can be used alone or as mixtures. As described in US 6,01,494 and US 6,136,576, carbohydrates, particularly monosaccharides, oligosaccharides, or polysaccharides, are used as carbon sources, as well as hydrocarbons, particularly alkanes, alkenes, and alkynes. Specifically, the carbon source can be selected from glucose, dextrose, sucrose, mannose, galactose, polysaccharides such as cellulose or hemicellulose, vegetable oils, animal fats, fatty acids, fatty acid esters, carbon-containing gases, alkanes, glycerol, acetates, ethanol, and methanol. More particularly, the carbon source can be selected from glucose, sucrose, glycerol, vegetable oils, methane, ethane, and butane. A significant advantage of this invention is that cells according to the invention can form lipids having general formulas I and / or II from the simplest carbon sources, such as glucose, sucrose, or glycerol, so that a long-chain C source is not necessarily required in the culture medium during the process according to any aspect of the invention. Specifically, the carbon source can be selected from glucose, dextrose, sucrose, xylose, mannose, galactose, arabinose, monosaccharides, polysaccharides, cellulose, hemicellulose, vegetable oil, animal fat, fatty acids, fatty acid esters, carbon-containing gases, alkanes, glycerol, acetate, ethanol, and methanol.

[0058] According to another aspect of the invention, a microbial cell is provided for producing at least one lipid having general formula II from at least one carbon substrate. Formula II Where R 1 and R 2 Each contains, independently, the same or different organic groups, each having 5 to 13 carbon atoms. The cells mentioned are non-pathogenic cells that have been genetically modified to introduce DNA sequences encoding the following enzymes: - Enzyme E1, a 3-(3-hydroxyalkanoyloxy)alkanoic acid (HAA) synthase, wherein the amino acid sequence of E1 is selected from SEQ ID NO: 1, SEQ ID NO: 7, SEQ ID NO: 11, SEQ ID NO: 15 and variants thereof, wherein said variants contain 60% sequence identity with SEQ ID NO: 1, 7, 11 or 15; and - Enzyme E2, glycosyltransferase (EC 2.4), wherein the amino acid sequence of E2 is selected from SEQ ID NO: 2, SEQ ID NO: 8, SEQ ID NO: 11 and variants thereof, wherein the variants contain 60% sequence identity with SEQ ID NO: 2, 8 or 11.

[0059] According to a further aspect of the present invention, a method for producing at least one lipid having general formula II is provided: Formula II Where R 1 and R 2 Independently, they are the same or different alkyl groups having 5 to 13 carbon atoms, and The method includes the step of contacting at least one cell according to any aspect of the invention with at least one carbon source.

[0060] In particular, R 1 and / or R 2 The alkyl groups can be saturated or unsaturated. More specifically, R 1 and / or R 2 The alkyl group can be a saturated alkyl group. More specifically, R 1 and / or R 2 It can be selected from pentyl, heptyl, nonyl, undecyl and (CH2). n -CH3, where n=4-12.

[0061] The method according to any aspect of the invention can also be used to produce other lipids having general formula I from carbon substrates. General Formula I Where R 1and R 2 Each group consists independently of the same or different alkyl groups having 5 to 13 carbon atoms. Specifically, R... 1 and / or R 2 The alkyl groups can be saturated or unsaturated. More specifically, R 1 and / or R 2 The alkyl group can be a saturated alkyl group. More specifically, R 1 and / or R 2 It can be selected from pentyl, heptyl, nonyl, undecyl, tridecyl and (CH2). n -CH3, where n=4-12.

[0062] The method according to any aspect of the invention can be used to produce lipid mixtures comprising lipids of general formulas I and II. Specifically, lipids of general formulas I and II are produced in ratios 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, lipids of formulas I and II may have alkyl groups of different lengths simultaneously present in subgroup R.

[0063] According to another aspect of the invention, the use of cells according to any aspect of the invention for producing at least one lipid having general formula I and / or II is provided: General Formula I Formula II Where R 1 and R 2 Each group consists independently of the same or different alkyl groups having 5 to 13 carbon atoms. Specifically, R... 1 and / or R 2 The alkyl groups can be saturated or unsaturated. More specifically, R 1 and / or R 2 The alkyl group can be a saturated alkyl group. More specifically, R 1 and / or R 2 It can be selected from pentyl, heptyl, nonyl, undecyl, tridecyl and (CH2). n -CH3, where n=4-12. Example

[0064] The preferred embodiments described above may be varied or modified in design, construction, or operation without departing from the scope of the claims, as will be understood by those skilled in the art. For example, these variations are intended to be covered by the scope of the claims.

