Patent Application
20180127791
The present invention relates to biotechnological methods and cells for producing at least one acyl amino acid from amino acids and/or fatty acids.
Acyl amino acids are a class of surface-active agents with a variety of uses, for example as detergents for washing purposes, emulsifiers in food products and as essential ingredients in various personal care products such as shampoos, soaps, moisturizing agents and the like. In addition to having both hydrophobic and hydrophilic regions, a prerequisite for use as a surfactant, the compounds (surfactants) are made of naturally occurring molecules, more specifically amino acids and fatty acids, which are not only non-hazardous and environmentally acceptable but may be readily produced at a large scale using inexpensive biological raw materials. In pharmacological research, acyl amino acids are used as neuromodulators and probes for new drug targets.
Acyl amino acids have been isolated from a multitude of biological sources and are believed to have a range of functions, for example as signalling molecules in mammalian tissues, as building blocks for antibiotics in bacterial cultures, as compounds involved in bacterial protein sorting and the like.
Traditionally acyl amino acids have been produced at an industrial scale starting with materials derived from tropical oils. More specifically, activated fatty acids provided in the form of acid chlorides may be used to acylate amino acids in an aqueous alkaline medium as described in GB1483500. Shortcomings of such approaches include the need to add hazardous chemicals such as sulphuric acid or anhydrides thereof. Other synthetic approaches are associated with the accumulation of by-products such as chloride salts which have undesirable effects on surfactancy.
Thus these very effective and high-foaming surfactants are gaining significant and increasing value in the chemical industry as an ingredient in home and personal care applications. This would be especially true, if an environmentally benign production process would be available, as the current chemical production processes involve compounds such as phosgene or phosphorus trichloride. Such an environmentally friendly process would be the biocatalytic conversion of amino and fatty acids to acyl amino acids by whole-cell biocatalysts not requiring any toxic chemicals.
A range of biotechnological routes towards production of acyl amino acids has been described. However, none of them is adequate for the commercial large-scale production of acyl amino acids owing to low yields, insufficient purities and the need for multi-step purification procedures. In particular, only a small proportion of the carbon substrates fed to biotechnologically useful organisms is actually converted to the sought-after product, whilst much of it is consumed by reactions of the primary metabolism. These carbon substrates may even include amino and fatty acids which may be used by the whole-cell biocatalysts as an energy source thus reducing product yield per substrate and, in turn, increasing manufacturing costs.
The present invention attempts to solve the problems above by providing at least one method of producing acyl amino acids using biocatalysts with attenuated capability to use amino acids and/or fatty acids as an energy source for cell growth and maintenance. Accordingly, the yield and purity of the product of the present invention, namely acyl amino acids with less catalysts or unwanted by-products, may be improved compared to the processes in the state of the art.
According to one aspect of the present invention there is provided a microbial cell for producing at least one acyl amino acid, wherein the cell is genetically modified to comprise:
The cell according to any aspect of the present invention may be capable of producing acyl amino acids and the cell may further comprise a second genetic mutation that reduces the use of amino acids and/or fatty acids as a carbon/ energy source for the cell. The second genetic mutation in the cell that reduces the use of available amino acids and/or fatty acids as a carbon source may be a genetic mutation in an enzyme that is involved in glutamate breakdown. In particular, the second genetic mutation may result in a decrease in the activity of at least one enzyme involved in glutamate breakdown relative to a wild type cell. More in particular, the second genetic mutation may result in a reduced expression relative to a wild type cell of at least one enzyme that catalyses the breakdown of glutamate via at least one compound selected from the group consisting of acetyl-CoA, acetate, pyruvate, succinate, succinyl-CoA, fumarate, malate, citrate, lactate, 2-oxoglutarate, and the like to ultimately carbon dioxide, biomass or a fermentation product.
In one example, the first genetic mutation may be in at least one amino acid-N-acyl-transferase (E1) and (ii) acyl-CoA synthetase (E2). In another example, the first genetic mutation may result in increased activity relative to the wild type cell of glycine-N-acyl transferase (E1a) that is capable of producing N-acyl glutamate. In yet another example, the cell according to any aspect of the present invention may be genetically modified to have a mutation in an enzyme that is involved in glutamate breakdown and a mutation in an enzyme that may result in increased activity relative to the wild type cell of glycine-N-acyl transferase (E1a).
According to another aspect of the present invention, there is provided a microbial cell for producing at least one acyl amino acid, wherein the cell is genetically modified to comprise:
wherein the first genetic mutation comprises at least a genetic mutation that results in increased activity of glycine-N-acyl transferase (E1a) relative to the wild type cell.
In particular, glutamate breakdown may involve the breakdown of glutamate via at least one compound selected from the group consisting of acetyl-CoA, acetate, pyruvate, succinate, succinyl-CoA, fumarate, malate, citrate, lactate, 2-oxoglutarate and the like. More in particular, the second genetic mutation may result in a reduced expression relative to a wild type cell of at least one enzyme that catalyses the breakdown of glutamate via at least one compound selected from the group consisting of acetyl-CoA, acetate, pyruvate, succinate, succinyl-CoA, fumarate, malate, citrate, lactate and 2-oxoglutarate, to ultimately carbon dioxide, biomass or a fermentation product.
As used herein the term “glutamate breakdown” may refer to the chemical processes which are part of glutamate metabolism whereby glutamate is degraded, including those steps wherein an enzyme catalyses the use of glutamate to form another material. Each step of the metabolic pathway is usually catalysed by an enzyme, whose structure is encoded by a gene. In particular, glutamate may be broken down to a variety of compounds including but not limited to acetyl-CoA, acetate, pyruvate, succinate, succinyl-CoA, fumarate, malate, citrate, lactate and 2-oxoglutarate, which are then further metabolized to carbon dioxide, a component of biomass, that is an amino acid, a protein, a phospholipid, a polysaccharide, a nucleic acid, or any other small or macromolecule being a constituent of microbial biomass, or a secreted fermentation product.
In particular, glutamate breakdown may include at least one degradation pathway selected from the following pathways: The assays that may be used to measure the activity of these enzymes are provided at least in brackets or are described in the journal articles provided in the brackets.
In one example, the cell according to any aspect of the present invention may be genetically modified to attenuate at least one degradation pathway from Degradation pathway I-VII to reduce glutamate breakdown relative to a wild type cell. In another example, the decrease in glutamate breakdown in a cell according to any aspect of the present invention may be due to attenuation in a combination of Degradation pathways I-VII. In one example, the decrease in glutamate breakdown in a cell according to any aspect of the present invention may be due to attenuation in Degradation pathway I and II, I and III; I and IV; I and V; I and VI; I and VII; II and III; II and IV; II and V; II and VI; II and VII; III and IV; III and V; III and VI; III and VII; IV and V; IV and VI; IV and VII; V and VI; V and VII; I, II and III; I, II and IV; I, II and V; I, II and VI; I, II and VII; I, III and V; I, III and VI; I, III and VII; I, IV and V; I, IV and VI; I, IV and VII; I, IV and VI; I, IV and VII; II, III and IV; II, III and V; II, III and VI; II, III and VII; III, IV and V; III, IV and VI; III, IV and VII; IV, V and VI; IV, V and VII; I, II, III and IV; I, II, III and V; I, II, III and VI; I, II, III and VII; II, III, IV and V; II, III, IV and VI; II, III, IV and VII; I, II, III, IV and V; I, II, III, IV and VI; I, II, III, IV and VII; I, II, III, IV, V and VI; I, II, III, IV, V and VII; I, II, III, IV, V and VI or I, II, III, IV, V, VI and VII. The cell according to any aspect of the present invention may comprise at least one genetic mutation in any of the enzymes mentioned above involved in any one of the Degradation pathways I-VII to result in attenuation in glutamate breakdown. In one example there may be a genetic mutation in a combination of enzymes that are part of any one of the Degradation pathways I-VII that may result in a decrease in glutamate breakdown in a cell according to any aspect of the present invention relative to a wild type cell.
