Biotechnical production of ω-functionalized carboxylic acids and their esters

Abstract

This invention relates to the biotechnological production of ω-functionalized carboxylic acids and their esters. Microbial cells are provided for the production of at least one ω-functionalized carboxylic acid ester from at least one alkane, wherein the cells are genetically modified relative to wild-type cells to increase the expression of: (i) an enzyme E1 capable of converting the alkane to the corresponding 1-alkanol; (ii) an enzyme E2 capable of converting the 1-alkanol of (i) to the corresponding 1-alkanal; (iii) an enzyme E3 capable of converting the 1-alkanal of (ii) to the corresponding alkanoic acid; (iv) an enzyme E4 capable of converting the alkanoic acid of (iii) to the corresponding alkanoic acid ester; and (iv) an enzyme E5 capable of converting the alkanoic acid ester of (iv) to the corresponding ω-hydroxy-alkanoic acid ester, and wherein the cells do not contain an increase relative to wild-type cells of the expression of the following enzymes selected from the group consisting of: 20 -E 24 Genetic modification of expression of at least one of them: -E 20 Acyl-ACP thioesterase, -E 21 Acyl-CoA thioesterase, -E 22 Acyl-CoA:ACP transacylase, -E 23 Polyketide synthase, and -E 24 Hexanoic acid synthase.

Description

Technical Field

[0001] This invention relates to a biotechnological method for producing ω-functionalized carboxylic acid esters. Specifically, the method uses alkanes as starting materials and genetically modified cells to convert alkanes into the corresponding ω-functionalized carboxylic acid esters. Background Technology

[0002] ω-functionalized carboxylic acids and their corresponding esters, including ω-aminocarboxylic acids and their corresponding lactams, such as ω-aminolauric acid, ω-aminoundecanoic acid, and laurolactam, are important monomers for the production of high-performance polyamides. Some existing chemical technologies used to produce these monomers from petrochemical or renewable feedstocks include:

[0003] i) Production of ω-aminolauric acid from methyl lauryl ester (a biodiesel fraction prepared from palm kernel or coconut oil)

[0004] ii) Production of ω-aminoundecanoic acid from ricinoleic acid (prepared from castor oil)

[0005] iii) Production of laurolactam from butadiene

[0006] Although all three production methods remain competitive, their competitiveness at a given location and time depends on many factors, including raw material costs and energy costs of the operating method.

[0007] Several biotechnical means for producing ω-functionalized carboxylic acids and / or their esters are known in the art. For example, genetically modified cells capable of producing ω-functionalized carboxylic acids from carboxylic acids used as substrates have been previously described at least in WO2009077461 and EP2322598. A very similar procedure using Candida albicans cells is described in WO2011008232, in which the β-oxidation pathway is blocked in the cells and ω-functionalized carboxylic acids are formed from fatty acids used as substrates. These methods have the disadvantage of using fatty acids as starting materials. This is because the fatty acids and their derivatives used are mainly obtained only from plant and animal oils or fats. Animal fats as raw materials are still rarely accepted by customers, and plant oils containing short and medium-length fatty acids are difficult to obtain or are only produced in tropical regions (as a result of rainforest destruction). Furthermore, specific plant and animal oil or fat raw materials have specific, but limited, fatty acid profiles, leading to coupled production.

[0008] WO2013024114 discloses a method for producing ω-functionalized carboxylic acids and / or their esters from simple carbon sources such as glucose, sucrose, arabinose, xylose, lactose, fructose, maltose, molasses, starch, cellulose, and hemicellulose, as well as glycerol or very simple organic molecules such as CO2, CO, or syngas. These simple sugars, especially glucose, are generally more expensive to obtain. Methods for producing ω-functionalized carboxylic acids and / or their esters from simple carbon sources can also be considered complex because the cells used in these methods must be genetically modified to increase the production of carboxylic acids from these simple carbon sources in the first place. This therefore increases production costs.

[0009] Therefore, there is a need in the art for a method to produce ω-functionalized carboxylic esters from another source of raw materials, which enables efficient and effective production. Summary of the Invention

[0010] This invention seeks to address the aforementioned problems by providing at least one genetically modified microbial cell capable of producing at least one ω-functionalized carboxylic acid ester from at least one alkane. The ω-functionalized carboxylic acid ester may be selected from ω-hydroxy, ω-oxo, ω-carboxyl, and ω-aminocarboxylic acid esters. Using these genetically modified cells in the production of ω-functionalized carboxylic acid esters increases the flexibility of the production of these compounds by enabling the use of readily available alternative petrochemical feedstocks. Furthermore, the use of a whole-cell biocatalyst capable of integrating a complete means of converting alkanes into fatty acid esters and their corresponding ω-functionalized derivatives simplifies the conversion process, as only a small number of process steps are involved. The dependence on fatty acids and simple carbon sources as carbon substrates is also eliminated.

[0011] According to a first aspect of the invention, a microbial cell for producing at least one ω-functionalized carboxylic acid ester from at least one alkane is provided, wherein the cell comprises genetic modifications that increase the expression of the following relative to wild-type cells:

[0012] (i) Enzyme E1 capable of converting alkanes into the corresponding 1-alkanols;

[0013] (ii) Enzyme E2 that can convert the 1-alkanol in (i) into the corresponding 1-alkanal;

[0014] (iii) Enzyme E3 that can convert the 1-alkane aldehyde of (ii) into the corresponding alkanoic acid;

[0015] (iv) Enzyme E4 capable of converting the alkyl acids of (iii) into the corresponding alkyl esters; and

[0016] (v) Enzyme E5, which can convert the alkyl esters of (iv) into the corresponding ω-hydroxyalkyl esters.

[0017] Microbial cells according to any aspect of the invention refer to cells that have not been previously modified to increase the formation of carboxylic acids or carboxylic esters from at least one simple carbon source. The term "simple carbon source" is understood to mean a carbon source in which at least one C / C bond in the carbon skeleton is broken. Specifically, the simple carbon source can be at least one carbohydrate, such as, for example, glucose, sucrose, arabinose, xylose, lactose, fructose, maltose, molasses, starch, cellulose, and hemicellulose, but the carbon source can also include glycerol or very simple organic molecules such as CO2, CO, or syngas. More specifically, microbial cells according to any aspect of the invention can be ungenetically modified to increase the expression of at least one of the following enzymes relative to wild-type cells:

[0018] - EC 3.1.2.14 or EC 3.1.2.22 E 20 Acyl-ACP thioesterase,

[0019] - E of EC 3.1.2.2, EC 3.1.2.18, EC 3.1.2.19, EC 3.1.2.20 or EC 3.1.2.22 21 Acyl-CoA thioesterase,

[0020] - E 22 Acyl-CoA:ACP transacylase

[0021] - E 23 Polyketide synthases catalyze reactions involved in the synthesis of carboxylic acids and carboxylic acid esters.

[0022] - E 24 Hexanoic acid synthase.

[0023] In one instance, a cell according to any aspect of the invention may contain further genetic modifications to increase the expression of the following relative to wild-type cells:

[0024] (vi) Enzyme E6, which can convert the ω-hydroxy-alkanoate of (v) to the corresponding ω-oxoalkanoate; and

[0025] (vii) is an enzyme E7 that can convert the ω-oxoalkyl ester of (vi) into the corresponding ω-aminoalkyl ester.

[0026] In another instance, cells according to any aspect of the invention may contain further genetic modifications to increase the expression of the following relative to wild-type cells:

[0027] (vi) Enzyme E6, which can convert the ω-hydroxy-alkanoate of (v) into the corresponding ω-oxoalkanoate;

[0028] (vii) Enzyme E capable of converting the ω-oxoalkyl ester of (vi) into the corresponding ω-carboxyalkyl ester. 13 ,and

[0029] (viii) Enzyme E capable of converting the ω-carboxyalkyl ester of (vi) into the corresponding ω-carboxyalkyl diester. 14 .

[0030] Cells according to any aspect of the invention can be used to produce ω-functionalized carboxylates from all alkanes in culture supernatants with high space-time yield, high carbon yield, and high concentration. As a result of these advantages, efficient workup is facilitated.

[0031] The phrase “wildtype,” as used herein in conjunction with cells or microorganisms, can refer to a cell having a genome composition that is naturally present in the form seen in the natural environment. This term can be applied to the whole cell and individual genes. Therefore, the term “wildtype” can also include cells that have been genetically modified in other respects (i.e., with respect to one or more genes), but not in relation to the target gene. Therefore, the term “wildtype” does not include cells in which the gene sequence of a specific target gene has been artificially at least partially altered using recombinant methods. Therefore, a wild-type cell according to any aspect of the invention refers to a cell that has no genetic mutations relative to the whole genome and / or a specific gene. Thus, in one example, a wild-type cell with respect to enzyme E1 can refer to a cell that has natural / unaltered expression of enzyme E1. Regarding enzymes E2, E3, E4, E5, E6, E7, E8, E9, E… 10 E 11 E 12 E 13 E 14 E 15 Wild-type cells can be explained in the same way, and can refer to cells that respectively possess enzymes E2, E3, E4, E5, E6, E7, E8, E9, and E 10 E 11 E 12 E 13 E 14 E 15 Cells with natural / unaltered expression, etc.

[0032] Any enzyme used according to any aspect of the invention may be an isolated enzyme. Specifically, the enzyme used according to any aspect of the invention may be used in its active state and in the presence of all cofactors, substrates, auxiliary and / or activating peptides or factors necessary for its activity. As used herein, the term "isolated" means that the target enzyme is enriched compared to the cells in which it is naturally present. The enzyme may be enriched by SDS-polyacrylamide electrophoresis and / or activity assays. For example, the target enzyme may constitute more than 5%, 10%, 20%, 50%, 75%, 80%, 85%, 90%, 95% or 99% of all peptides present in the preparation, as determined by visual examination of the polyacrylamide gel after staining with Coomassie blue dye.

[0033] The enzymes used according to any aspect of the invention can be recombinant. As used herein, the term "recombinant" refers to a molecule or encoded by such a molecule, particularly a polypeptide or nucleic acid, which is not naturally occurring but is the result of genetic engineering; 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 that promoter has been engineered to overexpress the catalytically active polypeptide at levels relative to the polypeptide in a corresponding wild-type cell containing the original, unaltered nucleic acid molecule.

[0034] Those skilled in the art will be able to genetically modify cells or microorganisms using any method known in the art. According to any aspect of the invention, genetically modified cells can be genetically modified such that, within a defined time interval, within 2 hours, particularly 8 hours or 24 hours, they form at least one or two times, particularly at least 10 times, at least 100 times, at least 1000 times, or at least 10000 times, ω-functionalized carboxylates of wild-type cells. The increase in product formation can be determined, for example, by culturing cells according to any aspect of the invention and wild-type cells separately 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 (ω-functionalized carboxylates) in the nutrient medium.

[0035] Genetically modified cells or microorganisms can be genetically different from wild-type cells or microorganisms. The genetic difference between a genetically modified microorganism according to any aspect of the invention and a wild-type microorganism may be the presence of complete genes, amino acids, nucleotides, etc., in the genetically modified microorganism, which may not be present in the wild-type microorganism. In one example, a genetically modified microorganism according to any aspect of the invention may contain an enzyme that enables the microorganism to produce more 1-alkanols, 1-alkanales, alkyl acids, alkyl esters, ω-hydroxyalkyl esters, etc., than wild-type cells. Wild-type microorganisms may lack the enzyme or have detectable enzyme activity relative to the genetically modified microorganism of the invention, which enables the genetically modified microorganism to produce 1-alkanols, 1-alkanales, alkyl acids, alkyl esters, ω-hydroxyalkyl esters, etc. As used herein, the term "genetically modified microorganism" may be used interchangeably with the term "genetically modified cell." Genetic modification according to any aspect of the invention is carried out on the cells of a microorganism.

[0036] Cells according to any aspect of the invention are genetically transformed according to any method known in the art. Specifically, the cells can be prepared according to the method disclosed in WO2013024114.

[0037] As used herein, the phrase “enzyme activity increased in genetically modified cells compared to their wild-type” means that the corresponding enzyme activity is increased by at least 2 times, particularly at least 10 times, more particularly at least 100 times, even more particularly at least 1000 times, or even more particularly at least 10000 times.

[0038] As used herein, the phrase "increased enzyme activity" should be understood as increased intracellular activity. Essentially, increased enzyme activity can be achieved by: increasing the copy number of one or more gene sequences encoding the enzyme; using a strong promoter or employing a gene or allele encoding a corresponding enzyme with increased activity; altering codon utilization of the gene; increasing the half-life of the mRNA or enzyme in various ways; modifying the regulation of gene expression; and optionally by combining these measures. Genetically modified cells used according to any aspect of 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 that gene, or a portion thereof, and a vector enabling gene expression. Heterologous expression is specifically achieved by integrating the gene or allele into a cellular chromosome or an extrachromosomal replication vector.

[0039] In the same context, the phrase "enzyme E" is used with respect to any aspect of the invention. x"Reduced activity" can be understood to mean a reduction in activity of at least 0.5-fold, particularly at least 0.1-fold, more particularly at least 0.01-fold, even more particularly at least 0.001-fold, and most particularly at least 0.0001-fold. The phrase "reduced activity" also encompasses the absence of detectable activity ("zero activity"). A reduction in the activity of a particular enzyme can be achieved, for example, through selective mutation or other measures known to those skilled in the art for reducing the activity of a particular enzyme. Specifically, those skilled in the art have found teachings on modifying and reducing protein expression and the accompanying reduction in enzyme activity by disrupting specific genes, for example, at least in Dubeau et al. 2009, Singh and Röhm. 2008, Lee et al. 2009, etc. The reduction of enzymatic activity in cells according to any aspect of the invention can be achieved by modifying a gene containing one of the nucleic acid sequences, wherein the modification is selected from the group consisting of: insertion of foreign DNA into a gene, deletion of at least a portion of a gene, point mutation in a gene sequence, RNA interference (siRNA), antisense RNA, or modification (insertion, deletion, or point mutation) of regulatory sequences (such as, for example, promoters and terminators) or ribosome binding sites, which are laterally attached to genes.

[0040] Foreign DNA should be understood in this context to mean any DNA sequence that is “foreign” to a gene (rather than to an organism), i.e., endogenous DNA sequences can also act as “foreign DNA” in this respect. In this regard, it is particularly preferred that the gene is disrupted by inserting a selection marker gene, and thus the foreign DNA is a selection marker gene, wherein the insertion is preferably achieved through homologous recombination at a locus.

[0041] The expression of the enzymes and genes mentioned above and all the enzymes and genes listed below can be determined by using 1-dimensional and 2-dimensional protein gel separation and then optically identifying the protein concentration in the gel with appropriate evaluation software.