[0065] Example 1 The construction of the expression vector for the NODE_2 gene rbwAB of Pantoea ananatis strain PNA 99-7 establish In order to come from Pantoea ananatis strain PNA 99-7 NODE_2 genes rbwA Pan997 is used as enzyme E1 and rbwB Pan997 was used as a heterologous expression of enzyme E2 to construct the plasmid pACYC {PRhaRS}[rhaRS_Ec]{PRha}[rbwA_Pan997rbwB_Pan997] {ter}. The plasmid was then used to express Pan997 as a heterologous expression of enzyme E2. rbwAB_ The synthetic operon composed of Pan997 (SEQ ID NO:3) is based on the rhamnose-inducible promoter P rha Under controlled conditions, the DNA was cloned into the pAYCY184-based vector pACYC{PRhaRS}[rhaRS_Ec]{PRha}[rhlA_Pa rbwB_Srub], as disclosed in example WO2019154984 (New England Biolabs, Frankfurt / Main, Germany). The termination sequence was located downstream of the synthetic operon. The synthetic DNA generated by Eurofins Genomics was used to amplify the gene by PCR. The plasmid pACYC{PRhaRS}[rhaRS_Ec]{PRha}[rhlA_Pa rbwB_Srub] carried P Rha The promoter sub-box (SEQ ID NO:4) and the termination sequence (SEQ ID NO:5) are used for the p15A replication origin of E. coli and for... Pseudomonas putida The pVS1 replication origin in KT2440. The pVS1 origin origins originate from the Pseudomonas plasmid pVS1 (Itoh Y et al., Plasmid 1984, 11(3), 206-20). For amplification, Q5® High-Fidelity 2X Master Mix from New England Biolabs (Frankfurt / Main, Germany) was used according to the manufacturer's manual. In the next step, restriction sites were used. Apa I / PspThe amplified fragment was cloned into the vector pACYC{PRhaRS}[rhaRS_Ec]{PRha}[rhlA_Pa rbwB_Srub] using XI and NEBuilder® HiFi DNA Assembly Master Mix, as disclosed in the example of WO2019154984. The assembled product was transformed into NEB® 10-β electroporated competent E. coli cells (New England Biolabs, Frankfurt / Main, Germany). 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{PRhaRS}[rhaRS_Ec]{PRha}[rbwA_Pan997 rbwB_Pan997]{ter} (SEQ ID NO:6).

[0066] Pseudomonas putida Strain KT2440 was transformed using plasmid pACYC{PRhaRS}[rhaRS_Ec]{PRha}[rbwA_Pan997 rbwB_Pan997]{ter} via electroporation (Iwasaki K et al., Biosci. Biotech. Biochem . 1994. 58(5):851-854)) and plated onto LB agar plates supplemented with kanamycin (50 µg / mL). The presence of the correct plasmid in the transformant was checked by plasmid preparation and analytical restriction analysis. The resulting strain was named BS-S-644 ( Pseudomonas putida KT2440 + pACYC {PRhaRS}[rhaRS_Ec]{PRha}[rbwA_Pan997rbwB_Pan997] {ter}).

[0067] Example 2 Construction of an expression vector for the Pantoea sp. SJZ147 gene rbwAB In order to come from Pantoea sp. SJZ147 genes rbwA _PanSJ as enzyme E1 and rbwB _PanSJ was used as a heterologous expression of enzyme E2, and the plasmid pACYC {PRhaRS}[rhaRS_Ec]{PRha}[rbwA_PanSJ rbwB_PanSJ] {ter}} was constructed. The plasmids encoding 3-(3'-hydroxyalkanoyloxy)alkanoic acid (HAA) synthase (RbwA, SEQ ID NO:7) and glucosyltransferase (RbwB, SEQ ID NO:8) were then used. rbwAB_The synthetic operon composed of _PanSJ (SEQ ID NO:9) is used in the rhamnose-inducible promoter P rha Under controlled cloning, the gene was inserted into the pAYCY184-based vector pACYC{PRhaRS}[rhaRS_Ec]{PRha}[rhlA_Pa rbwB_Srub] (WO2019154984) (New England Biolabs, Frankfurt / Main, Germany). The termination sequence was located downstream of the synthetic operon. The synthetic DNA generated by Eurofins Genomics was amplified by PCR. The plasmid pACYC{PRhaRS}[rhaRS_Ec]{PRha}[rhlA_Pa rbwB_Srub] carried P Rha The promoter cassette (SEQ ID NO:4) and the termination sequence (SEQ ID NO:5) are used for the p15A replication origin of E. coli and for... Pseudomonas putida The pVS1 replication origin in KT2440. The pVS1 origin origins originate from the Pseudomonas plasmid pVS1 (Itoh Y et al., Plasmid 1984, 11(3), 206-20). For amplification, Q5® High-Fidelity 2X Master Mix from New England Biolabs (Frankfurt / Main, Germany) was used according to the manufacturer's manual. In the next step, restriction sites were used. Apa I / Psp The amplified fragment was cloned into the vector pACYC{PRhaRS}[rhaRS_Ec]{PRha}[rhlA_Pa rbwB_Srub] using XI and NEBuilder® HiFi DNA Assembly Master Mix. The assembled product was transformed into NEB® 10-β electroporated competent E. coli cells (New England Biolabs, Frankfurt / Main, Germany). PCR purification, cloning, and transformation procedures were performed according to the manufacturer's manual. Restriction analysis was used to check for correct insertion of the target gene, and DNA sequencing was used to verify the authenticity of the introduced DNA fragment. The resulting plasmid was named pACYC{PRhaRS}[rhaRS_Ec]{PRha}[rbwA_PanSJ rbwB_PanSJ]{ter}} (SEQ ID NO: 10).