In another example, a cell according to any aspect of the present invention may comprise a second genetic mutation in at least one of the enzymes E11-E28 that may result in an overall decrease in glutamate breakdown relative to the wild type cell. In particular, the second genetic mutation may comprise a genetic mutation in a combination of enzymes that may be involved in the same or different pathways in glutamate breakdown. The final result will be a cell according to any aspect of the present invention with decreased glutamate breakdown thus higher concentration of glutamate.
As used herein the term “glutamate” refers to the amino acid α-glutamate or α-glutamic acid. A genetic mutation that decreases glutamate breakdown includes the inactivation of a gene which encodes for part of the glutamate metabolic pathway as shown above. Accordingly, inactivating at least one of the enzymes E11-E28 may result in a decrease in glutamate breakdown in the cell according to any aspect of the present invention relative to a wild type cell.
As used herein the term “ tricarboxylic acid cycle (TCA cycle)” may also refer to the citric acid cycle or the Krebs cycle. In particular, the TCA cycle is a series of chemical reactions used by all aerobic organisms to generate energy through the oxidation of acetate derived from carbohydrates, fats and proteins into carbon dioxide and chemical energy in the form of adenosine triphosphate (ATP). In addition, the cycle provides precursors of certain amino acids as well as the reducing agents NADH and NADPH that is used in numerous other biochemical reactions. The name of this metabolic pathway is derived from citric acid (a type of tricarboxylic acid) that is consumed and then regenerated by this sequence of reactions to complete the cycle. In addition, the cycle consumes acetate (in the form of acetyl-CoA) and water, reduces NAD(P)+ to NAD(P)H, and produces carbon dioxide as a waste byproduct. The NADH generated by the TCA cycle is fed into the oxidative phosphorylation (electron transport) pathway. The net result of these two closely linked pathways is the oxidation of nutrients to produce usable chemical energy in the form of ATP.
The cell according to any aspect of the present invention may comprise a genetic mutation that results in decreased glutamate breakdown wherein the mutation is in at least one enzyme selected from at least one of the group of enzymes consisting of:
In one example, the mutation may be in E11. In another example, the mutation may be in E12. In yet another example, the mutation may be in E16 or in E17 or in E24 or in E28. In a further example, the mutation may be in E13 and E14. In yet a further example, the mutation may be in at least one enzyme selected from E11, and E12-E28. The mutation that may result in reduced glutamate breakdown in the cell according to any aspect of the present invention may be in a combination of enzymes that may be part of a single pathway or multiple pathways that are part of glutamate breakdown. For example, the mutation may be in E11 and at least one enzyme selected from the group consisting of E12-E28.
According to any aspect of the present invention, the first genetic mutation may result in the cell having increased expression of (i) an amino acid-N-acyl-transferase (E1) that is capable of producing N-acyl glutamate and (ii) acyl-CoA synthetase (E2). The combination of amino acid-N-acyl transferase and an acyl-CoA synthetase, expressed according to any aspect of the present invention may be used to convert a variety of fatty acids and/or amino acids including a mixture comprising unsaturated and saturated fatty acids, to acyl amino acids. In particular, the amino acid-N-acyl transferases may be used to convert short unsaturated fatty acids such as lauroleic acid to an acyl amino acid. More in particular, the amino acid-N-acyl-transferase may be capable of converting a variety of fatty acids including short unsaturated fatty acids such as lauroleic acid to an acyl amino acid with an increase in the yields of acyl amino acids produced. Further, the composition of acyl amino acids produced in a cell, more specifically the length of fatty acids incorporated into such acyl amino acids, may be controlled by introducing into the cell one or more specific acyl-CoA thioesterases or altering the expression of one or more acyl-CoA thioesterases endogenously expressed by the cell. In particular, the first genetic mutation may result in the cell having increased expression of (i) glycine-N-acyl-transferase (E1a) and (ii) acyl-CoA synthetase (E2).
The term “amino acid-N-acyl transferase”, as used herein, refers to an enzyme capable of catalysing the conversion of acyl-CoA, for example the CoA ester of an acid, and an amino acid, for example a proteinogenic amino acid, for example glutamic acid, to an acyl amino acid. More in particular, the term “glycine N-acyl transferase” refers to an amino acid-N-acyl transferase that is capable of producing N-acyl glutamate alone and/or in combination with another acyl amino acid. In one example, the glycine N-acyl transferase used according to any aspect of the present invention may produce N-acyl glutamate only. In another example, the glycine N-acyl transferase used according to any aspect of the present invention may produce N-acyl glutamate and N-acyl glycinate. More in particular, the glycine N-acyl transferase used according to any aspect of the present invention may always produce at least N-acyl glutamate. Suitable amino acid-N-acyl transferases have been described in the prior art, for example in Waluk, D. P., Schultz, N., and Hunt, M. C. (2010). In particular, the amino acid sequence of glycine N-acyl transferase (E1a) used according to any aspect of the present invention may be selected from the sequences SEQ ID NO: 4, NP_001010904.1,NP_659453.3, XP_001147054.1, AAH16789.1, AA073139.1, XP_003275392.1, XP_002755356.1, XP_003920208.1, XP_004051278.1, XP_006147456.1, XP_006214970.1, XP_003801413.1, XP_006189704.1, XP_003993512.1, XP_005862181.1, XP_007092708.1, XP_006772167.1, XP_006091892.1, XP_005660936.1, XP_005911029.1, NP_001178259.1, XP_004016547.1, XP_005954684.1, ELR45061.1, XP_005690354.1, XP_004409352.1, XP_007519553.1, XP_004777729.1, XP_005660935.1, XP_004824058.1, XP_006068141.1, XP_006900486.1, XP_007497585.1, XP_002821801.2, XP_007497583.1, XP_003774260.1, XP_001377648.2, XP_003909843.1, XP_003801448.1, XP_001091958.1, XP_002821798.1, XP_005577840.1, XP_001092197.1, NP_001207423.1, NP_001207425.1, XP_003954287.1, NP_001271595.1, XP_003909848.1, XP_004087850.1, XP_004051279.1, XP_003920209.1, XP_005577835.1, XP_003774402.1, XP_003909846.1, XP_004389401.1, XP_002821802.1, XP_003774401.1, XP_007497581.1, EHH21814.1, XP_003909845.1, XP_005577839.1, XP_003774403.1, XP_001092427.1, XP_003275395.2, NP_542392.2, XP_001147271.1, XP_005577837.1, XP_003826420.1, XP_004051281.1, XP_001147649.2, XP_003826678.1, XP_003909847.1, XP_004682812.1, XP_004682811.1, XP_003734315.1, XP_004715052.1, BAG62195.1, XP_003777804.1, XP_003909849.1, XP_001092316.2, XP_006167891.1, XP_540580.2, XP_001512426.1, EAVV73833.1, XP_003464217.1, XP_007519551.1, XP_003774037.1, XP_005954680.1, XP_003801411.1, NP_803479.1, XP_004437460.1, XP_006875830.1, XP_004328969.1, XP_004264206.1, XP_004683490.1, XP_004777683.1, XP_005954681.1, XP_003480745.1, XP_004777682.1, XP_004878093.1, XP_007519550.1, XP_003421399.1, EHH53167.1, XP_006172214.1, XP_003993453.1,AAI12537.1, XP_006189705.1, Q2KIR7.2, XP_003421465.1, NP_001009648.1, XP_003464328.1, XP_001504745.1, ELV11036.1, XP_005