[0042] If the increase in enzyme activity is based solely on an increase in the expression of the corresponding gene, the quantification of the increase in enzyme activity can be easily determined by comparing one-way or two-way protein separation between wild-type and genetically modified cells. A common method for preparing protein gels and identifying proteins using bacteria is the method described by Hermann et al. (Electrophoresis, 22:1712-23 (2001)). Protein concentration can also be determined by Western blot hybridization with antibodies specific to the protein being measured (Sambrook et al., Molecular Cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NYUSA, 1989), followed by optical evaluation using appropriate software for concentration determination (Lohaus and Meyer (1989) Biospektrum, 5:32-39; Lottspeich (1999), Angewandte Chemie 111:2630-2647). This method is always an option when the possible products of the reaction catalyzed by the enzyme activity to be measured can be rapidly metabolized in microorganisms or when the activity in the wild type is too low to be sufficient to determine the enzyme activity based on production formation.

[0043] Specifically,

[0044] - Enzyme E1 can be selected from P450 alkane hydroxylase (E a AlkB alkane hydroxylase (E) and EC 1.14.15.3 b );

[0045] - Enzyme E2 can be selected from P450 alkane hydroxylase (E a AlkB alkane hydroxylase (E) of EC 1.14.15.3 b ), EC1.1.3.20 alcohol oxidase (E c ) and alcohol dehydrogenase (E d );

[0046] - Enzyme E3 is selected from P450 alkane hydroxylase (E) of EC 1.14.15.3. a AlkB alkane hydroxylase (E) of EC 1.14.15.3 b ), aldehyde dehydrogenase (E) e Bifunctional alcohol oxidase (E) of EC 1.1.3.20 c ), bifunctional AlkJ alcohol dehydrogenase (E diBifunctional alcohol dehydrogenases (E) of EC1.1.1.1 or EC1.1.1.2 dii ), where E c E di and E dii It can directly oxidize 1-alkanols to the corresponding alkanoates via 1-alkanal;

[0047] - Enzyme E4 can be selected from wax ester synthase (E f ) and alcohol O-acyltransferase (E g );

[0048] - Enzyme E5 can be selected from P450 alkane hydroxylase (E a AlkB alkane hydroxylase (E) and EC 1.14.15.3 b );

[0049] - Enzyme E6 can be selected from P450 alkane hydroxylase (E a AlkB alkane hydroxylase (E) of EC 1.14.15.3 b ), alcohol oxidase (E) c ) and alcohol dehydrogenase (E d );

[0050] - Enzyme E7 can be ω-transaminase (E h ).

[0051] In one instance, enzymes E1, E2, E3, and E5 can each be different enzymes capable of exercising their activities. For example, E1 could be AlkB alkane hydroxylase (E b E2 can be an alcohol oxidase (E c E3 can be a bifunctional AlkJ alcohol dehydrogenase, and E5 can be an AlkB alkane hydroxylase (E b In another instance, E1 could be P450 alkane hydroxylase (E... a E2 can be an alcohol dehydrogenase (E2). c E3 can be aldehyde dehydrogenase (E e E1, E2, E3, and E5 can be P450 alkane hydroxylases. Specifically, any combination of enzymes E1, E2, E3, and E5 can be used to perform their specific functions. In a further example, E1, E2, E3, and E5 can be the same enzyme. In this example, E1, E2, E3, and E5 can be selected from P450 alkane hydroxylases (E...). a ) and AlkB alkyl hydroxylase (E b In one instance, E1, E2, E3, and E5 could be P450 alkane hydroxylases (E...). aIn this example, the cells according to any aspect of the invention contain an increased amount of P450 alkane hydroxylase (E) relative to wild-type cells. a (This satisfies the functions of enzymes E1, E2, E3, and E5). In another example, E1, E2, E3, and E5 could be AlkB alkane hydroxylase (E b In this example, cells according to any aspect of the invention contain an increased amount of AlkB alkane hydroxylase (E) relative to wild-type cells. b (It satisfies the functions of enzymes E1, E2, E3 and E5) expression.

[0052] Enzyme E a To E h It may contain a polypeptide sequence in which up to 60%, preferably up to 25%, particularly up to 15%, particularly up to 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1% of amino acid residues compared to the following reference sequence (accession number) are modified by deletion, insertion, substitution or a combination thereof, and it still has at least 50%, preferably 65%, particularly preferably 80%, particularly greater than 90% of the activity of a protein having the corresponding following reference sequence, wherein 100% activity of the reference protein is understood to mean an increase in the activity of the cell used as a biocatalyst (i.e., the amount of substance converted per unit time based on the amount of cells used (units / gram dry cell weight [U / g CDW])) compared to the activity of the biocatalyst in the absence of the reference protein.

[0053] Modifications of amino acid residues in a given polypeptide sequence (which do not significantly alter the properties and function of the given polypeptide) are known to those skilled in the art. Therefore, many amino acids can often be interchanged without problem; examples of such suitable amino acid substitutions are: Ala replaced by Ser; Arg replaced by Lys; Asn replaced by Gln or His; Asp replaced by Glu; Cys replaced by Ser; Gln replaced by Asn; Glu replaced by Asp; Gly replaced by Pro; His replaced by Asn or Gln; Ile replaced by Leu or Val; Leu replaced by Met or Val; Lys replaced by Arg or Gln or Glu; Met replaced by Leu or Ile; Phe replaced by Met or Leu or Tyr; Ser replaced by Thr; Thr replaced by Ser; Trp replaced by Tyr; Tyr replaced by Trp or Phe; Val replaced by Ile or Leu. It is also known that modifications, particularly those at the N- or C-terminus of a polypeptide in the form of, for example, amino acid insertions or deletions, often have no significant effect on the function of the polypeptide.

[0054] Throughout this specification, the accession numbers described in connection with the invention correspond to NCBI ProteinBank database entries with the date July 26, 2011; typically, the version number of an entry is identified herein by a “number” such as, for example, “.1”.

[0055] Unless otherwise stated, all percentages (%) are mass percentages.

