Method for producing cis / trans fatty acid isomerase using escherichia coli

Abstract

The present invention relates to a method for mass-producing cis / trans fatty acid isomerase in an active form using Escherichia coli. In the present invention, it was confirmed that cis / trans fatty acid isomerase in an active form can be mass-produced by double-transforming Escherichia coli to express the cis / trans fatty acid isomerase and cytochrome C maturation system therein.

Description

Method for producing cis / trans fatty acid isomerase using Escherichia coli

[0001] The present invention relates to a method for mass-producing an active form of cis / trans fatty acid isomerase using E. coli.

[0002] The global industrial enzyme market, which includes microorganisms, fungi, and plants, is estimated to be worth approximately US$6.9 billion in 2022, a significant amount. This is projected to reach US$7.4 billion in 2023 and US$1.02 billion in 2028, representing a compound annual growth rate (CAGR) of 6.6%. However, with specific global companies accounting for over 70% of the existing industrial enzyme market, the discovery of innovative new enzyme materials that can pioneer new industrial enzyme markets is crucial.

[0003] As interest in lipid-related enzymes continues to grow globally, it is predicted that lipid-related enzymes will show high growth among various industrial enzyme materials (Markets and Markets, 2023). However, to date, no industrialization of enzymes that directly control cis / trans fatty acids in lipids has been attempted.

[0004] Meanwhile, some Gram-negative bacteria, including strains of the genus Pseudomonas, express cis / trans fatty acid isomerase as a short-term defense mechanism against changes in the external environment. When the cell membrane is affected by the external environment, the cis / trans fatty acid isomerase present in the periplasm penetrates into the phospholipid bilayer of the cell membrane and acts on the unsaturated fatty acid region in the phospholipid to change cis bonds to trans bonds, thereby forming a rigid cell membrane with less fluidity and making it resistant to changes in the external environment.

[0005] Cis / trans fatty acid isomerase is a relatively large protein of approximately 80 kDa that has a heme binding site (CXXCH). In order to be expressed as an active, complete enzyme protein (holoenzyme), the synthesis of heme molecules and cytochrome c maturation processes must occur during protein expression. In other words, cis / trans fatty acid isomerase is a heme protein in the form of cytochrome c to which one heme molecule is covalently bound.

[0006] However, when attempting to mass-produce cis / trans fatty acid isomerase using an E. coli strain, there is a problem in that the cytochrome c maturation process does not occur under aerobic culture conditions, making expression and mass production in an active form impossible.

[0007] The present invention aims to provide a method capable of producing an active form of cis / trans fatty acid isomerase with high productivity.

[0008] The present invention provides a method for producing cis / trans fatty acid isomerase, characterized by culturing Escherichia coli double-transformed to express cis / trans fatty acid isomerase and a cytochrome c maturation system.

[0009] In the method for producing a cis / trans fatty acid isomerase of the present invention, the cis / trans fatty acid isomerase may be expressed with a pelB signal sequence consisting of an amino acid sequence of sequence number 4 linked to the N-terminus.

[0010] In the method for producing a cis / trans fatty acid isomerase of the present invention, the cis / trans fatty acid isomerase may be expressed by replacing the signal sequence at the N-terminus with a pelB signal sequence consisting of the amino acid sequence of sequence number 4.

[0011] The method for producing a cis / trans fatty acid isomerase of the present invention may preferably be a method of transforming E. coli using a pEC86 plasmid (CCOS 891) into which a cytochrome c maturation system is introduced.

[0012] In the present invention, it was confirmed that cis / trans fatty acid isomerase in an active form can be mass-produced by double-transforming E. coli to express cis / trans fatty acid isomerase and a cytochrome c maturation system.

[0013] Figure 1 is a schematic diagram illustrating the process of inserting a cis / trans fatty acid isomerase gene into the Nco1 and Xho1 sites of the pET26b(+) plasmid using the in-fusion cloning technique.

[0014] Figure 2 is a schematic diagram of plasmids that are double-transformed into E. coli to produce an active recombinant cis / trans fatty acid isomerase. (a) is a schematic diagram of the pET26b(+) plasmid into which the cis / trans fatty acid isomerase gene is inserted, and (b) is a schematic diagram of the pEC86 plasmid for cytochrome c maturation.

[0015] Figure 3 shows the results of treating a plasmid extract extracted from a double-transformed E. coli with a restriction enzyme and checking the gene bands shown to confirm whether a double-transformed E. coli for producing an active recombinant cis / trans fatty acid isomerase was completely constructed, and it can be confirmed that bands (11.7 kb, 7.5 kb) matching the size of the introduced plasmid appear.

[0016] Figure 4 is a drawing showing the process of culturing the double-transformed E. coli and isolating and purifying the active recombinant cis / trans fatty acid isomerase. (a) shows the process of obtaining the eluted fraction using high-speed protein liquid chromatography and HisTrap HP column, (b) shows the result of SDS-PAGE analysis of the protein in each eluted fraction, and (c) shows the result of SDS-PAGE analysis after filtering and concentrating the final fraction protein containing the cis / trans fatty acid isomerase.