690351.1, XP_005216632.1, EPY77465.1, XP_005690352.1, XP_004016544.1, XP_001498276.2, XP_004264205.1, XP_005690353.1, XP_005954683.1, XP_004667759.1, XP_004479306.1, XP_004645843.1, XP_004016543.1, XP_002928268.1, XP_006091904.1, XP_005331614.1, XP_007196549.1, XP_007092705.1, XP_004620532.1, XP_004869789.1, EHA98800.1, XP_004016545.1, XP_004479307.1, XP_004093105.1, NP_001095518.1, XP_005408101.1, XP_004409350.1, XP_001498290.1, XP_006056693.1, XP_005216639.1, XP_007455745.1, XP_005352049.1, XP_004328970.1, XP_002709220.1, XP_004878092.1, XP_007196553.1, XP_006996816.1, XP_005331615.1, XP_006772157.1, XP_007196552.1, XP_004016546.1, XP_007628721.1, NP_803452.1, XP_004479304.1, DAA21601.1, XP_003920207.1, XP_006091906.1, XP_003464227.1, XP_006091903.1, XP_006189706.1, XP_007455744.1, XP_004585544.1, XP_003801410.1, XP_007124812.1, XP_006900488.1, XP_004777680.1, XP_005907436.1, XP_004389356.1, XP_007124811.1, XP_005660937.1, XP_007628724.1, XP_003513512.1, XP_004437813.1, XP_007628723.1, ERE78858.1, EPQ15380.1, XP_005862178.1, XP_005878672.1, XP_540581.1, XP_002928267.1, XP_004645845.1, EPQ05184.1, XP_003513511.1, XP_006214972.1, XP_007196545.1, XP_007196547.1, XP_006772160.1, XP_003801409.1, NP_001119750.1, XP_003801412.1, XP_006772159.1, EAVV73832.1, XP_006091897.1, XP_006772163.1, XP_006091898.1, XP_005408105.1, XP_006900487.1, XP_003993454.1, XP_003122754.3, XP_007455746.1, XP_005331618.1, XP_004585337.1, XP_005063305.1, XP_006091895.1, XP_006772156.1, XP_004051276.1, XP_004683488.1, NP_666047.1, NP_001013784.2, XP_006996815.1, XP_006996821.1, XP_006091893.1, XP_006173036.1, XP_006214971.1, EPY89845.1, XP_003826423.1, NP_964011.2, XP_007092707.1, XP_005063858.1, BAL43174.1, XP_001161154.2, XP_007124813.1, NP_083826.1XP_003464239.1, XP_003275394.1, ELK23978.1, XP_004878097.1, XP_004878098.1, XP_004437459.1, XP_004264204.1, XP_004409351.1, XP_005352047.1, Q5RFP0.1, XP_005408107.1, XP_007659164.1, XP_003909852.1, XP_002755355.1, NP_001126806.1, AAP92593.1, NP_001244199.1, BAA34427.1, XP_005063859.1, NP_599157.2, XP_004667761.1, XP_006900489.1, XP_006215013.1, XP_005408100.1, XP_007628718.1, XP_003514769.1, XP_006160935.1, XP_004683489.1, XP_003464329.1, XP_004921258.1, XP_003801447.1, XP_006167892.1, XP_004921305.1, AAH89619.1, XP_004706162.1, XP_003583243.1, EFB16804.1, XP_006728603.1, EPQ05185.1, XP_002709040.1, XP_006875861.1, XP_005408103.1, XP_004391425.1, EDL41477.1, XP_006772158.1, EGWO6527.1, AAH15294.1, XP_006772162.1, XP_005660939.1, XP_005352050.1, XP_006091901.1, XP_005878675.1, XP_004051323.1, EHA98803.1, XP_003779925.1, EDM12924.1, XP_003421400.1, XP_006160939.1, XP_006160938.1, XP_006160937.1, XP_006160936.1, XP_005702185.1, XP_005313023.1, XP_003769190.1, XP_002714424.1, XP_004715051.1, XP_007661593.1, XP_004590594.1, ELK23975.1, XP_004674085.1, XP_004780477.1, XP_006231186.1, XP_003803573.1, XP_004803176.1, EFB16803.1, XP_006056694.1, XP_005441626.1, XP_005318647.1XP_004605904.1, XP_005862182.1, XP_003430682.1, XP_004780478.1, XP_005239278.1, XP_003897760.1, XP_007484121.1, XP_004892683.1, XP_004414286.1, XP_006927013.1, XP_003923145.1, XP_852587.2, AAP97178.1, EHH53105.1, XP_005408113.1, XP_002915474.1, XP_005377590.1, XP_527404.2, XP_005552830.1, XP_004044211.1, NP_001180996.1, XP_003513513.2, XP_001498599.2, XP_002746654.1, XP_005072349.1, XP_006149181.1, EAX04334.1, XP_003833230.1, XP_005216635.1, XP_003404197.1, XP_007523363.1, XP_007433902.1, XP_003254235.1, XP_004471242.1, XP_005216634.1, XP_006860675.1, XP_004771956.1, XP_006038833.1, NP_001138534.1, XP_007068532.1, XP_003510714.1, ERE87950.1, XP_003986313.1, XP_006728644.1, XP_004878099.1, XP_003468014.1, XP_007095614.1, XP_004648849.1, XP_004869795.1, XP_004018927.1, XP_005696454.1, XP_006201985.1, XP_005960697.1, XP_004813725.1, XP_005496926.1, ELR45088.1, XP_004696625.1, XP_005860982.1, XP_005911003.1, XP_006260162.1, EPQ04414.1, XP_006099775.1, NP_001138532.1, XP_006190795.1, XP_004649775.1, XP_004424497.1, XP_004390885.1, XP_005911004.1, XP_003777803.1, XP_004312259.1, XP_005529140.1, XP_005314582.1, XP_006926523.1, XP_006926522.1, XP_004683491.1, XP_003826680.1, XP_003215018.1, XP_003215087.1, EGW12611.1, XP_006113023.1, XP_006882182.1, XP_007425200.1, XP_006041342.1, NP_001138533.1, EMP27694.1, XP_007497753.1, XP_006034252.1, or a variant thereof. Throughout this disclosure, any data base code, unless specified to the contrary, refers to a sequence available from the NCBI data bases, more specifically the version online on 5th Aug. 2013, and comprises, if such sequence is a nucleotide sequence, the polypeptide sequence obtained by translating the former.
In particular, the first genetic mutation may increase the activity of glycine N-acyl transferase (E1a) and acyl-CoA synthetase (E2) relative to the wild type cell. More in particular, the sequence of glycine N-acyl transferase (E1a) used according to any aspect of the present invention may have 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% sequence identity with SEQ ID NO:4 and/or the sequence of acyl-CoA synthetase (E2) used according to any aspect of the present invention may have 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% sequence identity with SEQ ID NO:1.
The cell according to any aspect of the present invention may comprise a genetic mutation in a second amino acid acyl transferase that may be capable of working on other amino acids or variants thereof related to the breakdown of glutamic acid and/or glutamate. In one example, the other amino acids or variants thereof may be selected from the group consisting of proline, arginine, glutathione, ornithine, spermidine, spermine, sarcosine and putrescine and sodium lauroyl sarcosinate. In another example, there may be more than 1, 2, 3, 4, 5, 6, 7 or the like mutations in the cell according to any aspect of the present invention that may result in the cell expressing more than one amino acid N-acyltransferase (E1) besides glycine-N-acyl transferase (E1a). In this example, the cell according to any aspect of the present invention may be capable of expressing an amino acid N-acyl-transferase, glycine-N-acyl transferase (E1a) and acyl-CoA synthetase (E2). This cell may then be capable of producing N-acyl glutamate and other relevant N-acyl amino acids depending on the amino acid-N-acyl-transferase expressed and the amino acid used as the substrate. For example, the cell according to any aspect of the present invention may be genetically modified in the following manner:
Glycine-N-acyl transferase (E1a) may catalyse the following reaction: glutamate+acyl-CoA→acylglutamate+CoA. More in particular, the cell according to any aspect of the present invention comprises a first genetic mutation that increases the activity of glycine-N-acyl transferase (E1a) and acyl-CoA synthetase (E2) relative to the wild type cell and a second genetic mutation that decreases the activity of at least one enzyme involved in glutamate breakdown relative to a wild type cell. Even more in particular, the enzyme involved in glutamate breakdown may be selected from the group consisting of E11-E28. The second genetic mutation in at least one of the enzymes E11-E28 may result in an overall decrease in glutamate breakdown relative to the wild type cell. In particular, the second genetic mutation may comprise a genetic mutation in a combination of enzymes that may be involved in the same or different pathways in glutamate breakdown. The final result will be a cell according to any aspect of the present invention with decreased glutamate breakdown thus higher concentration of glutamate.