[0056] According to any aspect of the invention, the microbial cells may be selected from the following bacterial species: *Atrophic bacteria* (*Bacteria* genus). Abiotrophia ), Acaryochloris , Accumulibacter acevibrio ( Acetivibrio Acetobacter spp. Acetobacter Acetobacter spp. Acetohalobium ), Acetobacter genus ( Acetonema ), Achromobacterium spp. Achromobacter ), amino acid cocci ( Acidaminococcus ), Acidilactones ( Acidimicrobium ), Acidophilus genus ( Acidiphilium ), genus *Thiobacillus* Acidithiobacillus ), Acidobacterium spp. Acidobacterium ), genus *Thermolytica* ( Acidothermus ), Acidobacteria ( Acidovorax Acinetobacter spp. Acinetobacter Actinobacterium spp. Actinobacillus Actinomycetes ( Actinomyces ), Actinomycetes genus ( Actinosynnema ), Balloonbacteria ( Aerococcus ), Aerophytes ( Aeromicrobium Aeromonas spp. Aeromonas ), Aphibotomyces ( Afipia ), genus *Agglutinosa* ( Aggregatibacter) Agrobacterium spp. Agrobacterium ), Ahrensia , Akkermansia Alkali-eating fungi ( Alcanivorax ), Cyclocycline Bacillus spp. Alicycliphilus) Cyclocycline Bacillus ( Alicyclobacillus ), Aliivibrio Alkali lake bacteria ( Alkalilimnicola ), Alkaliphilus Heterochromatids ( Allochromatium) Alternating Monocystes ( Alteromonadales Alternating Monoclonal bacteria () Alteromonas ), Aminobacterium Aminoaminomonas ( ) Aminomonas) Ammonia-producing bacteria ( ) Ammonifex ), Acidobacterium pseudoamycetes ( Amycolatopsis ), Amycolicicoccus Anabaena ( ) Anabaena) Anaerobic microbacteria ( Anaerobaculum ), Anaerobic cocci ( Anaerococcus) , Anaerofustis Anaerobic rosary bacteria ( Anaerolinea) Anaerobic myxobacteria ( Anaeromyxobacter) , Anaerostipes , Anaerotruncus Intangible entities ( Anaplasma ), Anaerobic Bacillus spp. ( Anoxybacillus) , genus of liquid-producing bacteria ( Aquifex Cryptobacterium spp. Arcanobacterium ), Toxoplasma spp. Arcobacter ), Aromatoleum Arthrobacter spp. Arthrobacter ), Arthrospira genus *Amygdalinum* ( ) Asticcacaulis ), Strange Bacteria genus ( Atopobium ), genus *Citronium* Aurantimonas ), *Azotobacter* spp. Azoarcus ), nitrogen-fixing rhizobia ( Azorhizobium ), Azospirobacter spp. Azospirillum ), nitrogen-fixing bacteria ( Azotobacter ), Bacillus ( Bacillus ), Bartonoidea ( Bartonella ), Basfia , Baumannia Bdellovibrio ( Bdellovibrio ), Bezatobacter spp. Beggiatoa ), spp. of Bayerlink ( Beijerinckia ), Bermanella genus *Syngonium* Beutenbergia) Bifidobacterium spp. Bifidobacterium ), bilirubin Bilophila ), Blastopirellula , Blautia , Blochmannia Bordetella spp. Bordetella ), genus *Treponema* ( Borrelia ), genus *Brachybacteria* ( Brachybacterium ), Short Spirochetes ( Brachyspira ), genus *Bacillus* ( Bradyrhizobium ), Bacillus pumilus ( Brevibacillus ), genus *Brucea* ( Brevibacterium ), Shortwave Monoclonal bacteria ( Brevundimonas Brucella ( ) Brucella ), Bukharia spp. Buchnera ), Bulleidia Burkholderia ( Burkholderia ), Vibrio butyricum ( Butyrivibrio ), Caldalkalibacillus , Caldanaerobacter pyrolytic cellulose bacteria ( Caldicellulosiruptor ), Calditerrivibrio , Caminibacter Campylobacter ( Campylobacter ), Carboxydibrachium Carbon monoxide thermophilic bacteria ( Carboxydothermus ), Cardiobacterium ( Cardiobacterium ), Clostridium botulinum ( Carnobacterium ), Carsonella , Catenibacterium , Catenulispora genus Cartozoella ( Catonella ), genus *Stenocephalum* Caulobacter ), Fibromospora ( Cellulomonas ), Vibrio filamentosa ( Cellvibrio Centipede Fungus ( ) Centipeda Rhizobium ( ) Chelativorans) Flexibrio spp. Chloroflexus Chromobacterium spp. Chromobacterium ), Halobacterium spp. Chromohalobacter ), Chthoniobacter , Citreicella Citrobacter spp. Citrobacter ), Citric acid microbes ( Citromicrobium ), genus *Clostridium* Clavibacter ), Cloacamonas Clostridium spp. Clostridium Collins spp. Collinsella) Collochia spp. Colwellia ), Trichomonas genus ( Comamonas ), Conexibacter Aggregates ( Congregibacter ), Coprobacillus Species of fecalith ( Coprococcus ), genus *Phaeophyte* Coprothermobacter ), Coraliomargarita genus Corynebacterium ( Coriobacterium ), corrodens Corynebacterium spp. Corynebacterium ), Coxsonia ( Coxiella ), genus *Cyclocarya* ( Crocosphaera ), Cronobacter sakazakii ( Cronobacter ), genus *Phyllostachys* ( Cryptobacterium ), Copper-loving bacteria ( Cupriavidus ), Cyanobacteria ( Cyanobium ), Blue filamentous fungi ( Cyanothece ), genus *Cymbidium* ( Cylindrospermopsis ), Dechloromonas Iron-reducing bacteria ( ) Deferribacter ), Dehalococcoides , Dehalogenimonas Abnormal cocci ( Deinococcus ), Delftella spp. Delftia ), Nitrogen-reducing Vibrio spp. Denitrovibrio ), genus *Dermococcus* Dermacoccus ), Desmospora Desulfurization bacteria ( ) Desulfarculus ), Desulphateibacillum Desulfurization bacteria ( ) Desulfitobacterium ), Desulfurized olive-shaped bacteria ( Desulfobacca ), Desulfobacterium spp. Desulfobacterium ), Desulfurized Leaf Fungus ( Desulfobulbus ), Desulfococcus spp. Desulfococcus ), Desulfurizing bacteria ( Desulfohalobium ), Desulfurizing Microbes ( Desulfomicrobium ), Desulfonatronospira , Desulforudis Desulfuric bacteria ( ) Desulfotalea ), Desulfurized Enterobacteriaceae ( Desulfurous stain ), Desulfuric Vibrio spp. Desulfovibrio ), ( Desulfurispirillum ), Desulfobacterium spp. Desulfurobacterium ), spp. of sulfurmonas ( Desulfuromonas ), Dethiobacter , Dethiosulfate Vibrio spp. Dethiosulfovibrio ), genus Alisteria ( Dialer ), Genus *Even-hoofed Fungi* Dichelobacter ), Dickey Tennis bacteria ( Dictyoglomus ), Dietrichia coli ( Dietzia ), Dinoroseobacter , Golden Edwardsiella spp. Edwardsiella ), Ehrlichia ( Ehrlichia ), Ekenella spp. Eikenella ), Elusimicrobium , Endoriftia Aquatic Fungi ( Enhydrobacter ), Enterobacteriaceae ( Enterobacter ), Enterococcus ( Enterococcus ), Fish soup Erwinia spp. Erwinia ), Erysipelothrix ( Erysipelothrix ), genus *Rhizobium* ( Erythrobacter Escherichia coli spp. Escherichia ), genus of hydrogen-producing bacteria ( Ethanoligen ), Eubacterium ( Eubacterium ), Eubacterium ( Eubacterium ), Microbacteria ( Small bacterium ), Faecalibacterium Iron-reducing monoclonal bacteria ( Ferrimonas ), Scintillans ( Fervidobacterium ), Filamentous Bacteria ( Fibrobacter ), Finegoldia genus Curved Branch Fungus ( Flexitypes Francisella spp. Francisella ), Franklinella spp. Frankish ), Fructobacillus , Fulvimarine Fusobacterium spp. Fusobacterium ), Gallic bacteria genus Trichophyton ( Gallinule Gardnerella spp. Gardnerella ), Geminids ( Twin ), budding fungi ( Gemstones ), Bacillus spp. Gemmatimonas ), Bacillus spp. ( Geobacillus ), genus *Typhonium* Geobacter ), genus *Geophilus* Geodermatophilus ), genus *Icefungus* ( Iceberg ), Colistinia spp. ( Gloeobacter ), genus *Tsetseus* ( Glossine ), Glucono-acetic acid bacteria ( Gluconacetobacter ), genus Gordonia ( Gordonia ), Granulibacter Streptococcus spp. Granulicatella ), genus Grimalomyces ( Grimontia Haemophilus spp. Haemophilus ), *Hypericum* genus ( Hahella ), Halanaerobiums , Haliangium genus Halomonas ( Halomonas ), genus *Haloxylon ammodendron* Halorhodospira ), Salt-heated fungi ( Halothermothrix ), genus *Salmonella* ( Halothiobacillus ), Hamiltonella Helicobacter spp. Helicobacter ), genus *Bacillus spp.* Heliobacterium ), genus *Streptococcus* Herbaspirillum ), Herminiimonas genus *Phyllostachys* Herpetosiphon ), genus *Hippophae* ( Hippolytus ), Hedyotis diffusa ( Deer Histophila ( ) Histophilus ), Hodgkin's disease , Hoeflea genus Haldemans ( Hold'em ), Hydrogen rod , Hydrogen stick , Hylemonella Microbes of the genus *Microbes* Hyphomicrobium ), genus *Firmium* ( Hyphomonas ), Idioms Coccidioides ( Ilyobacter ), genus *Mesocystis* ( Intrasporangium ), genus of termites ( Isoptericola ), and cocci ( Isosphaera ), genus Fasciola ( Janibacter ), genus *Citrus* ( Janthinobacterium Jones spp. Jonesia ), Jonquetella , Kangiella , Ketogulonicigenium Animalococcus ( Kineococcus ), genus *Aureobasidium* Kingella ), Klebsiella spp. Klebsiella ), Cochlea spp. Kocuria ), Koribacter , Kosmotoga , genus *Pyracantha* ( Kribbella ), Fibriobrina ( Ktedonobacter ), genus *Dermatococcus* Kytococcus ), Labrenzia Lactobacillus ( Lactobacillus Lactococcus spp. Lactococcus ), genus *Ophiobacterium* Laribacter Lauteropus spp. Lautropia Lawsonia spp. Lawsonia Legionella ( ) Legionella ), Reversonella spp. Leifsonia ), Lentisphaera genus *Cymbidium* ( Leptolyngbya Leptospira () Leptospira ), genus *Pteris* ( Leptothrix ), Ciliophytes ( Leptotrichia Leuconostoc ( ) Leuconostoc ), Bacillus spp. Liberibacter ), Limnobacter Listeria spp. Listeria ), Loktanella , Lutiella Cyanobacteria ( ) Lyngbya ), Lysinibacillus Megacoccus ( Macrococcus ), Magnetic Cocci ( Magnetococcus ), Magnetorhynchus ( Magnetospirillum ), Mahella Merhamella spp. Mannheimia Marine rod-shaped bacteria ( Maricaulis ), genus Thermopyridae ( Marinithermus ), Marinebacteria ( Marinobacter ), genus *Hymenospora* Marinomonas ), Deep-sea bacteria ( Mariprofundus ), Maritimibacter , Marvinbryantia genus *Gastromycium* Megasphaera ), genus *Pleurotus* ( Meiothermus ), genus *Apigeninella* ( Melissococcus ), Intermediate Rhizobium ( Mesorhizobium ), Methylacidiphilum , Methylibium Methylobacterium ( Methylobacillus ), Methylobacterium ( Methylobacter ), Methylobacterium ( Methylobacterium Methylcoccus ( Methylococcus ), Methylcystis ( Methylocystis ), Methylmicrobes ( Methylomicrobium ), Methylphages ( Methylophaga ), Methylophilales ( Methylophilales ), Methyl Aspergillus spp. Methylosinus ), Methyloversatilis Methylophilus ( Methylovorus Microbacteria ( Microbacterium Micrococcus ( Micrococcus ), Micrococcus genus ( Microcoleus ), Microcystis genus ( Microcystis ), genus *Malus* Microlunatus Micromonospora ( Micromonospora ), genus *Gymnospermum* ( Mitsuokella ), Molybditis spp. ( Mobiluncus ), Mullerella spp. Moorella Moraxella spp. Moraxella Antarctic psychrophiles ( Moritella ), Mycobacterium ( Mycobacterium ), genus Myxococcus Myxococcus ), Nakamurella Salt-alkali anaerobic bacteria ( Natranaerobius ), Neisseria spp. Neisseria ), Neorickettsiae ( Neorickettsia ), Neptuniibacter , Nitratifractor , Nitratiruptor Nitrifying bacteria ( Nitrobacter ), Nitrifying cocci ( Nitrococcus ), Nitrosomonas spp. Nitrosomonas ), Nitrostrophus ( Nitrosospira ), Nitrifying Spirulina ( Nitrospira Nocardia spp. Nocardia ), Nocardia spp. Nocardioides ), Nocardia spp. Nocardiopsis ), genus Arthrocyanobacteria ( Nodularia ), genus *Nostoc* ( Nostoc ), Neosphingolipids ( Novosphingobium ), Oceanibulbus , Oceanicaulis Marine fungi ( Oceanicola ), Marine thermophilus ( Oceanithermus Marine Bacillus ( ) Oceanobacillus ), genus *Pseudomonas* Ochrobactrum ), spp. of 18 Octadecabacter ), Odyssella Oligotrophic bacteria ( Oligotropha ), Olsenella , genus *Fengyou* ( Opitutus ), Oribacterium Oriental body ( Orientia ), Ornithinibacillus genus *Oscillatoria* Oscillatoria ), genus *Vibrio* ( Oscillochloris ), Oxalic acid bacteria ( Oxalobacter ), Bacillus spp. ( Paenibacillus Pantotheca ( ) Pantoea Paracoccus ( Paracoccus ), Parascardovia , Parasutterella , Parvibaculum Micromonas spp. Parvimonas ), genus *Short Box Fungus* Parvularcula Pasteurella ( Pasteurella ), Bacillus pastoris ( Pasteuria ), Pectinobacterium ( Pectobacterium ), Pediococcus ( Pediococcus ), Pedosphaera , Pelagibaca Oceanobacteria ( Pelagibacter ), genus *Cryptobacter* ( Pelobacter ), Pelotomaculum , Peptoniphilus Peptostreptococcus ( Peptostreptococcus ), Persephonella genus *Lithocarpus* Petrotoga ), Brownia ( Phaeobacter ), Koala spp. Phascolarctobacterium ), Phenyrobacterium ( Phenylobacterium ), genus *Luminobacterium* ( Photobacterium ), genus *Pyriformes* ( Pirellula ), *Plasmodium* genus ( Planctomyces ), Animalococcus spp. Planococcus ), Plesiocystis Species of Polar Cytomegalovirus ( Polaromonas ), genus Polar Cytozoa ( Polaromonas ), Polymorphum Multinucleobacillus spp. Polynucleobacter ), Spongiformes ( Poribacteria ), Prochlorococcus ( Prochlorococcus ), Propionibacterium spp. Propionibacterium ), Proteobacteria ( Proteus ), Providencia genus ( Providencia ), Pseudorabies spp. Pseudoalteromonas ), Pseudoflavonifractor Pseudomonas spp. Pseudomonas ), Nocardia spp. Pseudonocardia ), Pseudomonas ( Pseudoramibacter ), Pseudovibrio Pseudomonas spp. Pseudoxanthomonas ), Psychrophilic bacteria ( Psychrobacter ), Cryomonas spp. Psychromonas ), Puniceispirillum , Pusillimonas Species of Veillonella ( Pyramidobacter ), Rahn's genus ( Rahnella ), genus Rollstonella ( Ralstonia ), genus *Achnatherum* ( Raphidiopsis ), Queen , Reinecke Nephrobacter spp. Kidney bacteria Rhizobium ( ) Rhizobium ), Rhodobacteria ( Rhodobacter ), Rhodococcus spp. ( Rhodococcus ), Rhodotorula genus ( Rhodophera ), Rhodophyta ( Rhodomicrobium ), genus *Pycnodon* ( Rhodopyrellus ), Rhodopseudomonas spp. Rhodopseudomonas ), Rhodospirillum ( Rhodospirillum ), Rickettsia species ( Rickettsia ), Rickettsia genus ( Rickettsia ), Riesia Rosebury bacteria ( Rosemary ), genus Rosaceae ( Rosewood ), genus Curvularia ( Rose-flexed ), genus *Rosebacterium* Roseobacter ), genus Rosomonas ( Roseomonas ), genus *Rosechaete* ( Rose-ovarian ), genus Roselle ( Rothia ), genus *Lycoperdon* ( Red-haired ), spp. of Red Bacillus ( Rubrobacter ), Rugellella ( Ruegeria ), Rumenococcus ( Ruminococcus ), Ruthenia saccharomyces ( Saccharomonospora ), Sugar eater saccharopolysporum ( Saccharopolyspora ), genus *Arrowhead* ( Arrowhead ), Salinia spora Salmonella ( Salmonella ), genus *Hyperbacterium* Bloody ), Scardovia Sebarudella ( ) Sebaldella ), Small-sized genus Lunaemon ( Selenomonas ), Serratia ( Serratia Shewanella ( ) Shewanella ), Shigella spp. Shigella ), Shuttleworthia , Sideroxydans , Silicibacter Simons spp. Simonsiella ), *Rhizobium sinense* ( Sinorhizobium ), Shrekella spp. Slack ), genus *Amphibacterium* Member ), Solibacter , Solobacterium genus *Colombia* ( Sorangium ), Cocci ( Sphaerobacter ), Sphingolipids ( Sphingomyelophytum ), Sphingosomalmonella ( Sphingomyelomonas ), Sphingopyx Spirochetes ( Spirochaete ), Bacillus spp. ( Spore carriage ), Stackebrandia Staphylococcus spp. Staphylococcus ), Starkeya Oligotrophomonas spp. Stenotrophomonas ), Species of the genus ( Stigma ), Streptomyces ( Streptococcus Streptococcus ( Streptococcus Streptomyces ( Streptomyces ), genus *Neurospora* Streptosporangium ), Subdoligranule , subvibrio , Succinate , spp. of sulfite bacillus ( Sulfitobater ), serotypes of thiobacillus ( Sulfobacillus ), Sulfuric acid , Hydrogen sulfide , Sulfuric acid Species of sulfide ( Sulfurospirillum ), Sulfur Sartorius ( Sutterella ), Symbiobacterium Syncytosis bacteria ( Synechocystis ), Synergid bacteria ( Syntrophobacter ), commensal sausage-like bacteria ( Syntrophobotulus ), genus *Commonospora* ( Syntrophomonas ), genus *Hypertropha* ( Syntrophotherm ), Mutualistic bacteria ( Syntrophus ), Taiwanese Theylodes spp. Taylorella ), Teredinibacter , Terriglobus , Thalassiobium Solomonella ( Thauera ), thermophilic aerobic bacteria ( Thermaerobacter ), thermoanaerobic Vibrio spp. Thermanaerovibrio ), Thermincola , heat-resistant anaerobic bacteria ( Thermoanaerobacter ), genus *Heat-Aerobic Bacillus* Thermoanaerobacterium ), Thermobaculum Thermophilic schizocarpium ( Thermobifida ), thermophilic Diplosporum ( Thermobispora ), genus *Thermophyton* ( Thermocrinis ), Thermodesulphateator , Thermo-desulfuric bacteria ( Thermodesulfobacterium ), heat desulfurizing bacteria ( Thermodesulfobium ), genus *Vibrio spp.* (heat desulfurization) Thermodesulfovibrio ), Thermomicrobium ( Thermomicrobium ), Thermomonospora ( Thermomonospora ), Thermosediminibacter , Thermosinus Thermophyton genus ( Thermosipho ), Thermosynechococcus Thermospora genus ( Thermotoga ), Vibrio thermophilus ( Thermovibrio ), Thermophyton genus ( Thermus ), Thioalkalimicrobium , Thioalkalivibrio Thiobacillus spp. Thiobacillus ), genus *Thiospirillum* Thiomicrospira Thiomonas spp. Thiomonas ), Toxomonas spp. Tolumonas ), genus *Treponema* ( Treponema ), tribocorum genus *Bombyx mori* ( Trichodesmium ), Tropheryma Trüperella ( Truepera ), Tsukamura ( Tsukamurella ), Turicibacter Gluttonous bacteria ( Variovorax Veillonella ( ) Veillonella ), Verminephrobacter Verrucous microbes ( Verrucomicrobium ), Verrucous spp. ( Verrucosispora ), Vesicomyosocius Vibrio ( Vibrio ), Vibrioales ( Vibrionales ), Food cereal bacteria ( Victivallis ), Weissella spp. Weissella Wiggersworth spp. Wigglesworthia ), Wolbachia genus ( Wolbachia ), Warlinella spp. Wolinella ), Flavobacterium ( Xanthobacter Xanthomonas ( ) Xanthomonas ), pathogenic bacteria ( Xenorhabdus ), Xylanimonas genus *Trichobacterium* Xylella Yersinia spp. Yersinia ), Zinderia and fermentation monosporus ( Zymomonas ).

[0057] Specifically, the microbial cells may be derived from Escherichia coli (E. coli). E. coli ), species of the genus Pseudomonas ( Pseudomonas sp.), Pseudomonas fluorescens (sp.), Pseudomonas fluorescens ( Pseudomonas fluorescens ), Pseudomonas putida ( Pseudomonas putida ), Pseudomonas schrenckii ( Pseudomonas stutzeri Acinetobacter species ( Acinetobacter sp.), species of Burkholderia ( Burkholderia sp.), Burkholderia thailandensis Cyanobakterien, species of Klebsiella ( Klebsiella sp.), Klebsiella pneumoniae (sp.), Klebsiella oxytoca Salmonella species ( Salmonella sp. ), species of the genus Rhizobium ( Rhizobium sp .) and alfalfa rhizobia ( Rhizobium meliloti ), Bacillus species ( Bacillus sp.), Bacillus subtilis ( Bacillus subtilis Clostridium species ( Clostridium sp.), species of the genus Corynebacterium (sp.), Corynebacterium sp.), Corynebacterium glutamicum (sp.), Corynebacterium glutamicum ), species of the genus *Brevibacterium* ( Brevibacterium sp.), species of the genus Chlorella ( Chlorella sp.) and species of the genus Nostoc ( Nostoc More specifically, the microbial cells may be derived from *Escherichia coli*.

[0058] Alkanes are saturated hydrocarbons with a wide range of applications, depending on the number of carbon atoms and the structure of the alkane (i.e., branched, straight-chain, cyclic, etc.). Alkanes (technically, always acyclic or open-chain compounds) have the general chemical formula C1. n H 2n+2 The alkane used according to any aspect of the invention may contain at least 6 carbon atoms.

[0059] Specifically, the alkane used according to any aspect of the invention may contain 6-22, 6-20, 6-18, 6-17, 6-16, 6-15, 6-14, 6-13, 6-12, 6-11, 6-10, 8-20, 8-19, 8-18, 8-16, 8-15, 8-12, or 8-10 carbon atoms (including terminal values). The alkane may be selected from hexaane, heptane, octane, nonane, decane, undecane, dodecane, tridecane, tetradecane, pentadecane, hexadecane, and eicosane. Specifically, the alkane used may be decane, undecane, or dodecane.

[0060] Enzyme E1

[0061] Enzyme E1 can convert at least one alkane into the corresponding 1-alkanol. Specifically, E1 can be at least one P450 alkane hydroxylase of EC 1.14.15.1 (E a AlkB alkane hydroxylase (E) or EC 1.14.15.3 b P450 alkane hydroxylase (E) a ) is a component of the reaction system, the reaction system comprising

[0062] - Two enzyme components: cytochrome P450 alkane hydroxylase and NAD(P)H cytochrome P450 oxidoreductase of EC 1.6.2.4, or

[0063] - Three enzyme components: cytochrome P450 alkane hydroxylase of type CYP153, ferroredoxin NAD(P)+ reductase and ferroredoxin of EC 1.18.1.2 or EC 1.18.1.3.