[0068] Pseudomonas putidaStrain KT2440 was transformed using plasmid pACYC {PRhaRS}[rhaRS_Ec]{PRha}[rbwA_PanSJrbwB_PanSJ]{ter} via electroporation (Iwasaki K et al., Biosci. Biotech. Biochem 1994.58(5):851-854)) and plated onto LB agar plates supplemented with kanamycin (50 µg / mL). The presence of the correct plasmid in the transformant was checked by plasmid preparation and analytical restriction analysis. The resulting strain was named BS-S-645 ( Disgusting fake orders Cytomegalovirus KT2440 + pACYC {PRhaRS}[rhaRS_Ec]{PRha}[rbwA_PanSJrbwB_PanSJ] {ter}).

[0069] Example 3 Construction of an expression vector for the rbwAB gene of the Pantoea stewartii strain NS381 In order to come from Pantoea stewartii strain NS381 genes rbwA _Pst381 as enzyme E1 and rbwB _Pst381 was used as a heterologous expression of enzyme E2, and the plasmid pACYC {PRhaRS}[rhaRS_Ec]{PRha}[rbwA_Pst381rbwB_Pst381] {ter} was constructed. The plasmid was then used to express _Pst381 as a heterologous expression of enzyme E2. rbwAB The synthetic operon composed of _Pst381 (SEQ ID NO: 13) is used in the rhamnose-inducible promoter P rha Under controlled cloning, the gene was cloned into the pAYCY184-based vector pACYC{PRhaRS}[rhaRS_Ec]{PRha}[rhlA_Pa rbwB_Srub] (WO2019154984) (New England Biolabs, Frankfurt / Main, Germany). The termination sequence was located downstream of the synthetic operon. The synthetic DNA generated by EurofinsGenomics was amplified by PCR. The plasmid pACYC{PRhaRS}[rhaRS_Ec]{PRha}[rhlA_Pa rbwB_Srub] carried P Rha The promoter cassette (SEQ ID NO:4) and the termination sequence (SEQ ID NO:5) are used for the p15A replication origin of E. coli and for... Pseudomonas putidaThe pVS1 replication origin in KT2440. The pVS1 origin origins originate from the Pseudomonas plasmid pVS1 (Itoh Y et al., Plasmid 1984, 11(3), 206-20). For amplification, Q5® High-Fidelity 2X Master Mix from New England Biolabs (Frankfurt / Main, Germany) was used according to the manufacturer's manual. In the next step, restriction sites were used. Apa I / Psp The amplified fragment was cloned into the vector pACYC {PRhaRS}[rhaRS_Ec]{PRha}[rhlA_Pa rbwB_Srub] using XI and NEBuilder® HiFi DNAAssembly Master Mix. The assembled product was transformed into NEB® 10-β electroporated competent E. coli cells (NewEnglandBiolabs, Frankfurt / Main, Germany). PCR purification, cloning, and transformation procedures were performed according to the manufacturer's manual. Restriction analysis was used to check for correct insertion of the target gene, and DNA sequencing was used to verify the authenticity of the introduced DNA fragment. The resulting plasmid was named pACYC {PRhaRS}[rhaRS_Ec]{PRha}[rbwA_Pst381rbwB_Pst381]{ter} (SEQ ID NO:14).