The acyl-CoA substrate consumed in the cell according to any aspect of the present invention may be purified from a cell, chemically synthesised or produced using an acyl-CoA synthetase. The term “acyl-CoA synthetase”, as used herein, refers to an enzyme capable of catalysing the ATP- or GTP-dependent conversion of a fatty acid and CoA or ACP to acyl-CoA or acyl-ACP. In one example, the acyl-CoA synthetase may comprise nucleotide sequence SEQ ID NO:2 or amino acid sequence SEQ ID NO:1.
According to any aspect of the present invention, the term ‘acyl-CoA synthetase’ may refer to an acyl-CoA/ACP synthetase that may be capable of producing acyl thioester, i.e. acyl-CoA or acyl-ACP and/or catalysing the following reaction:
fatty acid+CoA/ACP+ATP/GTP→acyl-CoA/ACP+ADP/GDP+Pi
Examples of acyl-CoA synthetases may include EC 6.2.1.3, EC 6.2.1.10, EC 6.2.1.15, EC 6.2.1.20 and the like. The state of the art describes various methods to detect acyl-CoA synthetase activity. For example, the activity of an acyl-CoA synthetase may be assayed as follows: the standard reaction mixture for the spectrophotometric assay (total volume, 1 ml) is composed of 0.1 M Tris-HC1 buffer pH 8.0, 1.6 mM Triton X-100, 5 mM dithiothreitol, 0.15 M KCl, 15 mM MgCl2, 10 mM ATP, 0.1 mM potassium palmitate, 0.6 mM CoA, 0.2 mM potassium phosphoenolpyruvate, 0.15 mM NADH, 45 pg adenylate kinase per mL, 30 pg pyruvate kinase per mL and 30 pg lactate dehydrogenase per mL. The oxidation of NADH at 334 nm is followed with a recording spectrophotometer.
Alternatively, the activity of an acyl-CoA synthetase may be assayed as described in the state of the art, for example Kang, Y., et al, 2010. Briefly, the amount of free thiol in the form of unreacted CoASH may be determined by adding Ellmann's reagent and spectrophotometrically monitoring the absorbance at 410 nm, in a reaction buffer comprising 150 mM Tris-HCl (pH 7.2), 10 mM MgCl2, 2 mM EDTA, 0.1% Triton X-100, 5 mM ATP, 0.5 mM Coenzyme A (CoASH) and a fatty acid (30 to 300 mM).
Various acyl-CoA synthetases have been described in the state of the art, for example YP_001724804.1, WP_001563489.1 and NP_707317.1. In one example, the acyl-CoA synthetase comprises SEQ ID NO: 1 or YP_001724804.1 or a variant thereof.
The cell according to any aspect of the present invention may be genetically different from the wild type cell. The genetic difference between the cell according to any aspect of the present invention and the wild type cell may be in the presence of a complete gene, amino acid, nucleotide etc. in the cell according to any aspect of the present invention that may be absent in the wild type cell. In one example, the cell according to any aspect of the present invention may comprise enzymes that enable the cell to produce at least one acyl amino acid and cell may further comprise at least one mutation that results in decreased glutamate breakdown. The wild type cell relative to the cell according to any aspect of the present invention may have none or no detectable activity of the enzymes that enable the cell according to any aspect of the present invention to produce at least one acyl amino acid; and the wild type cell may be capable of metabolising glutamate as per normal.
The phrase “wild type” as used herein in conjunction with a cell may denote a cell with a genome make-up that is in a form as seen naturally in the wild. The term may be applicable for both the whole cell and for individual genes. The term “wild type” therefore does not include such cells or such genes where the gene sequences have been altered at least partially by man using recombinant methods.
A skilled person would be able to use any method known in the art to genetically modify a cell. According to any aspect of the present invention, the cell may be genetically modified so that in a defined time interval, within 2 hours, in particular within 8 hours or 24 hours, it forms at least twice, especially at least 10 times, at least 100 times, at least 1000 times or at least 10000 times more acyl amino acids than the wild-type cell. The increase in product formation can be determined for example by cultivating the cell according to any aspect of the present invention and the wild-type cell each separately under the same conditions (same cell density, same nutrient medium, same culture conditions) for a specified time interval in a suitable nutrient medium and then determining the amount of target product (acyl amino acid) in the nutrient medium.
The phrase ‘decreased activity of an enzyme’ and like may refer to a genetic modification that may be present in the cell according to any aspect of the present invention to decrease a specific enzymatic activity and this may be done by a gene disruption or a genetic modification. In particular, the decrease in activity of an enzyme relative to the wild type cell may be a 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100% less than the wild type cell.
The phrase “increased activity of an enzyme”, as used herein is to be understood as increased intracellular activity. Basically, an increase in enzymatic activity can be achieved by increasing the copy number of the gene sequence or gene sequences that code for the enzyme, using a strong promoter, increasing translation by improved ribosome binding sites or optimized codon usage or employing a gene or allele that code for a corresponding enzyme with increased activity and optionally by combining these measures. Genetically modified cells used according to any aspect of the present invention are for example produced by transformation, transduction, conjugation or a combination of these methods with a vector that contains the desired gene, an allele of this gene or parts thereof and a vector that makes expression of the gene possible. Heterologous expression is in particular achieved by integration of the gene or of the alleles in the chromosome of the cell or an extra-chromosomally replicating vector. In particular, an increase in an activity of an enzyme relative to the wild type cell may be a 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100% more than the wild type cell. More in particular, the increase in activity of at least one enzyme may be the result of overexpression of the gene.
In one example, the cell according to any aspect of the present invention may comprise a first genetic mutation that results in the overexpression of at least the enzymes amino acid-N-acyl transferase and acyl-CoA synthetase. In particular, the cell may over express enzymes glycine N-acyl transferase and acyl-CoA/ACP synthetase. The expression of glycine N-acyl transferase may be measured using the assay disclosed in Badenhorst CP, 2012. Namely, DTNB, water and cell lysate are mixed together and incubated at 37° C. for 10 min while monitoring absorbance at 412 nm. The 412 reading may be stable before adding in glutamate and C8-CoA (pre-mixed together) to initiate the reaction. The increase in absorbance at 412 nm is followed and the enzyme activity is calculated using ε412=14.15 mM−1cm−1
In particular, the amino acid-N-acyl-transferase (E1) may be a human glycine N-acyl-transferase (E1a). In one example, E1 may be SEQ ID NO:5 or a variant thereof. In another example, E1a may comprise SEQ ID NO:4 and the acyl-CoA synthetase (E2) may comprise SEQ ID NO: 1 or a variant thereof. More in particular, the cell according to any aspect of the present invention may comprise a first genetic mutation in E1a and E2, wherein E1a may comprise a nucleic acid sequence of SEQ ID NO: 5 and E2 may comprise nucleic acid sequence of SEQ ID NO: 2.
According to any aspect of the present invention, a formula referring to a chemical group that represents the dissociated or undissociated state of a compound capable of dissociating in an aqueous solution comprises both the dissociated and the undissociated state and the various salt forms of the group. For example, the residue —COOH comprises both the protonated (—COOH) as well as the unprotonated (—COO−) carboxylic acid.
The term “acyl amino acid”, as used herein, refers to the product of the reaction catalysed by an amino acid-N-acyl transferase, a compound represented by the formula acyl-CO—NH—CHR—COOH, wherein R is the side chain of a proteinogenic amino acid, and wherein the term “acyl” refers to the acyl residue of a fatty acid. In one example, the term “fatty acid”, as used herein, means a carboxylic acid, for example an alkanoic acid, with at least 6, 8, 10, or 12 carbon atoms. In one example, it is a linear fatty acid, in another example, it is branched. In one example it is a saturated fatty acid. In another example, it is unsaturated. In one example, it is a fatty acid with 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 carbon atoms. In particular, the acyl amino acid may be N-acyl glutamate. In another example, the acyl amino acid may be at least one lauroyl glutamate.