[0064] AlkB alkane hydroxylase (E 1b ) is a component of the reaction system, the reaction system comprising

[0065] - AlkB alkane hydroxylase of EC 1.14.15.3, which is a component of the reaction system, the reaction system comprising three enzyme components: AlkB alkane hydroxylase of EC 1.14.15.3, AlkT erythroredoxin NAD(P)+ reductase of EC 1.18.1.1 or EC 1.18.1.4, and erythroredoxin AlkG.

[0066] Specifically, E1 can be AlkB alkane hydroxylase (E), also known as alkane monooxygenase. b More specifically, E1 may contain at least 50% sequence identity with an alkane monooxygenase encoded by alkBGT from *Pseudomonas putida* GPo1. Even more specifically, E1 may contain at least 50% sequence identity with polypeptide YP_001185946.1. More specifically, E1 may contain a polypeptide having at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 94%, 95%, 98%, or 100% sequence identity with polypeptide YP_001185946.1.

[0067] Enzyme E a

[0068] Specifically, enzyme E1 can be at least one of the following P450 alkane hydroxylases (E a ):

[0069]

[0070]

[0071]

[0072]

[0073]

[0074]

[0075]

[0076]

[0077]

[0078]

[0079]

[0080]

[0081]

[0082]

[0083]

[0084]

[0085]

[0086]

[0087]

[0088]

[0089]

[0090]

[0091]

[0092]

[0093]

[0094]

[0095]

[0096] and .

[0097] Enzyme E b

[0098] In another example, enzyme E1 can be at least one AlkB alkane hydroxylase selected from the following: 1b ):

[0099]

[0100]

[0101]

[0102]

[0103]

[0104]

[0105]

[0106]

[0107]

[0108]

[0109]

[0110]

[0111]

[0112]

[0113]

[0114]

[0115]

[0116]

[0117]

[0118]

[0119]

[0120]

[0121]

[0122]

[0123] and .

[0124] Enzyme E2

[0125] Enzyme E2 can convert 1-alkanols to the corresponding 1-alkanal. Specifically, E2 can be at least one P450 alkane hydroxylase (EC1.14.15.3). a AlkB alkane hydroxylase (E) of EC 1.14.15.3 b Ethanol oxidase (EC 1.1.3.20) c Alcohol dehydrogenase (E) of EC 1.1.1.1 or EC 1.1.1.2 d More specifically, E2 can be selected from P450 alkane hydroxylase (E...). a AlkB alkyl hydroxylase (E) b Ethanol oxidase (EC 1.1.3.20) c AlkJ alcohol dehydrogenase (E) di ) and alcohol dehydrogenases (E) of EC1.1.1.1 or EC1.1.1.2 dii ).

[0126] Specifically, E2 can be AlkB alkane hydroxylase (E2), also known as alkane monooxygenase. b More specifically, E2 may contain at least 50% sequence identity with an alkane monooxygenase encoded by alkBGT from *Pseudomonas putida* GPo1. Even more specifically, E2 may contain at least 50% sequence identity with polypeptide YP_001185946.1. More specifically, E2 may contain a polypeptide having at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 94%, 95%, 98%, or 100% sequence identity with polypeptide YP_001185946.1.

[0127] Enzyme E c

[0128] The alcohol oxidase (E) c (Can be selected from:)

[0129]

[0130]

[0131]

[0132]

[0133]

[0134]

[0135]

[0136]

[0137]

[0138]

[0139] and

[0140] .

[0141] Specifically, the alcohol oxidase (E c (can be selected from)

[0142]

[0143] and .

[0144] Enzyme E di

[0145] Specifically, the AlkJ alcohol dehydrogenase (E di (Can be selected from:)

[0146]

[0147]

[0148]

[0149]

[0150]

[0151]

[0152]

[0153]

[0154]

[0155]

[0156]

[0157]

[0158]

[0159]

[0160]

[0161]

[0162]

[0163]

[0164]

[0165]

[0166] and .

[0167] Specifically, E di Can be selected

[0168] and .

[0169] Enzyme E dii

[0170] The alcohol dehydrogenase (E) dii It can be selected from bacteria, especially Escherichia coli.

[0171]

[0172] and .

[0173] Enzyme E3

[0174] Enzyme E3 can convert at least one 1-alkalaldehyde to the corresponding alkanoic acid. Specifically, E3 can be selected from P450 alkane hydroxylases of EC1.14.15.3-. a AlkB alkane hydroxylase (E) of EC 1.14.15.3 bBifunctional alcohol oxidase (E) of EC 1.1.3.20 c A bifunctional AlkJ alcohol dehydrogenase (E) capable of directly oxidizing 1-alkanols to the corresponding alkyl acids via 1-alkanaldehydes. di Bifunctional alcohol dehydrogenases (E) of EC 1.1.1.1 or EC 1.1.1.2 dii ) and aldehyde dehydrogenase (E e ).

[0175] Enzyme E e

[0176] Enzyme E e Aldehyde dehydrogenase can catalyze the following conversions

[0177] ω-Oxoalkyl acid (ester) = ω-Carboxyalkyl acid (ester).

[0178] In order to catalyze the above reaction, E e It can be an aldehyde dehydrogenase of EC 1.2.1.3, EC 1.2.1.4 or EC 1.2.1.5, an aliphatic alcohol oxidase of EC 1.1.3.20, an AlkJ alcohol dehydrogenase of EC 1.1.99.- and an alcohol dehydrogenase of EC 1.1.1.1 or EC 1.1.1.2.

[0179] In one instance, E e It can specifically catalyze the following reactions:

[0180] ω-Oxanoic acid (ester) + NAD(P) + = ω-Carboxyalkyl acid (ester) + NAD(P)H + H +

[0181] In this case, enzyme E e It can be an aldehyde dehydrogenase of EC 1.2.1.3, EC 1.2.1.4, or EC 1.2.1.5, and can be selected from bacteria, especially Escherichia coli.

[0182] and .

[0183] In another example, enzyme E e It can catalyze the following reactions:

[0184] ω-O-alkyl acid (ester) + O2 = ω-Carboxyalkyl acid (ester) + H2O2

[0185] In this case, E e It can be a fatty alcohol oxidase of EC 1.1.3.20, and can be selected from the above as enzyme E. cThe provided list.

[0186] In another instance, E e It can be at least one AlkJ alcohol dehydrogenase with EC 1.1.99, and can be selected from the above as E di The provided list.

[0187] In a further example, E e It can be selected from enzyme E dii The provided list includes alcohol dehydrogenases for EC 1.1.1.1 or EC 1.1.1.2.

[0188] Enzyme E4

[0189] Enzyme E4 can convert at least one alkyl acid to the corresponding alkyl ester. Specifically, E4 can be at least one wax ester synthase, also known as an alcohol O-acyltransferase (EC 2.3.1.20, EC 2.3.1.75) (E f ) or alcohol O-acetyltransferase (E g (EC 2.3.1.20, EC 2.3.1.75 or EC 2.3.1.84).

[0190] In one instance, E4 can be at least one wax ester synthase (E f More specifically, E4 may contain Acinetobacter calcifera ADP1 or... Hahella chejuensis The sequence identity of E4 with at least 50% of the sequence of the O-acetyltransferase. More specifically, E4 may contain at least 50% sequence identity with polypeptide YP_045555.1, WP_011398768.1, or NP_808414.2. More specifically, E4 may contain a polypeptide having at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 94%, 95%, 98%, or 100% sequence identity with a polypeptide selected from polypeptide YP_045555.1, WP_011398768.1, and NP_808414.2. In one instance, E4 may contain a polypeptide having at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 94%, 95%, 98%, or 100% sequence identity with SEQ ID NO:2.

[0191] Enzyme E f

[0192] Specifically, the enzyme E f You can choose from:

[0193]

[0194]

[0195]

[0196]

[0197]

[0198]

[0199]

[0200]

[0201]

[0202]

[0203]

[0204]

[0205]

[0206]

[0207]

[0208]

[0209]

[0210]

[0211]

[0212] and .

[0213] In another example, the enzyme E f You can select from the following NCBI gene identifiers:

[0214]

[0215]

[0216]

[0217] and .

[0218] Enzyme Eg

[0219] Specifically, the enzyme E g Can be selected

[0220]

[0221] and .

[0222] Enzyme E5

[0223] Enzyme E5 is capable of converting at least one alkyl ester to the corresponding ω-hydroxyalkyl ester. Specifically, E5 can be any enzyme listed as E1. Specifically, E5 can be at least one P450 alkyl hydroxylase (E1) of EC 1.14.15.3. a AlkB alkane hydroxylase (E) or EC 1.14.15.3 b ).

[0224] Enzyme E6

[0225] Enzyme E6 is capable of converting at least one ω-hydroxy-alkanoate to the corresponding ω-oxoalkanoate. Specifically, E6 can be any enzyme listed as E2. Specifically, E6 can be selected from P450 alkane hydroxylases (E6) of EC 1.14.15.3-. a AlkB alkane hydroxylase (E) of EC 1.14.15.3 b Ethanol oxidase (EC 1.1.3.20) c ) and alcohol dehydrogenases (E) of EC 1.1.1.1 or EC 1.1.1.2 d ).

[0226] The phrase "when present" used with respect to enzyme E6 refers to cells that have been genetically modified to produce ω-oxoalkyl esters. Cells according to any aspect of the invention may contain increased expression of enzyme E6 relative to wild-type cells, and are therefore capable of producing ω-oxoalkyl esters. In another example, cells according to any aspect of the invention may also contain no increased expression of enzyme E6 relative to wild-type cells, and therefore cannot produce ω-oxoalkyl esters. Thus, these cells may primarily produce ω-hydroxyalkyl esters. Therefore, when cells according to any aspect of the invention contain increased expression of enzyme E6 (i.e., when present), ω-oxoalkyl esters can be produced.

[0227] Enzyme E7

[0228] Enzyme E7 can convert at least one ω-oxoalkyl acid into the corresponding ω-aminoalkyl ester. Specifically, E7 can be the ω-transaminase (E7) of EC 2.6.1.h ).

[0229] Specifically, the enzyme E7 can be a transaminase selected from the following: h ): Pseudomonas putida ( Pseudomonas putida (WP_016502144; WP_016500675.1), Chromobacterium violaceum ( Chromobacterium violaceum (NP_901695.1), Rhodophyton floccosum ( Rhodobacter sphaeroides ) 2.4.1 (YP_353455.1) and

[0230]

[0231]

[0232]

[0233]

[0234]

[0235]

[0236]

[0237] Specifically, the enzyme E7 can be a transaminase selected from the following: h ):

[0238]

[0239]

[0240] and .

[0241] The phrase "when present" used with respect to enzyme E7 refers to cells that have been genetically modified to produce ω-aminoalkyl esters. Cells according to any aspect of the invention may contain increased expression of enzyme E7 relative to wild-type cells, and are therefore capable of producing ω-aminoalkyl esters. In another example, cells according to any aspect of the invention may also contain no increased expression of enzyme E7 relative to wild-type cells, and therefore cannot produce ω-aminoalkyl esters. Thus, these cells may primarily produce ω-oxoalkyl esters. In one example, cells containing increased expression of enzyme E6, but not E7, relative to wild-type cells may be capable of producing ω-oxoalkyl esters. In another example, when said cells are not genetically modified to increase the expression of E6, but not E7, said cells may produce ω-hydroxyalkyl esters.

[0242] Enzyme E h

[0243] Specifically, the enzyme E h You can choose from:

[0244]

[0245]

[0246]

[0247]

[0248]

[0249]

[0250] and .

[0251] Cells according to any aspect of the invention can be genetically modified to increase the expression of enzymes E1 to E5 relative to wild-type cells. The cells can be further genetically modified to increase the expression of at least enzymes E6 and / or E7. In one example, enzymes E1, E2, E3, E5, and E6 can be at least one AlkB alkane hydroxylase (E b Furthermore, enzyme E4 can be a wax ester synthase (E... f Specifically,

[0252] - The AlkB alkane hydroxylase (E b It contains at least 60% sequence identity relative to SEQ ID NO:1; and

[0253] - The wax ester synthase (E f It contains at least 60% sequence identity relative to SEQ ID NO:2.

[0254] In another example, when cells according to any aspect of the invention are genetically modified to produce at least one ω-aminoalkyl ester, the cells may be modified to express at least one ω-transaminase (E h (), which may contain at least 60% sequence identity relative to SEQ ID NO:3.

[0255] Enzyme E8

[0256] Cells according to any aspect of the invention can be further genetically modified to reduce the expression of at least one enzyme E8, which cleaves at least one intermediate in the conversion of alkanes to ω-functionalized carboxylic esters. Specifically, enzyme E8 can be an enzyme capable of functioning in the cell's fatty acid degradation capacity. Specifically, E8 can be selected from acyl-CoA dehydrogenases (E... i (FadE), enoyl-CoA hydratase (E) j (FadB), 3-hydroxyacyl-CoA dehydrogenase (E) k ) (FadB) and β-ketothiolase (FadA), also known as 3-ketoacyl-CoA thiolase (E l ).

[0257] Fatty acids are taken up and transported across the cell membrane via a transport / acyl activation mechanism. The first intracellular step involves the action of acyl-CoA dehydrogenase (E... i Acyl-CoA is converted to enoyl-CoA, and the acyl-CoA dehydrogenase (Ei) is referred to as FadE in the case of *E. coli*. The activity of the acyl-CoA dehydrogenase can be determined as described in the prior art, for example by monitoring the concentration of NADH at 340 nm spectrophotometry in 100 mM MOPS (pH 7.4), 0.2 mM enoyl-CoA, and 0.4 mM NADH. The resulting enoyl-CoA is converted to 3-ketoacyl-CoA via 3-hydroxyacyl-CoA through hydration and oxidation, wherein the hydration and oxidation are performed by enoyl-CoA hydratases referred to as FadB and FadJ in *E. coli*. R )-3-hydroxyacyl-CoA dehydrogenase (E j / E k Catalysis. The activity of enoyl-CoA hydratase / 3-hydroxyacyl-CoA dehydrogenase, more specifically, the formation of the product NADH, can be determined spectrophotometrically as described in the prior art, for example as outlined for FadE. Finally, 3-ketoacyl-CoA thiolylasase (E l FadA and Fad1 in *E. coli* catalyze the cleavage of 3-ketoacyl-CoA to yield acetyl-CoA, and the addition of acyl-CoA shortens the carbon atom by two atoms. The activity of ketoacyl-CoA thiolases can be determined as described in existing techniques (e.g., Antonenkov, V., et al., 1997).

[0258] In one instance, as used herein, the term "acyl-CoA dehydrogenase" can be a polypeptide capable of catalyzing the conversion of acyl-CoA to enoyl-CoA as part of a β-oxidation pathway. For example, the polypeptide FadE (accession number: BAA77891.2) in *E. coli* can be an acyl-CoA dehydrogenase. As used herein, the term "enoyl-CoA hydratase," also known as 3-hydroxyacyl-CoA dehydrogenase, refers to a polypeptide capable of catalyzing the conversion of enoyl-CoA to 3-ketoacyl-CoA through hydration and oxidation as part of a β-oxidation pathway.