[0070] Pseudomonas putida Strain KT2440 was transformed using plasmid pACYC{PRhaRS}[rhaRS_Ec]{PRha}[rbwA_Pst381 rbwB_Pst381]{ter} via electroporation (Iwasaki K et al., Biosci. Biotech. Biochem . 1994. 58(5):851-854)) and plated onto LB agar plates supplemented with kanamycin (50 µg / mL). The presence of the correct plasmid in the transformant was checked by plasmid preparation and analytical restriction analysis. The resulting strain was named BS-S-646 ( Pseudomonas putida KT2440 + pACYC {PRhaRS}[rhaRS_Ec]{PRha}[rbwA_Pst381rbwB_Pst381] {ter}).

[0071] Example 4 Pseudomonas aeruginosa gene rhlA and Pantoea ananatis strain PNA 99-7 NODE_2 gene Construction of rbwB expression vector In order to come from Pantoea ananatis strain PNA 99-7 NODE_2 genes rhlA_Pa as enzyme E1 and rbwB _Pan997 was used as a heterologous expression of enzyme E2 to construct the plasmid pACYC {PRhaRS}[rhaRS_Ec]{PRha}[rhlA_Pa rbwB_Pan997] {ter}. The plasmid was then used to express _Pan997 as a heterologous expression of enzyme E2. rhlA_Pa The synthetic operon composed of (SEQ ID NO:17) is used in the rhamnose-inducible promoter P rha Under controlled cloning, the gene was inserted into the pAYCY184-based vector pACYC{PRhaRS}[rhaRS_Ec]{PRha}[rhlA_ParbwB_Srub] (WO2019154984). The termination sequence was located downstream of the synthetic operon. The gene was synthesized by Eurofins Genomics. rbwB _Pan997 is a DNA fragment. The plasmid pACYC {PRhaRS}[rhaRS_Ec]{PRha}[rhlA_ParbwB_Srub] carries P Rha The promoter cassette (SEQ ID NO:4) and the termination sequence (SEQ ID NO:5) are used for the p15A replication origin of E. coli and for... Pseudomonas putida The pVS1 replication origin in KT2440. The pVS1 origin origins originate from the Pseudomonas plasmid pVS1 (Itoh Y et al., Plasmid 1984, 11(3), 206-20). Using restrictive sites. Psp The XI and NEBuilder® HiFi DNA Assembly Master Mix were used to clone the synthesized DNA fragment into the vector pACYC {PRhaRS}[rhaRS_Ec]{PRha}[rhlA_Pa rbwB_Srub]. The assembled product was transformed into NEB® 10-β electroporated competent E. coli cells (NewEngland Biolabs, Frankfurt / Main, Germany). PCR purification, cloning, and transformation procedures were performed according to the manufacturer's manual. Restriction analysis was used to check for correct insertion of the target gene, and DNA sequencing was used to verify the authenticity of the introduced DNA fragment. The resulting plasmid was named pACYC {PRhaRS}[rhaRS_Ec]{PRha}[rhlA_Pa rbwB_Pan997]{ter} (SEQ ID NO:17).

[0072] Pseudomonas putida Strain KT2440 was transformed using plasmid pACYC{PRhaRS}[rhaRS_Ec]{PRha}[rhlA_ParbwB_Pan997]{ter} via electroporation (Iwasaki K et al., Biosci. Biotech. Biochem 1994.58(5):851-854)) and plated onto LB agar plates supplemented with kanamycin (50 µg / mL). The presence of the correct plasmid in the transformant was checked by plasmid preparation and analytical restriction analysis. The resulting strain was named BS-S-647 ( Disgusting fake orders Cytomegalovirus KT2440 + pACYC {PRhaRS}[rhaRS_Ec]{PRha}[rhlA_ParbwB_Pan997] {ter}).