The teachings of the present invention may not only be carried out using biological macromolecules having the exact amino acid or nucleic acid sequences referred to in this application explicitly, for example by name or accession number, or implicitly, but also using variants of such sequences. The term “variant”, as used herein, comprises amino acid or nucleic acid sequences, respectively, that are at least 70, 75, 80, 85, 90, 92, 94, 95, 96, 97, 98 or 99% identical to the reference amino acid or nucleic acid sequence, wherein preferably amino acids other than those essential for the function, for example the catalytic activity of a protein, or the fold or structure of a molecule are deleted, substituted or replaced by insertions or essential amino acids are replaced in a conservative manner to the effect that the biological activity of the reference sequence or a molecule derived therefrom is preserved. The state of the art comprises algorithms that may be used to align two given nucleic acid or amino acid sequences and to calculate the degree of identity, see Arthur Lesk (2008), Thompson et al., 1994, and Katoh et al., 2005. The term “variant” is used synonymously and interchangeably with the term “homologue”. Such variants may be prepared by introducing deletions, insertions or substitutions in amino acid or nucleic acid sequences as well as fusions comprising such macromolecules or variants thereof. In one example, the term “variant”, with regard to amino acid sequence, comprises, in addition to the above sequence identity, amino acid sequences that comprise one or more conservative amino acid changes with respect to the respective reference or wild type sequence or comprises nucleic acid sequences encoding amino acid sequences that comprise one or more conservative amino acid changes. In one example, the term “variant” of an amino acid sequence or nucleic acid sequence comprises, in addition to the above degree of sequence identity, any active portion and/or fragment of the amino acid sequence or nucleic acid sequence, respectively, or any nucleic acid sequence encoding an active portion and/or fragment of an amino acid sequence. The term “active portion”, as used herein, refers to an amino acid sequence or a nucleic acid sequence, which is less than the full length amino acid sequence or codes for less than the full length amino acid sequence, respectively, wherein the amino acid sequence or the amino acid sequence encoded, respectively retains at least some of its essential biological activity. For example an active portion and/or fragment of a protease may be capable of hydrolysing peptide bonds in polypeptides. The phrase “retains at least some of its essential biological activity”, as used herein, means that the amino acid sequence in question has a biological activity exceeding and distinct from the background activity and the kinetic parameters characterising said activity, more specifically kcat and KM, are preferably within 3, 2, or 1 order of magnitude of the values displayed by the reference molecule with respect to a specific substrate. Similarly, the term “variant” of a nucleic acid comprises nucleic acids the complementary strand of which hybridises, preferably under stringent conditions, to the reference or wild type nucleic acid. A skilled person would be able to easily determine the amino acid-N-acyl-transferases that will be capable of making proteinogenic amino acids and/or fatty acids. In particular, the variants may include but are not limited to an amino acid-N-acyl-transferase selected from the group of organisms consisting of Nomascus leucogenys (NI, XP_003275392.1), Saimiri boliviensis (Sb, XP_003920208.1), Felis catus (Fc, XP_003993512.1), Bos taurus (Bt, NP_001178259.1), Mus musculus (Mm, NP_666047.1) and the like.
Stringency of hybridisation reactions is readily determinable by one ordinary skilled in the art, and generally is an empirical calculation dependent on probe length, washing temperature and salt concentration. In general, longer probes require higher temperatures for proper annealing, while shorter probes need lower temperatures. Hybridisation generally depends on the ability of denatured DNA to reanneal to complementary strands when present in an environment below their melting temperature. The higher the degree of desired homology between the probe and hybridisable sequence, the higher the relative temperature which may be used. As a result it follows that higher relative temperatures would tend to make the reaction conditions more stringent, while lower temperature less so. For additional details and explanation of stringency of hybridisation reactions, see F. M. Ausubel (1995). The person skilled in the art may follow the instructions given in the manual “The DIG System Users Guide for Filter Hybridization”, Boehringer Mannheim GmbH, Mannheim, Germany, 1993 and in Liebl et al., 1991 on how to identify DNA sequences by means of hybridisation. In one example, stringent conditions are applied for any hybridisation, i.e. hybridisation occurs only if the probe is 70% or more identical to the target sequence. Probes having a lower degree of identity with respect to the target sequence may hybridise, but such hybrids are unstable and will be removed in a washing step under stringent conditions, for example by lowering the concentration of salt to 2×SSC or, optionally and subsequently, to 0.5×SSC, while the temperature is, in order of increasing preference, approximately 50° C.-68° C., approximately 52° C.-68° C., approximately 54° C.-68° C., approximately 56° C.-68° C., approximately 58° C.-68° C., approximately 60° C.-68° C., approximately 62° C.-68° C., approximately 64° C.-68° C., approximately 66° C.-68° C. In a particularly preferred embodiment, the temperature is approximately 64° C.-68° C. or approximately 66° C.-68° C. It is possible to adjust the concentration of salt to 0.2×SSC or even 0.1×SSC. Polynucleotide fragments having a degree of identity with respect to the reference or wild type sequence of at least 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% may be isolated. The term “homologue” of a nucleic acid sequence, as used herein, refers to any nucleic acid sequence that encodes the same amino acid sequence as the reference nucleic acid sequence, in line with the degeneracy of the genetic code.
The cell according to any aspect of the present invention may comprise a further mutation in at least one enzyme selected from the group consisting of:
In particular, E3 may be a glutamate-translocating ABC transporter or permease; E4 may be an acyl-CoA:ACP transacylase; and E5 may be AlkL and/or FadL. AlkL and/or FadL may function as at least one transporter protein compared to the wild type cell. In one example, the cell may be genetically modified to overexpress both the fadL and the alkL gene. In particular, the alkL may comprise a sequence that may have 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% sequence identity with SEQ ID NO:6 or 7. In particular, the fadL may comprise a sequence that may have 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% sequence identity with SEQ ID NO:8.
The cell according to any aspect of the present invention may have reduced capacity of fatty acid degradation by beta-oxidation relative to the wild type cell. In particular, the reduced fatty acid degradation activity compared to the wildtype cell may be a result of decreased expression relative to the wild type cell of at least one enzyme selected from the group consisting of acyl-CoA dehydrogenase (FadE) (E6), enoyl-CoA hydratase (FadB) (E7), (R)-3-hydroxyacyl-CoA dehydrogenase (FadB) (E8) and 3-ketoacyl-CoA thiolase (FadA) (E9).
The term “having a reduced fatty acid degradation capacity”, as used herein, means that the respective cell degrades fatty acids, in particular those taken up from the environment, at a lower rate than a comparable cell or wild type cell having normal fatty acid degradation capacity would under identical conditions. In one example, the fatty acid degradation of such a cell is lower on account of deletion, inhibition or inactivation of at least one gene encoding an enzyme involved in the β-oxidation pathway. In one example, at least one enzyme involved in the β-oxidation pathway has lost, in order of increasing preference, 5, 10, 20, 40, 50, 75, 90 or 99% activity relative to the activity of the same enzyme under comparable conditions in the respective wild type microorganism. The person skilled in the art may be familiar with various techniques that may be used to delete a gene encoding an enzyme or reduce the activity of such an enzyme in a cell, for example by exposition of cells to radioactivity followed by accumulation or screening of the resulting mutants, site-directed introduction of point mutations or knock out of a chromosomally integrated gene encoding for an active enzyme, as described in Sambrook/Fritsch/Maniatis (1989). In addition, the transcriptional repressor FadR may be over expressed to the effect that expression of enzymes involved in the β-oxidation pathway is repressed (Fujita, Y., et al, 2007). The phrase “deletion of a gene”, as used herein, means that the nucleic acid sequence encoding said gene is modified such that the expression of active polypeptide encoded by said gene is reduced. For example, the gene may be deleted by removing in-frame a part of the sequence comprising the sequence encoding for the catalytic active centre of the polypeptide. Alternatively, the ribosome binding site may be altered such that the ribosomes no longer translate the corresponding RNA. It would be within the routine skills of the person skilled in the art to measure the activity of enzymes expressed by living cells using standard essays as described in enzymology text books, for example Cornish-Bowden, 1995.