[0259] For example, the E. coli peptides FadB and FadJ (accession numbers: BAE77457.1 and P77399.1, respectively) are enoyl-CoA hydratases. As used herein, the term "ketoacyl-CoA thiolylasase" can refer to a peptide capable of catalyzing the cleavage of 3-ketoacyl-CoA as the final step in the β-oxidation pathway, resulting in the production of acyl-CoA and acetyl-CoA with two shortened carbon atoms. For example, the E. coli peptides FadA and FadI (accession numbers: YP_491599.1 and P76503.1, respectively) are ketoacyl-CoA thiolylasases.

[0260] Enzymes E9 and E 10

[0261] Cells according to any aspect of the invention can be further genetically modified to increase the expression of the following relative to wild-type cells:

[0262] - The enzyme E9 is fatty acyl-CoA methyl esterase BioH (E m ); and / or

[0263] - The enzyme E 10 It is a fatty acyl-CoA thioesterase (E) selected from TesA, TesB, YciA, FadM, YbfF, and YbgC. n ).

[0264] Specifically, E m It can hydrolyze fatty acid esters into free fatty acids and the corresponding alcohols; and / or E n It can hydrolyze fatty acyl-CoA into free fatty acids and CoA.

[0265] Enzyme E 11

[0266] Cells according to any aspect of the invention may contain a further genetic mutation that increases the expression of at least one transporter protein relative to wild-type cells. This further mutation enables the cells to increase the uptake of at least one fatty acid. Specifically, the transporter protein may be AlkL (SEQ ID NO: 4 or 5) and / or FadL (SEQ ID NO: 6). AlkL and / or FadL can function as at least one transporter protein compared to wild-type cells. In one example, the cells may be genetically modified to overexpress both the fadL and alkL genes. Cells according to any aspect of the invention may be further genetically modified to increase the expression of AlkL and / or FadL relative to wild-type cells.

[0267] In one example, enzyme E 11 It can be FadL or BAA16205.1.

[0268] Enzyme E 12

[0269] Cells according to any aspect of the invention may contain further genetic mutations that increase the content of acyl-CoA synthase (enzyme E) at EC 6.2.1.3 and EC 2.3.1.86 relative to wild-type cells. 12 The expression of enzyme E. 12 Acyl-CoA esters can catalyze the conversion of fatty acids into acyl-CoA esters, i.e., molecules in which the carboxyl functional group -OH is replaced by -S-CoA. For example, the polypeptides FadD and FadK in *E. coli* (accession numbers: BAA15609.1 and NP_416216.4, respectively) are acyl-CoA synthases. In another example, E... 12 It could be a long-chain fatty acid-CoA ligase of YP_001724804.1.

[0270] Enzyme E 13

[0271] Enzyme E 13 It can convert ω-oxoalkyl esters into their corresponding ω-carboxyalkyl esters. Specifically, enzyme E... 13 It can be any enzyme E3 as defined above. More specifically, E... 13 P450 alkane hydroxylases (E) selected from EC 1.14.15.3- can be used. a AlkB alkane hydroxylase (E) of EC 1.14.15.3 b Bifunctional alcohol oxidase (E) of EC 1.1.3.20 c A bifunctional AlkJ alcohol dehydrogenase (E) capable of directly oxidizing ω-hydroxyalkanoates to the corresponding ω-carboxyalkanoates via ω-oxoalkanoates. diBifunctional alcohol dehydrogenases (E) of EC 1.1.1.1 or EC 1.1.1.2 dii ); and aldehyde dehydrogenase (E e ).

[0272] Enzyme E 14

[0273] Enzyme E 14 It can convert ω-carboxyalkyl esters into the corresponding ω-carboxyalkyl diesters. Specifically, enzyme E 14 It can be any enzyme E4 as defined above. More specifically, E... 14 It can be at least one wax ester synthase (E f ) or alcohol O-acyltransferase (E g (EC 2.3.1.20, EC 2.3.1.75 or EC 2.3.1.84).

[0274] Cells according to any aspect of the invention do not contain genetic modifications, said genetic modifications increasing the number of enzymes selected from the following enzymes E relative to wild-type cells. 20 - E 24 Expression of at least one of them:

[0275] - EC 3.1.2.14 or EC 3.1.2.22 E 20 Acyl-ACP thioesterase,

[0276] - E of EC 3.1.2.2, EC 3.1.2.18, EC 3.1.2.19, EC 3.1.2.20 or EC 3.1.2.22 21 Acyl-CoA thioesterase,

[0277] - E 22 Acyl-CoA:ACP transacylase

[0278] - E 23 Polyketide synthase, and

[0279] - E 24 Hexanoic acid synthase.

[0280] Specifically, cells according to any aspect of the invention have enzyme E 20 - E 24 Wild-type expression. Therefore, in cells according to the method of the present invention, enzyme E... 20 - E 24 It is neither expressed nor knocked out. More specifically, in cells according to any aspect of the invention, enzyme E... 20 - E 24 Any one of them, namely enzyme E 20 E21 E 22 E 23 or E 24 All enzymes E 20 E 21 E 22 E 23 and E 24 The expression of [the enzyme] is not genetically modified. Even more specifically, cells according to any aspect of the invention can contain enzyme E, which is naturally present in the initial cells. 20 -E 24 The natural or wild-type expression of any of the enzymes. Therefore, cells according to any aspect of the invention can be considered to not contain enzyme E. 20 - E 24 Recombinant expression of any of the enzymes. This is particularly advantageous because enzyme E can then be easily selected. 20 - E 24 None of them showed increased expression (i.e., they had enzyme E). 20 - E 24 Cells expressing any of the wild-type enzymes in the group were used to utilize alkanes as a carbon source for the formation of ω-functionalized carboxylates. Specifically, cells with enzyme E... 20 - E 24 Increased expression of any of these fatty acids in any cell can lead to increased production of such fatty acids, which can be used as a carbon source for the production of fatty acids by cells with enzyme E. 20 - E 24 Increased expression of any of these enzymes leads to the formation of ω-functionalized carboxylic acids and / or their esters. Enzyme E is present in cells. 20 - E 24 Cells with increased expression of any of these enzymes can therefore benefit from using high concentrations of fatty acids as substrates for the production of ω-functionalized carboxylates, and alkanes will therefore not be used for the formation of ω-functionalized carboxylates by the cells. Using carbon sources other than alkanes for ω-functionalized carboxylate formation would drastically increase production costs to produce more fatty acids, and the cells would require other carbon sources, such as glucose. Therefore, according to any aspect of the invention, cells using any aspect of the invention (which do not contain an increase in the following enzyme E relative to wild-type cells) 20 - E 24 (Genetic modification of the expression of at least one of the alkanes) is used to produce at least one ω-functionalized carboxylic acid ester from at least one alkane.

[0281] Enzyme E 20 -E 24

[0282] Enzyme E 20 -E 24In WO2013024114, pages 60-79 provide a detailed explanation of enzyme E. i To E iv .

[0283] According to another aspect of the invention, a method for producing at least one ω-functionalized carboxylic acid ester is provided, wherein the method comprises the step of contacting at least one cell according to any aspect of the invention with at least one alkane. Specifically, the ω-functionalized carboxylic acid ester formed may be selected from ω-hydroxy-alkanoates, ω-oxo-alkanoates, ω-carboxyl-alkanoates, and ω-amino-alkanoates. Specifically, the ω-functionalized carboxylic acid ester may be methyl 12-aminolaurate, methyl 12-hydroxylaurate, methyl (di) 12-carboxylaurate, and / or methyl laurate derived from the alkane dodecane. In another example, the ω-functionalized carboxylic acid ester produced may be methyl 11-aminoundecanoate, methyl 11-hydroxyundecanoate, methyl (di) 11-carboxyundecanoate, and / or methyl undecanoate derived from the alkane undecane. In at least one further example, monofunctional alcohols and / or aldehydes may be formed as byproducts.

[0284] As used herein, the term "contact" means direct contact between alkane and / or cells according to any aspect of the invention in an aqueous solution. For example, the cells and alkane may not be in different compartments separated by a barrier such as an inorganic membrane. If the alkane is soluble and can be absorbed by the cells or diffuse across a biological membrane, it can be simply added to the cells according to any aspect of the invention in an aqueous solution. In cases of insufficient dissolution, it can be dissolved in a suitable organic solvent before being added to the aqueous solution. Those skilled in the art can prepare aqueous solutions of poorly soluble alkane by adding suitable organic and / or polar solvents. Such solvents can be provided in the form of an organic phase comprising a liquid organic solvent. In one instance, an organic solvent or phase can be considered liquid when it is liquid at 25°C and standard atmospheric pressure. In another instance, fatty acids can be provided in the form of fatty acid esters such as the corresponding methyl or ethyl esters. In yet another instance, compounds and catalysts can be contacted in vitro, i.e., in a more or less enriched or even purified state, or they can be contacted in situ, i.e., they are prepared as part of cellular metabolism and subsequently reacted within the cells.

[0285] The terms "aqueous solution" and "aqueous medium" are used interchangeably and refer to any solution containing water, primarily water as a solvent, which can be used to at least temporarily maintain cells according to any aspect of the invention in a metabolically active and / or viable state, and contains (if necessary) any additional substrate. Those skilled in the art are familiar with the preparation of many aqueous solutions, commonly referred to as culture media suitable for maintaining cells of the invention, such as LB medium in the case of *E. coli*. It is advantageous to use a basic culture medium as an aqueous solution, i.e., a rationally simple composition of medium that contains only the minimum set of salts and nutrients essential for maintaining cells in a metabolically active and / or viable state compared to a complex medium, thus avoiding unnecessary product contamination with unwanted byproducts. For example, M9 medium can be used as a basic culture medium.

[0286] According to another aspect of the invention, a method for producing at least one ω-functionalized carboxylic acid ester from alkanes is provided, wherein the method comprises:

[0287] (a) Contact the alkane with the following enzyme:

[0288] (i) An enzyme E1 capable of converting the alkane into the corresponding 1-alkanol;

[0289] (ii) Enzyme E2 that can convert the 1-alkanol in (i) into the corresponding 1-alkanal;

[0290] (iii) Enzyme E3 that can convert the 1-alkane aldehyde of (ii) into the corresponding alkanoic acid;

[0291] (iv) Enzyme E4 capable of converting the alkyl acids of (iii) into the corresponding alkyl esters; and

[0292] (v) Enzyme E5, which can convert the alkyl esters of (iv) into the corresponding ω-hydroxyalkyl esters.

[0293] The method according to any aspect of the present invention may include the following steps:

[0294] (b) Contact the ω-hydroxy-alkyl ester with the following enzyme:

[0295] (vi) Enzyme E6 capable of converting the corresponding ω-hydroxy-alkanoate of (v) to the corresponding ω-oxoalkanoate; or

[0296] (vii) Enzyme E6, which can convert the corresponding ω-hydroxy-alkanoate of (v) to the corresponding ω-oxoalkanoate, and enzyme E7, which can convert ω-oxoalkanoate to the corresponding ω-aminoalkanoate; or

[0297] (viii) Enzyme E6, which can convert the corresponding ω-hydroxy-alkanoate of (v) to the corresponding ω-oxoalkanoate, and enzyme E, which can convert ω-oxoalkanoate to the corresponding ω-carboxyalkanoate. 13 And enzyme E, which can convert ω-carboxyalkyl esters into the corresponding ω-carboxyalkyl diesters. 14 .

[0298] The enzymes used according to any aspect of the invention may be the same as those disclosed in the context of the cells according to the invention. Detailed Implementation

[0299] Example

[0300] The foregoing describes preferred embodiments, and as those skilled in the art will understand, variations or modifications in design, construction, or operation may be made without departing from the scope of the claims. These variations may be intended to be covered by the scope of the claims, for example.

[0301] Example 1

[0302] Lauric acid (methyl ester) and undecane (and, in the case of methyl ester, methanol) are produced from dodecane and undecane, respectively, using an enzyme carrying alkane monooxygenase and wax ester synthase that catalyzes the degradation of fatty acids and an enzyme that weakens the enzyme that hydrolyzes fatty acid esters into free fatty acids and corresponding alcohols.

[0303] Example 2

[0304] Methyl 12-aminolaurate and methyl 11-aminoundecanoate are produced from dodecane and undecane, respectively, by whole-cell biocatalysts containing alkane monooxygenase, acetyl ester synthase, and ω-transaminase that catalyze the degradation of fatty acids and enzymes that hydrolyze fatty acid esters into free fatty acids and corresponding alcohols.

[0305] Example 3

[0306] The expression of the *E. coli* fadD gene and the *Hahella chejuensis* gene encoding wax ester synthase was used for... Construction of the carrier

[0307] The vector pCDF-fadD_Ec-wes_Hche (SEQ ID NO:7) carries a gene from Escherichia coli. fadD (encoding acyl-CoA synthase) and codon-optimized expression in E. coli from Hahella chejuensisThe wax ester synthase gene (SEQ ID NO: 2). Although acyl-CoA synthases are responsible for activating fatty acids to the corresponding CoA thioesters, wax ester synthases are required for ester formation between fatty acyl-CoA and alcohols, more specifically methanol. This vector is based on plasmid pCDFDuet-1 (Merck Biosciences; Nottingham, UK) and carries the gene for wax ester synthase. tac under the control of the promoter fadD Genes and coding under the control of the T5 promoter Hahella chejuensis The gene for wax ester synthase was obtained. Genomic DNA of *E. coli* W3110 was amplified by PCR. fadD as well as tac And the T5 promoter box, which obtains the encoding through DNA synthesis. Hahella chejuensis The gene for wax ester synthase. Using a commercially available kit for in vitro recombination (NEBuilder HiFi DNA Assembly Cloning Kit; NEB; Frankfurt / Main, Germany), the vector backbone and representative *E. coli* were... fadD , tac and T5 starter box and code Hahella chejuensis Four DNA fragments of the wax ester synthase gene were fused to obtain the vector pCDF-fadD_Ec-wes_Hche (SEQ ID NO:7).

[0308] Example 4

[0309] Construction of Escherichia coli strains capable of converting alkanes into corresponding ω-functionalized fatty acid methyl esters

[0310] The expression vector pBT10_alkL (for construction details and the listed SEQ ID NO: 8, see Example 1 of WO / 2011 / 131420) contains Pseudomonas oleifera ( Pseudomonas oleovorans The gene of the alk operator alkB, alkG, alkT, alkS and alkL The corresponding gene product catalyzes the oxidation of alkanes to the corresponding alkanols, alkanols, and fatty acids (AlkBGT), as well as substrate uptake (AlkL). Furthermore, the AlkBGT gene product also catalyzes the oxidation of fatty acid methyl esters once fatty acids and methanol are formed from them via the action of acyl-CoA synthase and wax ester synthase (see Example 1). The vector pJ294_alaDH_B.s._TA_C.v.(Ct) (for construction details and the listed SEQ ID NO: 17, see Example 1 of WO / 2013 / 024114) carries a vector from Bacillus subtilis (… Bacillus subtilis ) genes ald(encoding alanine dehydrogenase) and from *Cyperus violaceum* ( Chromobacterium violaceum Cv_2505 (encoding ω-transaminase). Although ω-transaminase is responsible for converting OLAME and OUAME into the corresponding amines ALAME and AUAME, it requires alanine dehydrogenase to provide the amine donor alanine from pyruvate and inorganic ammonia.