[0073] Example 5 Construction of expression vectors for Pseudomonas aeruginosa gene rhlA and Pantoea sp. SJZ147 gene rbwB establish In order to come from Pantoea sp. SJZ147 genes rhlA _Pa as enzyme E1 and rbwB _PanSJ was used as a heterologous expression of enzyme E2, and the plasmid pACYC{PRhaRS}[rhaRS_Ec]{PRha}[rhlA_Pa rbwB_PanSJ]{ter} was constructed. The plasmid was then used to express _PanSJ as a heterologous expression of enzyme E2. rhlA_Pa The synthetic operon composed of (SEQ ID NO:18) is used in the rhamnose-inducible promoter P rha Under controlled cloning, the gene was inserted into the pAYCY184-based vector pACYC{PRhaRS}[rhaRS_Ec]{PRha}[rhlA_Pa rbwB_Srub] (WO2019154984) (New England Biolabs, Frankfurt / Main, Germany). The termination sequence was located downstream of the synthetic operon. The gene was synthesized by Eurofins Genomics. rbwB _PanSJ is a DNA fragment. The plasmid pACYC {PRhaRS}[rhaRS_Ec]{PRha}[rhlA_Pa rbwB_Srub] carries P Rha The promoter cassette (SEQ ID NO:4) and the termination sequence (SEQ ID NO:5) are used for the p15A replication origin of E. coli and for... Disgusting fake orders Cytomegalovirus The pVS1 replication origin in KT2440. The pVS1 origin origins originate from the Pseudomonas plasmid pVS1 (Itoh Y et al., Plasmid 1984, 11(3), 206-20). Using restrictive sites. Psp The XI and NEBuilder® HiFi DNAAssembly Master Mix were used to clone the synthesized DNA fragment into the vector pACYC {PRhaRS}[rhaRS_Ec]{PRha}[rhlA_Pa rbwB_Srub]. The assembled product was transformed into NEB® 10-β electroporated competent E. coli cells (NewEngland Biolabs, Frankfurt / Main, Germany). PCR purification, cloning, and transformation procedures were performed according to the manufacturer's manual. Restriction analysis was used to check for correct insertion of the target gene, and DNA sequencing was used to verify the authenticity of the introduced DNA fragment. The resulting plasmid was named pACYC {PRhaRS}[rhaRS_Ec]{PRha}[rhlA_Pa rbwB_PanSJ]{ter} (SEQ ID NO:19).

[0074] Pseudomonas putida strain KT2440 was transformed using plasmid pACYC{PRhaRS}[rhaRS_Ec]{PRha}[rhlA_ParbwB_PanSJ]{ter} via electroporation (Iwasaki K et al., Biosci. Biotech. Biochem 1994.58(5):851-854)) and plated onto LB agar plates supplemented with kanamycin (50 µg / mL). The presence of the correct plasmid in the transformant was checked by plasmid preparation and analytical restriction analysis. The resulting strain was named BS-S-648 ( Disgusting fake orders Cytomegalovirus KT2440 + pACYC {PRhaRS}[rhaRS_Ec]{PRha}[rhlA_ParbwB_PanSJ] {ter}).

[0075] Example 6 The rhlA gene of *Pseudomonas aeruginosa* and the rbwB gene of *Pantoea stewartii* strain NS381. Construction of expression carrier In order to come from Pantoea stewartii strain NS381 genes rhlA _Pa as enzyme E1 and rbwB_Pst381 was used as a heterologous expression of enzyme E2 to construct the plasmid pACYC{PRhaRS}[rhaRS_Ec]{PRha}[rhlA_ParbwB_Pst381]{ter}. The plasmid was then used to express _Pst381 as a heterologous expression of enzyme E2. rhlA_Pa The synthetic operon composed of (SEQ ID NO:20) is used in the rhamnose-inducible promoter P rha Under controlled cloning, the gene was inserted into the pAYCY184-based vector pACYC{PRhaRS}[rhaRS_Ec]{PRha}[rhlA_Pa rbwB_Srub] (WO2019154984) (New England Biolabs, Frankfurt / Main, Germany). The termination sequence was located downstream of the synthetic operon. The gene was synthesized by Eurofins Genomics. rbwB _Pst381 is a DNA fragment. The plasmid pACYC {PRhaRS}[rhaRS_Ec]{PRha}[rhlA_Pa rbwB_Srub] carries P Rha The promoter cassette (SEQ ID NO:4) and the termination sequence (SEQ ID NO:5) are used for the p15A replication origin of E. coli and for... Pseudomonas putida The pVS1 replication origin in KT2440. The pVS1 origin origins originate from the Pseudomonas plasmid pVS1 (Itoh Y et al., Plasmid 1984, 11(3), 206-20). Using restrictive sites. Psp The XI and NEBuilder® HiFi DNA Assembly Master Mix were used to clone the synthesized DNA fragment into the vector pACYC {PRhaRS}[rhaRS_Ec]{PRha}[rhlA_Pa rbwB_Srub]. The assembled product was transformed into NEB® 10-β electroporated competent E. coli cells (NewEngland Biolabs, Frankfurt / Main, Germany). PCR purification, cloning, and transformation procedures were performed according to the manufacturer's manual. Restriction analysis was used to check for correct insertion of the target gene, and DNA sequencing was used to verify the authenticity of the introduced DNA fragment. The resulting plasmid was named pACYC {PRhaRS}[rhaRS_Ec]{PRha}[rhlA_Pa rbwB_Pst381]{ter} (SEQ ID NO:21).