Degradation of fatty acids is accomplished by a sequence of enzymatically catalysed reactions. First of all, fatty acids are taken up and translocated across the cell membrane via a transport/acyl-activation mechanism involving at least one outer membrane protein and one inner membrane-associated protein which has fatty acid-CoA ligase activity, referred to in the case of E. coli as FadL and FadD/FadK, respectively. Inside the cell, the fatty acid to be degraded is subjected to enzymes catalysing other reactions of the β-oxidation pathway. The first intracellular step involves the conversion of acyl-CoA to enoyl-CoA through acyl-CoA dehydrogenase, the latter referred to as FadE in the case of E. coli. The activity of an acyl-CoA dehydrogenase may be assayed as described in the state of art, for example by monitoring the concentration of NADH spectrophotometrically at 340 nm in 100 mM MOPS, pH 7.4, 0.2 mM Enoyl-CoA, 0.4 mM NADH. The resulting enoyl-CoA is converted to 3-ketoacyl-CoA via 3-hydroxyacyl-CoA through hydration and oxidation, catalysed by enoyl-CoA hydratase/(R)-3-hydroxyacyl-CoA dehydrogenase, referred to as FadB and FadJ in E. coli. Enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase activity, more specifically formation of the product NADH may be assayed spectrophotometrically as described in the state of the art, for example as outlined for FadE. Finally, 3-ketoacyl-CoA thiolase, FadA and FadI in E. coli, catalyses the cleavage of 3-ketoacyl-CoA, to give acetyl-CoA and the input acyl-CoA shortened by two carbon atoms. The activity of ketoacyl-CoA thiolase may be assayed as described in the state of the art, for example in Antonenkov, V., et al, 1997.
The phrase “a cell having a reduced fatty acid degradation capacity”, as used herein, refers to a cell having a reduced capability of taking up and/or degrading fatty acids, particularly those having at least eight carbon chains. The fatty acid degradation capacity of a cell may be reduced in various ways. In particular, the cell according to any aspect of the present invention has, compared to its wild type, a reduced activity of an enzyme involved in the β-oxidation pathway. The term “enzyme involved in the β-oxidation pathway”, as used herein, refers to an enzyme that interacts directly with a fatty acid or a derivative thereof formed as part of the degradation of the fatty acid via the β-oxidation pathway. The β-oxidation pathway comprises a sequence of reactions effecting the conversion of a fatty acid to acetyl-CoA and the CoA ester of the shortened fatty acid. The enzyme involved in the β-oxidation pathway may by recognizing the fatty acid or derivative thereof as a substrate, converts it to a metabolite formed as a part of the β-oxidation pathway. For example, the acyl-CoA dehydrogenase is an enzyme involved in the β-oxidation pathway as it interacts with fatty acid-CoA and converts fatty acid-CoA ester to enoyl-CoA, which is a metabolite formed as part of the β-oxidation. In another example, the term “enzyme involved in the β-oxidation pathway”, as used herein, comprises any polypeptide from the group comprising acyl-CoA dehydrogenase, enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase and 3-keto-acyl-CoA thiolase. The acyl-CoA synthetase may catalyse the conversion of a fatty acid to the CoA ester of a fatty acid, i.e. a molecule, wherein the functional group —OH of the carboxy group is replaced with —S—CoA and introducing the fatty acid into the β-oxidation pathway. For example, the polypeptides FadD and FadK in E. coli (accession number: BAA15609.1 and NP_416216.4, respectively) are acyl-CoA synthetases. In one example, the term “acyl-CoA dehydrogenase”, as used herein, may be a polypeptide capable of catalysing the conversion of an acyl-CoA to enoyl-CoA, as part of the β-oxidation pathway. For example, the polypeptide FadE in E. coli (accession number: BAA77891.2) may be an acyl-CoA dehydrogenase. The term “enoyl-CoA hydratase”, as used herein, also referred to as 3-hydroxyacyl-CoA dehydrogenase, refers to a polypeptide capable of catalysing the conversion of enoyl-CoA to 3-ketoacyl-CoA through hydration and oxidation, as part of the β-oxidation pathway. For example, the polypeptides FadB and FadJ in E. coli (accession numbers: BAE77457.1 and P77399.1, respectively) are enoyl-CoA hydratases. The term “ketoacyl-CoA thiolase”, as used herein, may refer to a polypeptide capable of catalysing the cleaving of 3-ketoacyl-CoA, resulting in an acyl-CoA shortened by two carbon atoms and acetyl-CoA, as the final step of the β-oxidation pathway. For example, the polypeptides FadA and FadI in E. coli (accession number: YP_491599.1 and P76503.1, respectively) are ketoacyl-CoA thiolases.
Any of the enzymes used according to any aspect of the present invention, may be an isolated enzyme. In particular, the enzymes used according to any aspect of the present invention may be used in an active state and in the presence of all cofactors, substrates, auxiliary and/or activating polypeptides or factors essential for its activity. The term “isolated”, as used herein, means that the enzyme of interest is enriched compared to the cell in which it occurs naturally. The enzyme may be enriched by SDS polyacrylamide electrophoresis and/or activity assays. For example, the enzyme of interest may constitute more than 5, 10, 20, 50, 75, 80, 85, 90, 95 or 99 percent of all the polypeptides present in the preparation as judged by visual inspection of a polyacrylamide gel following staining with Coomassie blue dye.
The enzyme used according to any aspect of the present invention may be recombinant. The term “recombinant” as used herein, refers to a molecule or is encoded by such a molecule, particularly a polypeptide or nucleic acid that, as such, does not occur naturally but is the result of genetic engineering or refers to a cell that comprises a recombinant molecule. For example, a nucleic acid molecule is recombinant if it comprises a promoter functionally linked to a sequence encoding a catalytically active polypeptide and the promoter has been engineered such that the catalytically active polypeptide is overexpressed relative to the level of the polypeptide in the corresponding wild type cell that comprises the original unaltered nucleic acid molecule.
Whether or not a nucleic acid molecule, polypeptide, more specifically an enzyme used according to any aspect of the present invention, is recombinant or not has not necessarily implications for the level of its expression. However, in one example one or more recombinant nucleic acid molecules, polypeptides or enzymes used according to any aspect of the present invention may be overexpressed. The term “overexpressed”, as used herein, means that the respective polypeptide encoded or expressed is expressed at a level higher or at higher activity than would normally be found in the cell under identical conditions in the absence of genetic modifications carried out to increase the expression, for example in the respective wild type cell. The person skilled in the art is familiar with numerous ways to bring about overexpression. For example, the nucleic acid molecule to be overexpressed or encoding the polypeptide or enzyme to be overexpressed may be placed under the control of a strong inducible promoter such as the lac promoter. The state of the art describes standard plasmids that may be used for this purpose, for example the pET system of vectors exemplified by pET-3a (commercially available from Novagen). Whether or not a nucleic acid or polypeptide is overexpressed may be determined by way of quantitative PCR reaction in the case of a nucleic acid molecule, SDS polyacrylamide electrophoreses, Western blotting or comparative activity assays in the case of a polypeptide. Genetic modifications may be directed to transcriptional, translational, and/or post-translational modifications that result in a change of enzyme activity and/or selectivity under selected and/or identified culture conditions. Thus, in various examples of the present invention, to function more efficiently, a microorganism may comprise one or more gene deletions. Gene deletions may be accomplished by mutational gene deletion approaches, and/or starting with a mutant strain having reduced or no expression of one or more of these enzymes, and/or other methods known to those skilled in the art.