[0311] Plasmids pBT10_alkL and pCDF-fadD_Ec-wes_Hche, along with (if appropriate) pJ294_alaDH_B.s._TA_C.v.(Ct), were transformed into *E. coli* W3110∆bioH ∆fadE via electroporation and plated on LB agar plates containing kanamycin (50 µg / ml), ampicillin (100 µg / ml), and spectinomycin (100 µg / ml) (if applicable). The presence and authenticity of the transformants were screened by plasmid preparation and restriction digestion analysis. The following strains were generated:

[0312] ● Escherichia coli W3110 ∆bioH ∆fadE pBT10_alkL / pCDF-fadD_Ec-wes_Hche

[0313] ● Escherichia coli W3110 ∆bioH ∆fadE pBT10_alkL / pJ294_alaDH_B.s._TA_C.v.(Ct) / pCDF-fadD_Ec-wes_Hche.

[0314] Example 5

[0315] Bioconversion for converting alkanes into corresponding ω-functionalized fatty acid methyl esters

[0316] The strains *Escherichia coli* W3110 ∆bioH ∆fadE pBT10_alkL / pCDF-fadD_Ec-wes_Hche* and *Escherichia coli* W3110 ∆bioH ∆fadE pBT10_alkL / pJ294_alaDH_B.s._TA_C.v.(Ct) / pCDF-fadD_Ec-wes_Hche were subjected to fed-batch fermentation followed by biotransformation to investigate their ability to produce methyl ω-hydroxylaurate (HLAME), methyl ω-oxolaurate (OLAME), methyl ω-aminolaurate (ALAME), monomethyl dodecanoate (DDAME), and dimethyl dodecanoate (DDADME) from dodecane. The strains were also subjected to fed-batch fermentation followed by biotransformation to investigate their ability to produce methyl ω-hydroxyundecanoate (HUAME), methyl ω-oxoundecanoate (OUAME), methyl ω-aminoundecanoate (AUAME), monomethyl undecanoate (UDAME), and dimethyl undecanoate (UDADME) from undecane. This was carried out in an eight-parallel fermentation system from DASGIP.

[0317] For fermentation, a 1L reactor equipped with a top-mounted agitator and impeller turbine was used. pH and pO2 were measured online to monitor the process. OTR / CTR measurements were used, in particular, to estimate cellular metabolic activity and fitness.

[0318] The pH probe was calibrated using two-point calibration with measurement solutions of pH 4.0 and pH 7.0, according to the technical references provided by DASGIP. A reactor with the required sensor and connections was prepared according to the technical references provided by DASGIP, 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. After connecting to the measurement amplifier, the pO2 probe was polarized overnight (at least 6 h). The water was then removed under a clean bench and replaced with a high-cell-density culture 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% ferric ammonium citrate solution, and 5 ml / l of filter-sterilized trace element stock solution (composed of HCl (37%) 36.50 g / l, MnCl2*4 H2O 1.91 g / l, ZnSO4*7 H2O 1.87 g / l, ethylenediaminetetraacetic acid dihydrate 0.84 g / l, H3BO3 0.30 g / l, Na2MoO4*2 H2O 0.25 g / l, CaCl2*2 H2O 4.70 g / l. The formula consists of 17.80 g / l FeSO4*7H2O and 0.15 g / l CuCl2*2 H2O, with 15 g / l glucose as a carbon source (added by metering 30 ml / l of sterile feed solution, which consists of 500 g / l glucose, 1% (w / v) MgSO4*7H2O and 2.2% (w / v) NH4Cl), and 50 mg / l kanamycin.

[0319] Subsequently, following the technical references provided by DASGIP, the pO2 probe was calibrated to 100% using single-point calibration (stirrer: 600 rpm / aeration: 10 sL / h air), and the feed, correction agent, and stretches were cleaned in place. For this purpose, the tubes were first rinsed with 70% ethanol, then with 1 M NaOH, followed by sterile demineralized water, and finally filled with various culture media.

[0320] All of the aforementioned Escherichia coli strains were first cultured overnight for approximately 18 h at 37°C and 200 rpm in LB medium (25 ml in a 100 ml shake flask with baffle) containing 50 mg / L kanamycin from frozen cultures. Then, 2 ml of the culture was transferred to 25 ml of high cell density medium in a 100 ml shake flask and incubated at 37°C / 200 rpm for 6 h for a second pre-culture stage. The high cell density medium consisted of the following: (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 filter-sterilized 1% ferric ammonium citrate solution, and 5 ml / L filter-sterilized trace element stock solution (composed of HCl (37%) 36.50 g / L, MnCl2*4 H2O 1.91 g / L, ZnSO4*7 H2O 1.87 g / L, ethylenediaminetetraacetic acid dihydrate 0.84 g / L, and H3BO3 0.30 g / L. The mixture consists of 0.25 g / l Na2MoO4*2 H2O, 4.70 g / l CaCl2*2 H2O, 17.80 g / l FeSO4*7 H2O, and 0.15 g / l CuCl2*2 H2O, with 15 g / l glucose as a carbon source (added by metering 30 ml / l of sterile feed solution, which consists of 500 g / l glucose, 1% (w / v) MgSO4*7 H2O and 2.2% (w / v) NH4Cl), and the described antibiotic.

[0321] To inoculate the reactor at an optical density of 0.1, the OD of the second pre-culture stage was measured. 600 The required amount of culture for inoculation was calculated. Using a 5 ml syringe, the required amount of culture was added through a septum into a heat-treated and aerated reactor.

[0322] Use the following standard procedures:

[0323] .

[0324] The pH was adjusted to pH 6.8 using a 12.5% ​​ammonia solution on one side. During cultivation and biotransformation, dissolved oxygen (pO2 or DO) in the culture was adjusted to at least 30% using agitator feeding and aeration rates. After inoculation, DO was reduced from 100% to 30%, where it remained stable for the remainder of the fermentation process.

[0325] Fermentation was carried out in batches using a fed-batch method. The feeding phase began after the DO peak at the end of the induction batch period, with a feed consisting of 5 g / L glucose, 1% (w / v) MgSO4·7 H2O, and 2.2% (w / v) NH4Cl. The temperature was lowered from 37°C to 30°C upon initiation of feeding. Ten hours after feeding began, oxidative gene expression was induced using 0.025% (v / v) DCPK. Production commenced 14 hours after feeding began (i.e., the start of biotransformation). For this purpose, 150 ml of dodecane or undecane was added to the fermentation broth as a batch.

[0326] To quantify LSME and HLS in the fermentation samples, samples were taken at 1 / 2 / 4 / 20 / 22 h after the start of biotransformation. These samples were prepared for the analysis provided in Example 6.

[0327] Example 6

[0328] The product based on LC-ESI / MS 2 Quantitative

[0329] Quantification of HLAME, OLAME, ALAME, DDAME, DDADME, HUAME, OUAME, AUAME, UDAME, and UDADME in fermentation samples was performed using LC-ESI / MS. 2 Refer to the external calibration of all analytes (0.1–50 mg / L) and use internal standards aminoundecanoic acid (for HLSME, AUA) and d3-LSME (for LSME) to perform the calibration.

[0330] The following instruments are used here:

[0331] ● HPLC system 1260 (Agilent; Böblingen) with an autosampler (G1367E), a binary pump (G1312B), and a column oven (G1316A)

[0332] ● TripelQuad 6410 mass spectrometer with ESI source (Agilent; Böblingen)

[0333] ● HPLC column: Kinetex C18, 100 x 2.1 mm, particle size: 2.6 µm, pore size 100 Å (Phenomenex; Aschaffenburg)

[0334] ● Pre-column: KrudKatcher Ultra HPLC tandem filter; 0.5 µm filter depth and 0.004 mm inner diameter (Phenomenex; Aschaffenburg).

[0335] Samples were prepared by transferring 1900 µl of solvent (80% (v / v) acetonitrile, 20% double-distilled H2O (v / v), and 0.1% formic acid) and 100 µl of sample into a 2 mL reaction vessel. The mixture was vortexed for approximately 10 seconds and then centrifuged at approximately 13,000 rpm for 5 min. The clear supernatant was pipetted and appropriately diluted with diluent (80% (v / v) ACN, 20% double-distilled H2O (v / v), and 0.1% formic acid) for analysis. 100 µl of ISTD was transferred to each 900 µl sample (10 µl for a 90 µl sample volume).

[0336] HPLC separation was performed using the column or pre-column described above. The injection volume was 0.7 µL, the column temperature was 50 °C, and the flow rate was 0.6 mL / min. The mobile phase consisted of eluent A (0.1% (v / v) aqueous formic acid) and eluent B (acetonitrile containing 0.1% (v / v) formic acid). The following gradient profile was used:

[0337] .

[0338] ESI-MS2 analysis was performed in positive ionization mode using the following parameters of the ESI source:

[0339] ● Gas temperature 280℃

[0340] ● Gas flow rate 11 L / min

[0341] ● Atomization pressure 50 psi

[0342] ● Capillary voltage 4000 V.

[0343] The detection and quantification of compounds HLAME, OLAME, ALAME, DDAME, DDADME, HUAME, OUAME, AUAME, UDAME, and UDADME were performed using the following MRM parameters, in which one product ion was used as the qualifier and another as the quantifier in each case:

[0344]

[0345] Table 1. In SIM mode ( m / z 201 and 215) Detection of analytes LA and LAME.

[0346] Example 7

[0347] Through E. coli W3110 ∆bioH ∆fadE pBT10_alkL / pCDF-fadD_Ec-wes_Hche and Escherichia coli W3110 ∆bioH ∆fadE pBT10_alkL / pJ294_alaDH_B.s._TA_C.v.(Ct) / pCDF- fadD_Ec-wes_Hche converts alkanes into the corresponding ω-functionalized fatty acid methyl esters.

[0348] Using the above scheme, it can be shown that *E. coli* W3110 ∆bioH ∆fadE pBT10_alkL / pCDF-fadD_Ec-wes_Hche produces DDAME and DDADME from dodecane and UDAME and UDADME from undecane (see Tables 1 and 2). Furthermore, it can be shown that *E. coli* W3110 ∆bioH ∆fadE pBT10_alkL / pJ294_alaDH_B.s._TA_C.v.(Ct) / pCDF-fadD_Ec-wes_Hche produces HLAME and ALAME from dodecane and HUAME and AUAME from undecane (see Tables 2 and 3).

[0349]

[0350] Table 2. Concentrations of ω-functionalized fatty acid methyl esters formed from dodecane by strains Escherichia coli W3110 ∆bioH ∆fadE pBT10_alkL / pCDF-fadD_Ec-wes_Hche (strain 1) and Escherichia coli W3110 ∆bioH ∆fadE pBT10_alkL / pJ294_alaDH_B.s._TA_C.v.(Ct) / pCDF-fadD_Ec-wes_Hche (strain 2).

[0351]