[0076] Pseudomonas putida Strain KT2440 was transformed using plasmid pACYC{PRhaRS}[rhaRS_Ec]{PRha}[rhlA_ParbwB_Pst381]{ter} via electroporation (Iwasaki K et al., Biosci. Biotech. Biochem . 1994.58(5):851-854)) and plated onto LB agar plates supplemented with kanamycin (50 µg / mL). The presence of the correct plasmid in the transformant was checked by plasmid preparation and analytical restriction analysis. The resulting strain was named BS-S-649 ( Disgusting fake orders Cytomegalovirus KT2440 + pACYC {PRhaRS}[rhaRS_Ec]{PRha}[rhlA_ParbwB_Pst381] {ter}).

[0077] Example 7 Production of Rubiwettin from Pseudomonas putida derivatives For the production of rubiwettin, we used the BioLector I system (Beckman Coulter Life Sciences, Baesweiler, Germany). The following strains were analyzed: BS-S-644: *Pseudomonas putida* KT2440 + pACYC {PRhaRS}[rhaRS_Ec]{PRha}[rbwA_Pan997 rbwB_Pan997] {ter} BS-S-645: *Pseudomonas putida* KT2440 + pACYC {PRhaRS}[rhaRS_Ec]{PRha}[rbwA_PanSJ rbwB_PanSJ] {ter} BS-S-646: *Pseudomonas putida* KT2440 + pACYC {PRhaRS}[rhaRS_Ec]{PRha}[rbwA_Pst381 rbwB_Pst381] {ter} BS-S-647: *Pseudomonas putida* KT2440 + pACYC {PRhaRS}[rhaRS_Ec]{PRha}[rhlA_ParbwB_Pan997] {ter} BS-S-648: *Pseudomonas putida* KT2440 + pACYC {PRhaRS}[rhaRS_Ec]{PRha}[rhlA_ParbwB_PanSJ] {ter} BS-S-649: *Pseudomonas putida* KT2440 + pACYC {PRhaRS}[rhaRS_Ec]{PRha}[rhlA_ParbwB_Pst381] {ter} The medium contained 1 ml of seed culture medium (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), and 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 4 H₂O, 0.01 g / L CuCl₂ x 2 H₂O, 0.03 g / L Na₂MoO₄ x 2 H₂O, 0.02 g / L NiCl₂ x 6 H₂O). Precultures derived from glycerol stock solution were inoculated into light-free FlowerPlates (48-well MTP, flower, Beckman Coulter LifeSciences, Baesweiler, Germany, Art.-No.: MTP-48-B) supplemented with kanamycin (50 µg / mL) and H2O. The precultures were incubated at 1000 rpm, 32°C, and 85% relative humidity for approximately 19 hours.

[0078] FlowerPlate (48-well MTP, flower, Beckman Coulter LifeSciences, Baesweiler, Germany, Art.-No.: M2P-MTP-48-BOH1) with pH and dissolved oxygen photosensitive electrodes was prepared in M12-FIT medium (2.2 g / L (NH4)2SO4, 0.02 g / L NaCl, 0.4 g / L MgSO4 x 7H2O, 0.05 g / L CaCl2 x 2H2O, 3 g / L KH2PO4, 8.51 g / L Na2HPO4*2H2O, 5 g / L glucose, 25 g / L maltodextrin (C*Dry MD 01955), 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...). The mixture consisted of 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, and 1 g / L FeSO4 x 7 H2O, supplemented with kanamycin (50 µg / mL). The pre-culture was used to inoculate the major culture to achieve an initial OD of 0.5. 600 The main culture was incubated at 800 rpm, 32°C, and 85% relative humidity for approximately 50 hours.

[0079] The feed was initiated by adding a 1% (v / v) sterile filtered amylase solution (100,000 U / L) from Aspergillus niger, triggered by the pO2 peak indicating the end of the batching phase. Simultaneously, target gene expression was induced with 0.2% (w / v). After culturing, the concentrations (mg / L) of rubiwettin with different chain lengths were determined (see Table 1).

[0080] Table 1: Using different Pseudomonas putida The concentration of rubiwettin obtained from the strain. Indicates the concentration of each rubiwettin class having the indicated chain length and saturation of the 3-hydroxy fatty acid moiety.

[0081] Table 2: Using different Pseudomonas putida The proportions of each rubiwettin class produced by the strain. The proportions of each rubiwettin class with the indicated chain length and saturation of the 3-hydroxy fatty acid moiety are given as a percentage of total rubiwettin.

[0082] The results showed that using from Pantoea spec. SEQ-ID 1, 7 and 11 replace those from verdigris Monoclonal bacteria The SEQ-ID as E1 causes rubiwettin compounds to shift from C10:0-C12:1 and C10:0-C12:0 to C8:0-C10:0. Rubiwettin compounds with shorter 3-hydroxy fatty acid chain lengths will have different applications than those with longer 3-hydroxy fatty acid chain lengths.