The cell according to any aspect of the present invention may refer to a wide range of microbial cells. In particular, the cell may be selected from the group consisting of Pseudomonas, Corynebacterium, Bacillus and Escherichia. In one example, the cell may be Escherichia coli. In another example, the cell may be a lower eukaryote, such as a fungus from the group comprising Saccharomyces, Candida, Pichia, Schizosaccharomyces and Yarrowia, particularly, Saccharomyces cerevisiae. The microorganism may be an isolated cell, in other words a pure culture of a single strain, or may comprise a mixture of at least two strains. Biotechnologically relevant cells are commercially available, for example from the American Type Culture Collection (ATCC) or the German Collection of Microorganisms and Cell Cultures (DSMZ). Particles for keeping and modifying cells are available from the prior art, for example Sambrook/Fritsch/Maniatis (1989).
The cell according to any aspect of the present invention may be capable of producing acyl amino acids and may have reduced glutamate breakdown. In one example, the cell may be capable of producing acyl amino acids due to an increased expression of glycine-N-acyl transferase. In particular, the cell may further be capable of making N-acyl glutamate or lauroyl glutamate. In one example, the cell may also be capable of reduced fatty acid degradation.
The cell according to any aspect of the present invention may thus be genetically modified to:
The term “proteinogenic amino acid”, as used herein, refers to an amino acid selected from the group comprising alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine. Proteinogenic amino acids and fatty acids are synthesized as part of the primary metabolism of many wild type cells in reactions and using enzymes that have been described in detail in biochemistry textbooks, for example Jeremy M Berg, et al., 2002. In particular, the proteinogenic amino acid may be selected from the group consisting of glycine, glutamine, glutamate, asparagine and alanine. More in particular, the proteinogenic amino acid may be glutamate.
The cell according to any aspect of the present invention may comprise at least one genetic mutation that enables the cell to produce at least one fatty acid according to any method known in the art. In one example, the genetic mutation may enable the cell to produce at least one fatty acid by means of a malonyl-CoA dependent and malonyl-ACP independent fatty acyl-CoA metabolic pathway. This is further described in US20140051136.
The cell according to any aspect of the present invention may comprise a further genetic mutation that enables the cell to have increased expression of acyl-CoA thioesterase (E10). The term “acyl-CoA thioesterase”, as used herein, refers to an enzyme capable of hydrolysing acyl-CoA. In particular, the acyl-CoA thioesterase comprises a sequence selected from the group consisting of AEM72521.1 and AAC49180.1 or a variant thereof. In one example, the amino acid sequence of E10 may comprise SEQ ID NO:3. The activity of acyl-CoA thioesterase may be assayed using various assays described in the state of the art. Briefly, the reaction of Ellman's reagent, which reacts with free thiol groups associated with CoASH formed upon hydrolysis of acyl-CoA may be detected by spectophotometrically monitoring absorbance at 412 nm.
According to another aspect of the present invention, there is provided a method of producing at least one acyl amino acid, comprising contacting the cell according to any aspect of the present invention with at least one fatty acid and/or amino acid.
The term “contacting”, as used herein, means bringing about direct contact between the amino acid, the fatty acid and/or the cell according to any aspect of the present invention in an aqueous solution. For example, the cell, the amino acid and the fatty acid may not be in different compartments separated by a barrier such as an inorganic membrane. If the amino acid or fatty acid is soluble and may be taken up by the cell or can diffuse across biological membranes, it may simply be added to the cell according to any aspect of the present invention in an aqueous solution. In case it is insufficiently soluble, it may be solved in a suitable organic solvent prior to addition to the aqueous solution. The person skilled in the art is able to prepare aqueous solutions of amino acids or fatty acids having insufficient solubility by adding suitable organic and/or polar solvents. Such solvents may be provided in the form of an organic phase comprising liquid organic solvent. In one example, the organic solvent or phase may be considered liquid when liquid at 25° C. and standard atmospheric pressure. In another example, a fatty acid may be provided in the form of a fatty acid ester such as the respective methyl or ethyl ester. In another example, the compounds and catalysts may be contacted in vitro, i.e. in a more or less enriched or even purified state, or may be contacted in situ, i.e. they are made as part of the metabolism of the cell and subsequently react inside the cell.
The term “an aqueous solution” comprises any solution comprising water, mainly water as solvent that may be used to keep the cell according to any aspect of the present invention, at least temporarily, in a metabolically active and/or viable state and comprises, if such is necessary, any additional substrates. The person skilled in the art is familiar with the preparation of numerous aqueous solutions, usually referred to as media that may be used to keep inventive cells, for example LB medium in the case of E. coli. It is advantageous to use as an aqueous solution a minimal medium, i.e. a medium of reasonably simple composition that comprises only the minimal set of salts and nutrients indispensable for keeping the cell in a metabolically active and/or viable state, by contrast to complex mediums, to avoid dispensable contamination of the products with unwanted side products. For example, M9 medium may be used as a minimal medium.
The method according to any aspect of the present invention may be used to convert both saturated and unsaturated fatty acids, to acyl amino acids. In case the end product sought-after is to comprise a higher yield of saturated acyl residues than is present, it may be possible to complement the method according to any aspect of the present invention by hydrogenating the acyl residues of the acyl amino acids. The resultant composition would thus comprise a mixture of saturated acyl residues. The hydrogenation may be carried out according to various state of the art processes, for example those described in U.S. Pat. No. 5,734,070. Briefly, the compound to be hydrogenated may be incubated at 100° C. in the presence of hydrogen and a suitable catalyst, for example a nickel catalyst on silicon oxide as a support.
The fatty acids that are to be converted to acyl amino acids may be produced by the cell according to the present invention. In one example, the cell that produces the acyl amino acids is capable of producing the fatty acids from which the acyl amino acids are produced. In particular, the cells may be genetically modified to be able to produce fatty acids. In one example, the genetic modification may be to decrease a specific enzymatic activity and this may be done by a gene disruption or a genetic modification. The genetic modification may also increase a specific enzymatic activity. In particular, the genetic modification may increase microbial synthesis of a selected fatty acid or fatty acid derived chemical product above a rate of a control or wild type cell. This control or wild type cell may lack this genetic modification to produce a selected chemical product.
The method according to any aspect of the present invention may result in the production of fatty acyl glutamate with the amino acid being glutamic acid.
According to a further aspect of the present invention, there is provided a use of at least one cell according to any aspect of the present invention for the production of at least one acyl amino acid. In particular, the acyl amino acid may be N-acyl glutamate and/or lauroyl glutamate.
The foregoing describes preferred embodiments, which, as will be understood by those skilled in the art, may be subject to variations or modifications in design, construction or operation without departing from the scope of the claims. These variations, for instance, are intended to be covered by the scope of the claims.
Generation of Vectors for Deletion of Glutamate Degrading Pathway-Enzymes in Escherichia Coli W3110 ΔfadE
To reduce glutamate degradation, enzymes of different degradation pathways (I, II, III and IV as provided above) is deleted (Table 1).
Generation of a Vector for Deletion of the gdhA in Escherichia Coli W3110 ΔfadE
To generate a vector for the deletion of gdhA of E. coli W3110, approximately 500 bp upstream and downstream of gdhA is amplified via PCR. The upstream region of gdhA is amplified using the oligonucleotides gdhA_Up_fw (SEQ ID NO:9) and gdhA_Up_rev (SEQ ID NO: 10). The downstream region of gdhA is amplified using the oligonucleotides gdhA-DOWN_fw (SEQ ID NO: 11) and gdhA-DOWN_rev (SEQ ID NO: 12).
The following parameters are used for PCR: 1×: initial denaturation, 98° C., 3:00 min; 35×denaturation, 98° C., 0:10 min; annealing, 65° C., 0:20 min; elongation, 72° C., 0:17 min; 1×: final elongation, 72° C., 10 min. For amplification the Phusion™ High-Fidelity Master Mix from New England Biolabs (Frankfurt) is used according to manufacturer's manual. 50 μL of the PCR reaction is analyzed on a 1% TAE agarose gel. Procedure of PCR, agarose gel electrophoresis, ethidium bromide staining of DNA and determination of PCR fragment size is carried out as known to those skilled in the art.