[0352] Table 3. Concentrations of ω-functionalized fatty acid methyl esters formed from undecane by strains Escherichia coli W3110 ∆bioH ∆fadE pBT10_alkL / pCDF-fadD_Ec-wes_Hche (strain 1) and Escherichia coli W3110 ∆bioH ∆fadE pBT10_alkL / pJ294_alaDH_B.s._TA_C.v.(Ct) / pCDF-fadD_Ec-wes_Hche (strain 2). sequence list <110> Evonik Degussa GmbH <120> Biotechnical production of ω-functionalized carboxylic acids and their esters <130> 201500273EP <150> EP15196180 <151> 2015‑11‑25 <160> 7 <170> PatentIn version 3.5 <210> 1 <211> 401 <212> PRT <213> Bad bacteria <400> 1 Met Leu Glu Lys His Arg Val Leu Asp Ser Ala Pro Glu Tyr Val Asp 1 5 10 15 Lys Lys Lys Tyr Leu Trp Ile Leu Ser Thr Leu Trp Pro Ala Thr Pro 20 25 30 Met Ile Gly Ile Trp Leu Ala Asn Glu Thr Gly Trp Gly Ile Phe Tyr 35 40 45 Gly Leu Val Leu Leu Val Trp Tyr Gly Ala Leu Pro Leu Leu Asp Ala 50 55 60 Met Phe Gly Glu Asp Phe Asn Asn Pro Pro Glu Glu Val Val Pro Lys 65 70 75 80 Leu Glu Lys Glu Arg Tyr Tyr Arg Val Leu Thr Tyr Leu Thr Val Pro 85 90 95 Met His Tyr Ala Ala Leu Ile Val Ser Ala Trp Trp Val Gly Thr Gln 100 105 110 Pro Met Ser Trp Leu Glu Ile Gly Ala Leu Ala Leu Ser Leu Gly Ile 115 120 125 Val Asn Gly Leu Ala Leu Asn Thr Gly His Glu Leu Gly His Lys Lys 130 135 140 Glu Thr Phe Asp Arg Trp Met Ala Lys Ile Val Leu Ala Val Val Gly 145 150 155 160 Tyr Gly His Phe Phe Ile Glu His Asn Lys Gly His His Arg Asp Val 165 170 175 Ala Thr Pro Met Asp Pro Ala Thr Ser Arg Met Gly Glu Ser Ile Tyr 180 185 190 Lys Phe Ser Ile Arg Glu Ile Pro Gly Ala Phe Ile Arg Ala Trp Gly 195 200 205 Leu Glu Glu Gln Arg Leu Ser Arg Arg Gly Gln Ser Val Trp Ser Phe 210 215 220 Asp Asn Glu Ile Leu Gln Pro Met Ile Ile Thr Val Ile Leu Tyr Ala 225 230 235 240 Val Leu Leu Ala Leu Phe Gly Pro Lys Met Leu Val Phe Leu Pro Ile 245 250 255 Gln Met Ala Phe Gly Trp Trp Gln Leu Thr Ser Ala Asn Tyr Ile Glu 260 265 270 His Tyr Gly Leu Leu Arg Gln Lys Met Glu Asp Gly Arg Tyr Glu His 275 280 285 Gln Lys Pro His His Ser Trp Asn Ser Asn His Ile Val Ser Asn Leu 290 295 300 Val Leu Phe His Leu Gln Arg His Ser Asp His His Ala His Pro Thr 305 310 315 320 Arg Ser Tyr Gln Ser Leu Arg Asp Phe Pro Gly Leu Pro Ala Leu Pro 325 330 335 Thr Gly Tyr Pro Gly Ala Phe Leu Met Ala Met Ile Pro Gln Trp Phe 340 345 350 Arg Ser Val Met Asp Pro Lys Val Val Asp Trp Ala Gly Gly Asp Leu 355 360 365 Asn Lys Ile Gln Ile Asp Asp Ser Met Arg Glu Thr Tyr Leu Lys Lys 370 375 380 Phe Gly Thr Ser Ser Ala Gly His Ser Ser Ser Thr Ser Ala Val Ala 385 390 395 400 Ser <210> 2 <211> 458 <212> PRT <213> Hahella chejuensis <400> 2 Met Thr Pro Leu Ser Pro Val Asp Gln Ile Phe Leu Trp Leu Glu Lys 1 5 10 15 Arg Gln Gln Pro Met His Val Gly Gly Leu His Ile Phe Ser Phe Pro 20 25 30 Asp Asp Ala Asp Ala Lys Tyr Met Thr Glu Leu Ala Gln Gln Leu Arg 35 40 45 Ala Tyr Ala Thr Pro Gln Ala Pro Phe Asn Arg Arg Leu Arg Gln Arg 50 55 60 Trp Gly Arg Tyr Tyr Trp Asp Thr Asp Ala Gln Phe Asp Leu Glu His 65 70 75 80 His Phe Arg His Glu Ala Leu Pro Lys Pro Gly Arg Ile Arg Glu Leu 85 90 95 Leu Ala His Val Ser Ala Glu His Ser Asn Leu Met Asp Arg Glu Arg 100 105 110 Pro Met Trp Glu Cys His Leu Ile Glu Gly Ile Arg Gly Arg Arg Phe 115 120 125 Ala Val Tyr Tyr Lys Ala His His Cys Met Leu Asp Gly Val Ala Ala 130 135 140 Met Arg Met Cys Val Lys Ser Tyr Ser Phe Asp Pro Thr Ala Thr Glu 145 150 155 160 Met Pro Pro Ile Trp Ala Ile Ser Lys Asp Val Thr Pro Ala Arg Glu 165 170 175 Thr Gln Ala Pro Ala Ala Gly Asp Leu Val His Ser Leu Ser Gln Leu 180 185 190 Val Glu Gly Ala Gly Arg Gln Leu Ala Thr Val Pro Thr Leu Ile Arg 195 200 205 Glu Leu Gly Lys Asn Leu Leu Lys Ala Arg Asp Asp Ser Asp Ala Gly 210 215 220 Leu Ile Phe Arg Ala Pro Pro Ser Ile Leu Asn Gln Arg Ile Thr Gly 225 230 235 240 Ser Arg Arg Phe Ala Ala Gln Ser Tyr Ala Leu Glu Arg Phe Lys Ala 245 250 255 Ile Gly Lys Ala Phe Gln Ala Thr Val Asn Asp Val Val Leu Ala Val 260 265 270 Cys Gly Ser Ala Leu Arg Asn Tyr Leu Leu Ser Arg Gln Ala Leu Pro 275 280 285 Asp Gln Pro Leu Ile Ala Met Ala Pro Met Ser Ile Arg Gln Asp Asp 290 295 300 Ser Asp Ser Gly Asn Gln Ile Ala Met Ile Leu Ala Asn Leu Gly Thr 305 310 315 320 His Ile Ala Asp Pro Val Arg Arg Leu Glu Leu Thr Gln Ala Ser Ala 325 330 335 Arg Glu Ser Lys Glu Arg Phe Arg Gln Met Thr Pro Glu Glu Ala Val 340 345 350 Asn Tyr Thr Ala Leu Thr Leu Ala Pro Ser Gly Leu Asn Leu Leu Thr 355 360 365 Gly Leu Ala Pro Lys Trp Gln Ala Phe Asn Val Val Ile Ser Asn Val 370 375 380 Pro Gly Pro Asn Lys Pro Leu Tyr Trp Asn Gly Ala Arg Leu Glu Gly 385 390 395 400 Met Tyr Pro Val Ser Ile Pro Val Asp Tyr Ala Ala Leu Asn Ile Thr 405 410 415 Leu Val Ser Tyr Arg Asp Gln Leu Glu Phe Gly Phe Thr Ala Cys Arg 420 425 430 Arg Thr Leu Pro Ser Met Gln Arg Leu Leu Asp Tyr Ile Glu Gln Gly 435 440 445 Ile Ala Glu Leu Glu Lys Ala Ala Gly Val 450 455 <210> 3 <211> 459 <212> PRT <213> purple color <400> 3 Met Gln Lys Gln Arg Thr Thr Ser Gln Trp Arg Glu Leu Asp Ala Ala 1 5 10 15 His His Leu His Pro Phe Thr Asp Thr Ala Ser Leu Asn Gln Ala Gly 20 25 30 Ala Arg Val Met Thr Arg Gly Glu Gly Val Tyr Leu Trp Asp Ser Glu 35 40 45 Gly Asn Lys Ile Ile Asp Gly Met Ala Gly Leu Trp Cys Val Asn Val 50 55 60 Gly Tyr Gly Arg Lys Asp Phe Ala Glu Ala Ala Arg Arg Gln Met Glu 65 70 75 80 Glu Leu Pro Phe Tyr Asn Thr Phe Phe Lys Thr Thr His Pro Ala Val 85 90 95 Val Glu Leu Ser Ser Leu Leu Ala Glu Val Thr Pro Ala Gly Phe Asp 100 105 110 Arg Val Phe Tyr Thr Asn Ser Gly Ser Glu Ser Val Asp Thr Met Ile 115 120 125 Arg Met Val Arg Arg Tyr Trp Asp Val Gln Gly Lys Pro Glu Lys Lys 130 135 140 Thr Leu Ile Gly Arg Trp Asn Gly Tyr His Gly Ser Thr Ile Gly Gly 145 150 155 160 Ala Ser Leu Gly Gly Met Lys Tyr Met His Glu Gln Gly Asp Leu Pro 165 170 175 Ile Pro Gly Met Ala His Ile Glu Gln Pro Trp Trp Tyr Lys His Gly 180 185 190 Lys Asp Met Thr Pro Asp Glu Phe Gly Val Val Ala Ala Arg Trp Leu 195 200 205 Glu Glu Lys Ile Leu Glu Ile Gly Ala Asp Lys Val Ala Ala Phe Val 210 215 220 Gly Glu Pro Ile Gln Gly Ala Gly Gly Val Ile Val Pro Pro Ala Thr 225 230 235 240 Tyr Trp Pro Glu Ile Glu Arg Ile Cys Arg Lys Tyr Asp Val Leu Leu 245 250 255 Val Ala Asp Glu Val Ile Cys Gly Phe Gly Arg Thr Gly Glu Trp Phe 260 265 270 Gly His Gln His Phe Gly Phe Gln Pro Asp Leu Phe Thr Ala Ala Lys 275 280 285 Gly Leu Ser Ser Gly Tyr Leu Pro Ile Gly Ala Val Phe Val Gly Lys 290 295 300 Arg Val Ala Glu Gly Leu Ile Ala Gly Gly Asp Phe Asn His Gly Phe 305 310 315 320 Thr Tyr Ser Gly His Pro Val Cys Ala Ala Val Ala His Ala Asn Val 325 330 335 Ala Ala Leu Arg Asp Glu Gly Ile Val Gln Arg Val Lys Asp Asp Ile 340 345 350 Gly Pro Tyr Met Gln Lys Arg Trp Arg Glu Thr Phe Ser Arg Phe Glu 355 360 365 His Val Asp Asp Val Arg Gly Val Gly Met Val Gln Ala Phe Thr Leu 370 375 380 Val Lys Asn Lys Ala Lys Arg Glu Leu Phe Pro Asp Phe Gly Glu Ile 385 390 395 400 Gly Thr Leu Cys Arg Asp Ile Phe Phe Arg Asn Asn Leu Ile Met Arg 405 410 415 Ala Cys Gly Asp His Ile Val Ser Ala Pro Pro Leu Val Met Thr Arg 420 425 430 Ala Glu Val Asp Glu Met Leu Ala Val Ala Glu Arg Cys Leu Glu Glu 435 440 445 Phe Glu Gln Thr Leu Lys Ala Arg Gly Leu Ala 450 455 <210> 4 <211> 230 <212> PRT <213> Bad bacteria <400> 4 Met Ser Phe Ser Asn Tyr Lys Val Ile Ala Met Pro Val Leu Val Ala 1 5 10 15 Asn Phe Val Leu Gly Ala Ala Thr Ala Trp Ala Asn Glu Asn Tyr Pro 20 25 30 Ala Lys Ser Ala Gly Tyr Asn Gln Gly Asp Trp Val Ala Ser Phe Asn 35 40 45 Phe Ser Lys Val Tyr Val Gly Glu Glu Leu Gly Asp Leu Asn Val Gly 50 55 60 Gly Gly Ala Leu Pro Asn Ala Asp Val Ser Ile Gly Asn Asp Thr Thr 65 70 75 80 Leu Thr Phe Asp Ile Ala Tyr Phe Val Ser Ser Asn Ile Ala Val Asp 85 90 95 Phe Phe Val Gly Val Pro Ala Arg Ala Lys Phe Gln Gly Glu Lys Ser 100 105 110 Ile Ser Ser Leu Gly Arg Val Ser Glu Val Asp Tyr Gly Pro Ala Ile 115 120 125 Leu Ser Leu Gln Tyr His Tyr Asp Ser Phe Glu Arg Leu Tyr Pro Tyr 130 135 140 Val Gly Val Gly Val Gly Arg Val Leu Phe Phe Asp Lys Thr Asp Gly 145 150 155 160 Ala Leu Ser Ser Phe Asp Ile Lys Asp Lys Trp Ala Pro Ala Phe Gln 165 170 175 Val Gly Leu Arg Tyr Asp Leu Gly Asn Ser Trp Met Leu Asn Ser Asp 180 185 190 Val Arg Tyr Ile Pro Phe Lys Thr Asp Val Thr Gly Thr Leu Gly Pro 195 200 205 Val Pro Val Ser Thr Lys Ile Glu Val Asp Pro Phe Ile Leu Ser Leu 210 215 220 Gly Ala Ser Tyr Val Phe 225 230 <210> 5 <211> 230 <212> PRT <213> artificial sequence <220> <223> ALK L <400> 5 Val Ser Phe Ser Asn Tyr Lys Val Ile Ala Met Pro Val Leu Val Ala 1 5 10 15 Asn Phe Val Leu Gly Ala Ala Thr Ala Trp Ala Asn Glu Asn Tyr Pro 20 25 30 Ala Lys Ser Ala Gly Tyr Asn Gln Gly Asp Trp Val Ala Ser Phe Asn 35 40 45 Phe Ser Lys Val Tyr Val Gly Glu Glu Leu Gly Asp Leu Asn Val Gly 50 55 60 Gly Gly Ala Leu Pro Asn Ala Asp Val Ser Ile Gly Asn Asp Thr Thr 65 70 75 80 Leu Thr Phe Asp Ile Ala Tyr Phe Val Ser Ser Asn Ile Ala Val Asp 85 90 95 Phe Phe Val Gly Val Pro Ala Arg Ala Lys Phe Gln Gly Glu Lys Ser 100 105 110 Ile Ser Ser Leu Gly Arg Val Ser Glu Val Asp Tyr Gly Pro Ala Ile 115 120 125 Leu Ser Leu Gln Tyr His Tyr Asp Ser Phe Glu Arg Leu Tyr Pro Tyr 130 135 140 Val Gly Val Gly Val Gly Arg Val Leu Phe Phe Asp Lys Thr Asp Gly 145 150 155 160 Ala Leu Ser Ser Phe Asp Ile Lys Asp Lys Trp Ala Pro Ala Phe Gln 165 170 175 Val Gly Leu Arg Tyr Asp Leu Gly Asn Ser Trp Met Leu Asn Ser Asp 180 185 190 Val Arg Tyr Ile Pro Phe Lys Thr Asp Val Thr Gly Thr Leu Gly Pro 195 200 205 Val Pro Val Ser Thr Lys Ile Glu Val Asp Pro Phe Ile Leu Ser Leu 210 215 220 Gly Ala Ser Tyr Val Phe 225 230 <210> 6 <211> 446 <212> PRT <213> Artificial sequence <220> <223> FADL <400> 6 Met Ser Gln Lys Thr Leu Phe Thr Lys Ser Ala Leu Ala Val Ala Val 1 5 10 15 Ala Leu Ile Ser Thr Gln Ala Trp Ser Ala Gly Phe Gln Leu Asn Glu 20 25 30 Phe Ser Ser Ser Gly Leu Gly Arg Ala Tyr Ser Gly Glu Gly Ala Ile 35 40 45 Ala Asp Asp Ala Gly Asn Val Ser Arg Asn Pro Ala Leu Ile Thr Met 50 55 60 Phe Asp Arg Pro Thr Phe Ser Ala Gly Ala Val Tyr Ile Asp Pro Asp 65 70 75 80 Val Asn Ile Ser Gly Thr Ser Pro Ser Gly Arg Ser Leu Lys Ala Asp 85 90 95 Asn Ile Ala Pro Thr Ala Trp Val Pro Asn Met His Phe Val Ala Pro 100 105 110 Ile Asn Asp Gln Phe Gly Trp Gly Ala Ser Ile Thr Ser Asn Tyr Gly 115 120 125 Leu Ala Thr Glu Phe Asn Asp Thr Tyr Ala Gly Gly Ser Val Gly Gly 130 135 140 Thr Thr Asp Leu Glu Thr Met Asn Leu Asn Leu Ser Gly Ala Tyr Arg 145 150 155 160 Leu Asn Asn Ala Trp Ser Phe Gly Leu Gly Phe Asn Ala Val Tyr Ala 165 170 175 Arg Ala Lys Ile Glu Arg Phe Ala Gly Asp Leu Gly Gln Leu Val Ala 180 185 190 Gly Gln Ile Met Gln Ser Pro Ala Gly Gln Thr Gln Gln Gly Gln Ala 195 200 205 Leu Ala Ala Thr Ala Asn Gly Ile Asp Ser Asn Thr Lys Ile Ala His 210 215 220 Leu Asn Gly Asn Gln Trp Gly Phe Gly Trp Asn Ala Gly Ile