[0083] Example 7 The *Pseudomonas aeruginosa* gene *rhlA* and the *rbwB* gene from *Serratia rubidaea*. Construction of expression carrier For genes rhlA _Pa as enzyme E1 and from Serratia rubidaea The gene rbwB_Srub was expressed heterologously as part of the E2 enzyme combination using a pACYC5 vector backbone as described in Example 3 (WO2019154984) to position these genes in the rhamnose-inducible promoter P. rha Under their control.

[0084] Example 8 Production of Rubiwettin from Pseudomonas putida derivatives For the production of rubiwettin, we used the BioLector I system (Beckman Coulter Life Sciences, Baesweiler, Germany). The following strains were analyzed: BS-S-368: *Pseudomonas putida* KT2440 + pACYC_rhlA_Pa_rbwB_Srub (Example 7) BS-S-647: *Pseudomonas putida* KT2440 + pACYC {PRhaRS}[rhaRS_Ec]{PRha}[rhlA_ParbwB_Pan997] {ter} (Example 4) BS-S-648: *Pseudomonas putida* KT2440 + pACYC {PRhaRS}[rhaRS_Ec]{PRha}[rhlA_ParbwB_PanSJ] {ter} (Example 5) BS-S-649: *Pseudomonas putida* KT2440 + pACYC {PRhaRS}[rhaRS_Ec]{PRha}[rhlA_ParbwB_Pst381] {ter} (Example 6) The medium contained 1 ml of seed culture medium (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), and 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 4 H₂O, 0.01 g / L CuCl₂ x 2 H₂O, 0.03 g / L Na₂MoO₄ x 2 H₂O, 0.02 g / L NiCl₂ x 6 H₂O). Precultures derived from glycerol stock solution were inoculated into light-free FlowerPlates (48-well MTP, flower, Beckman Coulter LifeSciences, Baesweiler, Germany, Art.-No.: MTP-48-B) supplemented with kanamycin (50 µg / mL) and H2O. The precultures were incubated at 1000 rpm, 32°C, and 85% relative humidity for approximately 19 hours.

[0085] FlowerPlate (48-well MTP, flower, Beckman Coulter LifeSciences, Baesweiler, Germany, Art.-No.: M2P-MTP-48-BOH1) with pH and dissolved oxygen photosensitive electrodes was prepared in M12-FIT medium (2.2 g / L (NH4)2SO4, 0.02 g / L NaCl, 0.4 g / L MgSO4 x 7H2O, 0.05 g / L CaCl2 x 2H2O, 3 g / L KH2PO4, 8.51 g / L Na2HPO4*2H2O, 5 g / L glucose, 25 g / L maltodextrin (C*Dry MD 01955), 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...). The mixture consisted of 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, and 1 g / L FeSO4 x 7 H2O, supplemented with kanamycin (50 µg / mL). The pre-culture was used to inoculate the major culture to achieve an initial OD of 0.5. 600 The main culture was incubated at 800 rpm, 32°C, and 85% relative humidity for approximately 50 hours.

[0086] The feed was initiated by adding a 1% (v / v) sterile filtered amylase solution (100,000 U / L) from Aspergillus niger, triggered by the pO2 peak indicating the end of the batching phase. Simultaneously, target gene expression was induced with 0.2% (w / v). After culture, the concentrations (mg / L) of rubiwettin with different chain lengths were determined (see Table 3).

[0087] Table 3: Using different Pseudomonas putida The concentration of rubiwettin obtained from the strain. Indicates the concentration of each rubiwettin class having the indicated chain length and saturation of the 3-hydroxy fatty acid moiety.

[0088] The results show that the source of this application is used. Pantoea spec. The SEQ ID NO: 2, SEQ ID NO: 8, or SEQ ID NO: 12 replaces the source from WO2019154984. Serratia marcescens rubidaea)SEQ ID NO: 4, as enzyme E2, leads to the production of higher amounts of rubiwettin.

[0089] Example 9 HPLC-based quantification of rubiwettin Lipids R1 and RG1 were quantified using HPLC. Using a Combitip, 400 µl of n-propanol was introduced into a 2 mL reaction vessel, which was immediately closed to minimize evaporation. Then, 400 µl of fermentation broth was added. After shaking at 30 Hz for 1 min in a Retsch mill, the resulting crude extract mixture was centrifuged at 13,000 rpm for 5 min, sterilely filtered through a 0.2 µm PVDF filter, and 800 µl of the clear supernatant was transferred to an HPLC vial. Further dilution with cell broth was performed in 55% (v / v) propanol. Samples were stored at -20 °C before measurement.