In each case PCR fragments of the expected size is amplified (PCR 1, 523 bp, (SEQ ID NO: 13); PCR 2, 544 bp, SEQ ID NO: 14). The PCR samples are separated via agarose gel electrophoresis and DNA fragments are isolated with QiaQuick Gel extraction Kit (Qiagen, Hilden). The purified PCR fragments are cloned into the Notl cut vector pKO3 (SEQ ID NO:15), using the NEBuilder® HiFi DNA Assembly Cloning Kit (New England Biolabs, Frankfurt). The assembled product is transformed into NEB® 10-beta electrocompetent E. coli cells (New England Biolabs, Frankfurt). Procedure of PCR purification, in-vitro cloning and transformation are carried out according to manufacturer's manual. The correct insertion of the target genes is checked by restriction analysis and the authenticity of the introduced DNA fragments is verified by DNA sequencing. The resulting knock-out vector is named pKO3_KO-gdhA (SEQ ID NO:16).
The construction of strain E. coli W3110 ΔfadEAgdhA is carried out with the help of pKO3_KO-gdhA using the method described in Link et al., 1997. The DNA sequence after the deletion step of gdhA is SEQ ID NO: 17.
Generation of a vector for deletion of the gadA/gadB and aspC in Escherichia coli W3110 ΔfadE To generate vectors for the deletion of enzymes of the degradation pathways II to IV approximately 500 bp upstream and downstream of the corresponding genes listed in Table 1 (gadA/gadB and aspC) are amplified via PCR and the pKO3-vectors are constructed analogous as described for pKO3_KO-gdhA and named pKO3_KO-gadA/gadB and pKO3_KO-aspC respectively.
Transformation of Vectors into E. Coli Cells
The E. coli strains W3110 ΔfadEAgdhA, ΔfadEΔgadA/gadB and ΔfadEΔgdhA are each transformed with the plasmid of Example 2 of WO2015/028423 pJ294{Ptac}[synUcTE]/pCDF{Ptac}[hGLYAT3(co_Ec)— fadD_Ec] by means of electroporation and plated onto LB-agar plates and is supplemented with spectinomycin and ampicillin (both 100 μg/mL). Transformants are checked for the presence of the correct plasmids by plasmid preparation and analytic restriction analysis as known in the art. The resulting strains were named:
Production of Lauroyl Glutamate by E. Coli Strains Expressing hGLYAT3
The strains generated in Example 1 may be used to study their ability to produce fatty acid/amino acid adducts, as described in Example 9 of WO2015/028423. The results may be compared to the results of culturing the strains of Example 4 of WO2015/028423. A brief description of determining the results (i.e. measuring fatty acid/amino acid adduct production) is provided below. Starting from a −80° C. glycerol culture, the strains to be studied are first plated on an LB-agar plate supplemented with 100 μg/mL ampicillin and 100 μg/mL spectinomycin and incubated overnight at 37° C. Starting from a single colony in each case, the strains are then grown as a 5 mL preculture in Luria-Bertani broth, Miller (Merck, Darmstadt) supplemented with 100 μg/mL ampicillin and 100 μg/mL spectinomycin. The further culture steps are performed in M9 medium (38 mM disodium hydrogenphosphate dihydrate, 22 mM potassium dihydrogenphosphate, 8.6 mM sodium chloride, 37 mM ammonium chloride, 2% (w/v) glucose, 2 mM magnesium sulphate heptahydrate (all chemicals from Merck, Darmstadt) and 0.1% (v/v) trace element solution, is brought to pH 7.4 with 25% strength ammonium hydroxide solution). The trace element solution to be added, composed of 9.7 mM manganese(II) chloride tetrahydrate, 6.5 mM zinc sulphate heptahydrate, 2.5 mM sodium-EDTA (Titriplex III), 4.9 mM boric acid, 1 mM sodium molybdate dihydrate, 32 mM calcium chloride dihydrate, 64 mM iron(II) sulphate heptahydrate and 0.9 mM copper(II) chloride dihydrate, to be dissolved in 1 M hydrochloric acid (all chemicals from Merck, Darmstadt), is filter-sterilized before being added to the M9 medium. 20 mL of M9 medium is supplemented with 100 μg/mL spectinomycin and 100 μg/mL ampicillin is introduced into baffled 100-mL Erlenmeyer flasks and is inoculated with 0.5 mL preculture. The flasks are cultured at 37° C. and 200 rpm in a shaker-incubator. After a culture time of 8 hours, 50 mL of M9 medium is supplemented with 100 μg/mL spectinomycin and 100 μg/mL ampicillin is introduced into a baffled 250-mL Erlenmeyer flask and is inoculated with the 10 mL culture to achieve an optical density (00600) of 0.2. The flasks are incubated at 37° C. and 200 rpm in a shaker-incubator. When an OD600 of 0.7 to 0.8 is reached, gene expression is induced by addition of 1 mM IPTG. The induced strains are cultured for a further 48 hours at 30° C. at 200 rpm. During culturing, samples are taken, and the fatty acid/amino acid adducts present are analysed. It was demonstrated that E. coli strains W3110 ΔfadE pJ294{Ptac}[synUcTE]/pCDF{Ptac}[hGLYAT3(co_Ec)_fadD_Ec] was capable of forming various fatty acid/amino acid adducts, for example lauroyl glutamic acid, from glucose. By contrast, no such adducts can be found in a cell that lacks the plasmids (negative control). All strains of Example 1 produce more lauroyl glutamic acid compared to strain W3110 ΔfadE pJ294{Ptac}[synUcTE]/pCDF{Ptac}[hGLYAT3(co_Ec)_fadD_Ec], due to the reduced capability of glutamate degradation.
Production of Lauroyl Glutamate by E. Coli Strains Expressing hGLYAT3 in a Parallel Fermentation System
The strains generated in Example 1, E. coli W3110 ΔfadEΔgdhA pJ294{Ptac}[synUcTE]/pCDF{Ptac}[hGLYAT3(co_Ec)_fadD_Ec], E. coli W3110 ΔfadEΔgadA/gadB pJ294{Ptac}[synUcTE]/pCDF{Ptac}[hGLYAT3(co_Ec)_fadD_Ec] and E. coli W3110 ΔfadEΔaspC pJ294{Ptac}[synUcTE]/pCDF{Ptac}[hGLYAT3(co_Ec)_fadD_Ec] are fermented in a fed-batch fermentation to study the ability of linking lauric acid and glutamic acid to produce more lauroyl glutamate compared to the strain of Example 4 of WO2015/028423 (where no second genetic mutation that results in a decrease in activity relative to a wild type cell of at least one enzyme involved in glutamate breakdown is present). This fermentation is carried out in a parallel fermentation system from DASGIP (https://online-shop.eppendorf.de/DE-de/Bioprozesstechnik-44559/Bioprozess-Systeme-60767/DASGIP-Parallele-Bioreaktorsysteme-PF-133597.html) with 8 bioreactors as described in Example 9 of WO2015/028423 except that 100 g/L glutamic acid instead of glycine is used. To quantify lauroyl, myristoyl and palmitoyl glutamate in the fermentation, samples are taken 23 h and 42 h after the start of the fermentation. These samples are prepared for analysis, and analysed using chromatography as described in Example 7 of WO2015/028423.
During the fermentation the strains of Example 1, produce more lauroyl-glutamic acid compared to the strain W3110 ΔfadE pJ294{Ptac}[synUcTE]/pCDF{Ptac}[hGLYAT3(co_Ec)_fadD_Ec.
| Number | Date | Country | Kind |
|---|---|---|---|
| 15163499.5 | Apr 2015 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2016/057333 | 4/4/2016 | WO | 00 |