Leu Tyr 225 230 235 240 Glu Leu Asp Lys Asn Asn Arg Tyr Ala Leu Thr Tyr Arg Ser Glu Val 245 250 255 Lys Ile Asp Phe Lys Gly Asn Tyr Ser Ser Asp Leu Asn Arg Ala Phe 260 265 270 Asn Asn Tyr Gly Leu Pro Ile Pro Thr Ala Thr Gly Gly Ala Thr Gln 275 280 285 Ser Gly Tyr Leu Thr Leu Asn Leu Pro Glu Met Trp Glu Val Ser Gly 290 295 300 Tyr Asn Arg Val Asp Pro Gln Trp Ala Ile His Tyr Ser Leu Ala Tyr 305 310 315 320 Thr Ser Trp Ser Gln Phe Gln Gln Leu Lys Ala Thr Ser Thr Ser Gly 325 330 335 Asp Thr Leu Phe Gln Lys His Glu Gly Phe Lys Asp Ala Tyr Arg Ile 340 345 350 Ala Leu Gly Thr Thr Tyr Tyr Tyr Asp Asp Asn Trp Thr Phe Arg Thr [[ID=​​​​​​​​​​​​​​​​​​​​​​​​​​​​​<220> <223> Vector pCDF-fadD_Ec-wes_Hche <400> 7 cgggatctcg acgctctccc ttatgcgact cctgcgttta gggaaagagc atttgtcaga 60 atatttaagg gcgcctgtca ctttgcttga tatatgagaa ttatttaacc ttataaatga 120 gaaaaaagca acgcacttta aataagatac gttgcttttt cgattgatga acacctataa 180 ttaaactatt catctattat ttatgatttt ttgtatatac aatatttcta gtttgttaaa 240 gagaattaag aaaataaatc tcgaaaataa taaagggaaa atcagttttt gatatcaaaa 300 ttatacatgt caacgataat acaaaatata atacaaacta taagatgtta tcagtattta 360 ttatgcattt agaatacctt ttgtgtcgcc cttattcgac tccctataga agttcctatt 420 ctctagaaag tataggaact tcccttcatt ttggatccaa ttgtgagcgg ataacaatta 480 cgagcttcat gcacagtgat cgacgctgtt gacaattaat catcggctcg tataatgtgt 540 ggatgtggaa ttgtgagcgc tcacaattcc acaacggttt ccctctagaa ataattttgt 600 ttaacaggag gtaaaacata tgttaacggc atgtatatca tttggggttg cgatgacgac 660 gaacacgcat tttagaggtg aagaattgaa gaaggtttgg cttaaccgtt atcccgcgga 720 cgttccgacg gagatcaacc ctgaccgtta tcaatctctg gtagatatgt ttgagcagtc 780 ggtcgcgcgc tacgccgatc aacctgcgtt tgtgaatatg ggggaggtaa tgaccttccg 840 caagctggaa gaacgcagtc gcgcgtttgc cgcttatttg caacaagggt tggggctgaa 900 gaaaggcgat cgcgttgcgt tgatgatgcc taatttattg caatatccgg tggcgctgtt 960 tggcattttg cgtgccggga tgatcgtcgt aaacgttaac ccgttgtata ccccgcgtga 1020 gcttgagcat cagcttaacg atagcggcgc atcggcgatt gttatcgtgt ctaactttgc 1080 tcacacactg gaaaaagtgg ttgataaaac cgccgttcag cacgtaattc tgacccgtat 1140 gggcgatcag ctatctacgg caaaaggcac ggtagtcaat ttcgttgtta aatacatcaa 1200 gcgtttggtg ccgaaatacc atctgccaga tgccatttca tttcgtagcg cactgcataa 1260 cggctaccgg atgcagtacg tcaaacccga actggtgccg gaagatttag cttttctgca 1320 atacaccggc ggcaccactg gtgtggcgaa aggcgcgatg ctgactcacc gcaatatgct 1380 ggcgaacctg gaacaggtta acgcgaccta tggtccgctg ttgcatccgg gcaaagagct 1440 ggtggtgacg gcgctgccgc tgtatcacat ttttgccctg accattaact gcctgctgtt 1500 tatcgaactg ggtgggcaga acctgcttat cactaacccg cgcgatattc cagggttggt 1560 aaaagagtta gcgaaatatc cgtttaccgc tatcacgggc gttaacacct tgttcaatgc 1620 gttgctgaac aataaagagt tccagcagct ggatttctcc agtctgcatc tttccgcagg 1680 cggtgggatg ccagtgcagc aagtggtggc agagcgttgg gtgaaactga ccggacagta 1740 tctgctggaa ggctatggcc ttaccgagtg tgcgccgctg gtcagcgtta acccatatga 1800 tattgattat catagtggta gcatcggttt gccggtgccg tcgacggaag ccaaactggt 1860 ggatgatgat gataatgaag taccaccagg tcaaccgggt gagctttgtg tcaaaggacc 1920 gcaggtgatg ctgggttact ggcagcgtcc cgatgctacc gatgaaatca tcaaaaatgg 1980 ctggttacac accggcgaca tcgcggtaat ggatgaagaa ggattcctgc gcattgtcga 2040 tcgtaaaaaa gacatgattc tggtttccgg ttttaacgtc tatcccaacg agattgaaga 2100 tgtcgtcatg cagcatcctg gcgtacagga agtcgcggct gttggcgtac cttccggctc 2160 cagtggtgaa gcggtgaaaa tcttcgtagt gaaaaaagat ccatcgctta ccgaagagtc 2220 actggtgact ttttgccgcc gtcagctcac gggatacaaa gtaccgaagc tggtggagtt 2280 tcgtgatgag ttaccgaaat ctaacgtcgg aaaaattttg cgacgagaat tacgtgacga 2340 agcgcgcggc aaagtggaca ataaagcctg agcgaattcg gatccatgca cagtgaaatc 2400 atgaaaaatt tatttgcttt gtgagcggat aacaattata atagcatgct ggtcagtatt 2460 gagcgatgca tgcacggttt ccctctagaa ataattttgt ttaactttta ggaggtaaaa 2520 accatgggta gctctcacca tcatcatcat cacagctctg gcctggttcc gcgcggttcc 2580 cacatgacgc cgctgagccc ggtcgatcaa atctttctgt ggctggagaa gcgtcagcag 2640 ccgatgcacg tcggtggctt gcacattttc agcttccctg atgacgcaga cgcgaagtat 2700 atgaccgagc tggcgcagca actgcgtgca tacgcgacgc cgcaggcacc attcaaccgt 2760 cgcctgcgtc agcgctgggg ccgttactat tgggacaccg atgctcagtt cgacctggag 2820 catcattttc gtcacgaagc gctgccgaaa ccgggtcgca ttcgcgaact gttggcccac 2880 gttagcgcgg agcattctaa tctgatggat cgtgaacgtc cgatgtggga gtgccatctg 2940 atcgaaggca tccgtggtcg ccgtttcgcg gtttactaca aggcgcatca ctgtatgctg 3000 gacggtgtag ccgccatgcg tatgtgcgtg aaatcctaca gctttgatcc gaccgcaacg 3060 gagatgccgc cgatttgggc tatcagcaaa gacgttaccc cggctcgtga aactcaagca 3120 ccggcagcgg gtgacctggt gcactccctg tcccagctgg ttgagggtgc cggtcgtcaa 3180 ctggcgaccg tcccgaccct gattcgtgag ctgggcaaaa acttgctgaa ggcgcgtgac 3240 gactctgacg cgggtctgat ttttcgcgct ccgccaagca ttctgaacca acgcatcacc 3300 ggtagccgcc gttttgcggc gcagagctac gcgttggaac gctttaaggc gatcggtaag 3360 gcattccagg ctacggttaa cgatgtggtg ctggcggtgt gcggttccgc actgcgtaac 3420 tatttgctga gccgccaagc cctgccggat caaccgctga ttgcaatggc ccctatgagc 3480 atccgtcagg acgatagcga cagcggcaat cagatcgcga tgatcctggc gaatctgggc 3540 acccacatcg cggacccggt ccgtcgtttg gaactgacgc aagcaagcgc tcgcgagagc 3600 aaagagcgct tccgtcagat gacgccggaa gaggcagtga actataccgc gctgaccctg 3660 gccccgagcg gtctgaatct gctgacgggt ttggccccga aatggcaggc cttcaatgtc 3720 gtgattagca acgttccagg cccgaataag ccgctgtact ggaacggtgc gcgcctggaa 3780 ggcatgtatc cggtttctat tcctgtcgat tatgcggcat tgaatatcac tctggttagc 3840 taccgtgatc aactggaatt tggtttcacc gcatgtcgcc gtaccctgcc gtcgatgcaa 3900 cgtctgttgg attacattga gcaaggcatt gccgagctgg agaaagcggc tggcgtgtaa 3960 ctcgagatcg aatgagcaat aactagcata accccttggg gcctctaaac gggtcttgag 4020 gctcgagtct ggtaaagaaa ccgctgctgc gaaatttgaa cgccagcaca tggactcgtc 4080 tactagcgca gcttaattaa cctaggctgc tgccaccgct gagcaataac tagcataacc 4140 ccttggggcc tctaaacggg tcttgagggg ttttttgctg aaacctcagg catttgagaa 4200 gcacacggtc acactgcttc cggtagtcaa taaaccggta aaccagcaat agacataagc 4260 ggctatttaa cgaccctgcc ctgaaccgac gaccgggtca tcgtggccgg atcttgcggc 4320 ccctcggctt gaacgaattg ttagacatta tttgccgact accttggtga tctcgccttt 4380 cacgtagtgg acaaattctt ccaactgatc tgcgcgcgag gccaagcgat cttcttcttg 4440 tccaagataa gcctgtctag cttcaagtat gacgggctga tactgggccg gcaggcgctc 4500 cattgcccag tcggcagcga catccttcgg cgcgattttg ccggttactg cgctgtacca 4560 aatgcgggac aacgtaagca ctacatttcg ctcatcgcca gcccagtcgg gcggcgagtt 4620 ccatagcgtt aaggtttcat ttagcgcctc aaatagatcc tgttcaggaa ccggatcaaa 4680 gagttcctcc gccgctggac ctaccaaggc aacgctatgt tctcttgctt ttgtcagcaa 4740 gatagccaga tcaatgtcga tcgtggctgg ctcgaagata cctgcaagaa tgtcattgcg 4800 ctgccattct ccaaattgca gttcgcgctt agctggataa cgccacggaa tgatgtcgtc 4860 gtgcacaaca atggtgactt ctacagcgcg gagaatctcg ctctctccag gggaagccga 4920 agtttccaaa aggtcgttga tcaaagctcg ccgcgttgtt tcatcaagcc ttacggtcac 4980 cgtaaccagc aaatcaatat cactgtgtgg cttcaggccg ccatccactg cggagccgta 5040 caaatgtacg gccagcaacg tcggttcgag atggcgctcg atgacgccaa ctacctctga 5100 tagttgagtc gatacttcgg cgatcaccgc ttccctcata ctcttccttt ttcaatatta 5160 ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa 5220 aaataaacaa atagctagct cactcggtcg ctacgctccg ggcgtgagac tgcggcgggc 5280 gctgcggaca catacaaagt tacccacaga ttccgtggat aagcagggga ctaacatgtg 5340 aggcaaaaca gcagggccgc gccggtggcg tttttccata ggctccgccc tcctgccaga 5400 gttcacataa acagacgctt ttccggtgca tctgtgggag ccgtgaggct caaccatgaa 5460 tctgacagta cgggcgaaac ccgacaggac ttaaagatcc ccaccgtttc cggcgggtcg 5520 ctccctcttg cgctctcctg ttccgaccct gccgtttacc ggatacctgt tccgcctttc 5580 tcccttacgg gaagtgtggc gctttctcat agctcacaca ctggtatctc ggctcggtgt 5640 aggtcgttcg ctccaagctg ggctgtaagc aagaactccc cgttcagccc gactgctgcg 5700 ccttatccgg taactgttca cttgagtcca acccggaaaa gcacggtaaa acgccactgg 5760 cagcagccat tggtaactgg gagttcgcag aggatttgtt tagctaaaca cgcggttgct 5820 cttgaagtgt gcgccaaagt ccggctacac tggaaggaca gatttggttg ctgtgctctg 5880 cgaaagccag ttaccacggt taagcagttc cccaactgac ttaaccttcg atcaaaccac 5940 ctccccaggt ggtttttcg tttacagggc aaaagattac gcgcagaaaa aaaggatctc 6000 aagaagatcc tttgatcttt tctactgaac cgctctagat ttcagtgcaa tttatctctt 6060 caaatgtagc acctgaagtc agccccatac gatataagtt gtaattctca tgttagtcat 6120 gccccgcgcc caccggaagg agctgactgg gttgaaggct ctcaagggca tcggtcgaga 6180 tcccggtgcc taatgagtga gctaacttac attaattgcg ttgcgctcac tgcccgcttt 6240 ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg 6300 cggtttgcgt attgggcgcc agggtggtttt ttcttttcac cagtgagacg ggcaacagct 6360 gattgccctt caccgcctgg ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc 6420 ccagcaggcg aaaatcctgt ttgatggtgg ttaacggcgg gatataacat gagctgtctt 6480 cggtatcgtc gtatcccact accgagatgt ccgcaccaac gcgcagcccg gactcggtaa 6540 tggcgcgcat tgcgcccagc gccatctgat cgttggcaac cagcatcgca gtgggaacga 6600 tgccctcatt cagcatttgc atggtttgtt gaaaaccgga catggcactc cagtcgcctt 6660 cccgttccgc tatcggctga atttgattgc gagtgagata tttatgccag ccagccagac 6720 gcagacgcgc cgagacagaa cttaatgggc ccgctaacag cgcgatttgc tggtgaccca 6780 atgcgaccag atgctccacg cccagtcgcg taccgtcttc atgggagaaa ataatactgt 6840 tgatgggtgt ctggtcagag acatcaagaa ataacgccgg aacattagtg caggcagctt 6900 ccacagcaat ggcatcctgg tcatccagcg gatagttaat gatcagccca ctgacgcgtt 6960 gcgcgagaag attgtgcacc gccgctttac aggcttcgac gccgcttcgt tctaccatcg 7020 acaccaccac gctggcaccc agttgatcgg cgcgagattt aatcgccgcg acaatttgcg 7080 acggcgcgtg cagggccaga ctggaggtgg caacgccaat cagcaacgac tgtttgcccg 7140 ccagttgttg tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc gcttccactt 7200 tttcccgcgt tttcgcagaa acgtggctgg cctggttcac cacgcgggaa acggtctgat 7260 aagagacacc ggcatactct gcgacatcgt ataacgttac tggtttcaca ttcaccaccc 7320 tgaattgact ctcttccggg cgctatcatg ccataccgcg aaaggttttg cgccattcga 7380 tggtgtc 7387

Claims

1. Microbial cells for producing monomethyl dodecanoate (DDAME), dimethyl dodecanoate (DDADME), monomethyl undecanoate (UDAME), dimethyl undecanoate (UDADME), methyl ω-hydroxylaurate (HLAME), methyl ω-aminolaurate (ALAME), methyl ω-hydroxyundecanoate (HUAME) or methyl ω-aminoundecanoate (AUAME) from dodecane or undecane, wherein said cells are *Escherichia coli* W3110ΔbioHΔfadE comprising SEQ ID NO:7 and SEQ ID NO:8 in WO / 2011 / 131420.

2. The microbial cell according to claim 1, further comprising SEQ ID NO:17 of WO / 2013 / 024114.

3. A method for producing monomethyl dodecanoate (DDAME), dimethyl dodecanoate (DDADME), monomethyl undecanoate (UDAME), dimethyl undecanoate (UDADME), methyl ω-hydroxylaurate (HLAME), methyl ω-aminolaurate (ALAME), methyl ω-hydroxyundecanoate (HUAME) or methyl ω-aminoundecanoate (AUAME), wherein the method comprises the step of contacting the cells according to claim 1 or 2 with dodecane or undecane.

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