[0090] To detect and quantify lipids, an evaporative light scattering detector (Sedex LT-ELSD model 85LT) was used. Measurements were performed using an Agilent Technologies 1200 series (Santa Clara, Calif.) and a Zorbax SB-C8 RapidResolution column (4.6 x 150 mm, 3.5 µm, Agilent). The injection volume was 5.0 µl, and the run time was 20 min. Mobile phase A: 0.1% TFA aqueous solution (trifluoroacetic acid, solution); mobile phase B: methanol. The column temperature was 40 °C. An ELSD (detector temperature 60 °C) and a DAD (diode array, 210 nm) were used as detectors.

[0091] gradient: .

[0092] The gradient used started with 70% B / A, reaching 100% B within 15 minutes at a flow rate of 1 mL / min, and then reequilibrated with 70% B / A for 5 minutes (see Table 2). The identity and purity of the reference material were verified by HPLC-MS / MS and NMR.

Claims

1. A microbial cell for producing at least one lipid having general formula II from at least one carbon substrate, Formula II Where R 1 and R 2 Each contains, independently, the same or different organic groups, each having 5 to 13 carbon atoms. The cells mentioned above are non-pathogenic cells that have been genetically modified to increase the heterologous expression of the following enzymes relative to wild-type cells: - Enzyme E1, comprising a 3-(3-hydroxyalkanoyloxy)alkyl acid (HAA) synthase of SEQ ID NO: 1, SEQ ID NO: 7, SEQ ID NO: 11, SEQ ID NO: 15 or variants thereof, wherein said variant comprises 60% sequence identical to SEQ ID NO: 1, 7, 11 or 15; and - Enzyme E2, comprising a glycosyltransferase (EC2.4) of SEQ ID NO: 2, SEQ ID NO: 8 or SEQ ID NO: 12 or a variant thereof, wherein the variant comprises 60% sequence identical to SEQ ID NO: 2, 8 or 12.

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

3. The cell according to claim 2, wherein the alkyl group is selected from pentyl, heptyl, nonyl, undecyl, and tridecyl.

4. The cell according to any one of the preceding claims, wherein the cell produces additional lipids having general formula I from a carbon substrate. General Formula I Where R 1 and R 2 Each contains, independently, the same or different organic groups, each having 5 to 13 carbon atoms.

5. The cell according to claim 4, wherein R in the lipid having general formula I is a saturated alkyl group.

6. The cell according to any one of the preceding claims, wherein the carbon source is selected from glucose, dextrose, sucrose, xylose, mannose, galactose, arabinose, monosaccharides, polysaccharides, cellulose, hemicellulose, vegetable oil, animal fat, fatty acids, fatty acid esters, carbon-containing gases, alkanes, glycerol, acetates, ethanol, and methanol.

7. The cell according to any one of the preceding claims, wherein the cell is selected from... Acinetobacter spp., Bacillus spp. Genus *Bryophytes*, *Burkholderia* sp., *Chlorella*, *Clostridium*, *Corynebacterium*, cyanobacteria, Escherichia coli, Pseudomonas, Klebsiella, Salmonella, Rhizobium, Yeast, Pichia pastoris and Candida genus.

8. The cell according to any one of the preceding claims, wherein the cell is selected from... Bacillus subtilis, Burke of Thailand Burkholderia thailandensis, Corynebacterium glutamicum, Escherichia coli, Klebsiella pneumoniae, *Pseudomonas fluorescens*, *Pseudomonas putida*, *Pseudomonas schrenckii*, *Rhizobium alfalfa*, *Saccharomyces cerevisiae* and Pichia pastoris.

9. A method for producing at least one lipid having general formula II and / or general formula I: Formula II, General Formula I Where R 1 and R 2 Each independently contains the same or different organic groups, each having 5 to 13 carbon atoms, and The method includes the step of contacting at least one cell according to any one of claims 1 to 8 with at least one carbon source.

10. The method according to claim 9, wherein the carbon source is selected from glucose, dextrose, sucrose, xylose, mannose, galactose, arabinose, monosaccharides, polysaccharides, cellulose, hemicellulose, vegetable oil, animal fat, fatty acids, fatty acid esters, carbon-containing gases, alkanes, glycerol, acetates, ethanol, and methanol.

11. Use of the cells according to any one of claims 1 to 8 for the production of at least one lipid having general formula I and / or II: Formula II, General Formula I Where R 1 and R 2 Each contains, independently, the same or different organic groups, each having 5 to 13 carbon atoms.

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