[0017] Figure 5 shows the results of processing cis-unsaturated fatty acids (oleic acid, palmitoleic acid, cis-vaccenic acid) as a substrate to confirm the activity of a recombinant cis / trans fatty acid isomerase produced by the method of the present invention, and separating the cis-form unsaturated fatty acids and the produced trans-form unsaturated fatty acids (elaidic acid, palmitoleic acid, vaccenic acid).

[0018] Figure 6 shows the results of predicting protein domains using SignalP to confirm the structure of a recombinant cis / trans fatty acid isomerase produced by the method of the present invention.

[0019] Figure 7 shows the results of predicting the protein tertiary structure using deep learning-based RoseTTAFold to confirm the structure of a recombinant cis / trans fatty acid isomerase produced by the method of the present invention.

[0020] The present invention provides a method for producing cis / trans fatty acid isomerase, characterized by culturing Escherichia coli double-transformed to express cis / trans fatty acid isomerase and a cytochrome c maturation system.

[0021] When mass producing foreign proteins using E. coli, a method is used to produce them by culturing recombinant E. coli into which a gene expressing the foreign protein has been introduced.

[0022] However, when the inventors of the present invention recombined E. coli to express cis / trans fatty acid isomerase and cultured it, they confirmed that the cytochrome c maturation process did not occur under aerobic conditions, resulting in the production of cis / trans fatty acid isomerase in an inactive form.

[0023] Accordingly, the present inventors have confirmed that when double-transformed E. coli to express cis / trans fatty acid isomerase and cytochrome c maturation protein, the recombinant E. coli can mass-produce an active form of cis / trans fatty acid isomerase, and have completed the present invention.

[0024] Meanwhile, in the present invention, cis / trans fatty acid isomerase refers to an enzyme that regulates the conversion of cis / trans forms resulting from carbon double bonds in unsaturated fatty acids. In this case, the unsaturated fatty acid may be an unsaturated fatty acid in the form of a free fatty acid, a neutral fat, a fatty acid ester, or a phospholipid.

[0025] In the following examples, experiments were conducted to convert cis-form free unsaturated fatty acids such as oleic acid, palmitoleic acid, and cis-vaccenic acid into trans-form using cis / trans fatty acid isomerase derived from Pseudomonas putida KT2440 as substrates. Depending on the characteristics of the transformed cis / trans fatty acid isomerase, various forms of unsaturated fatty acids can serve as substrates.

[0026] Meanwhile, in the present invention, the cis / trans fatty acid isomerase may be expressed by replacing the N-terminal signal sequence with a pelB signal sequence consisting of the amino acid sequence of sequence number 4.

[0027] In order for cis / trans fatty acid isomerase to be active, it must be expressed in the periplasm, where it must synthesize heme molecules and undergo cytochrome c maturation. Cis / trans fatty acid isomerase can be divided into a signal sequence region and an enzymatic activity region. Here, the signal sequence region is a sequence located at the N-terminus of cis / trans fatty acid isomerase and plays a role in expressing the protein in the periplasm.

[0028] Cis / trans fatty acid isomerases derived from various strains may contain various signal sequence regions. When directly introduced into E. coli, the efficiency of cis / trans fatty acid isomerase expression into the periplasm may be reduced, resulting in expression in an inactive form. In cases where this problem occurs, the N-terminal signal sequence region can be replaced with the pelB signal sequence to induce expression into the periplasm, thereby producing an active form of cis / trans fatty acid isomerase with high efficiency.

[0029] Meanwhile, in the present invention, the cytochrome c maturation system refers to a system that enables the cis / trans fatty acid isomerase, which is a cytochrome c type heme protein (a protein having a CXXCH type motif to which heme C is bound) to mature and exhibit activity under aerobic conditions. The cytochrome c maturation system is known to bind and associate heme during the expression process of a cytochrome c type protein, and to exhibit activity by cleaving and folding the protein. In the examples below, the pEC86 plasmid (CCOS 891) into which the cytochrome c maturation system is introduced so as to enable the maturation of the cis / trans fatty acid isomerase under aerobic conditions was used.

[0030] Meanwhile, in the production method of the present invention, when culturing double-transformed Escherichia coli, it is preferable to further include IPTG (Isopropyl β-d-1-thiogalactopyranoside) and 5-aminolevulinic acid in the medium, and more preferably, it is more preferable to further include 0.001 to 0.5 mM IPTG and 0.1 to 1.0 mM 5-aminolevulinic acid in the medium, and the culture is performed. According to the following example, when IPTG and 5-aminolevulinic acid are added to the medium as described above and the culture is performed, cis / trans fatty acid isomerase can be produced with high productivity.

[0031]

[0032] Hereinafter, the present invention will be described in more detail through the following examples. However, the present invention is not limited to the following examples, and includes variations of equivalent concepts.

[0033]

[0034] [Example 1: Preparation of a plasmid for expression of cis / trans fatty acid isomerase]

[0035] 1-1. Preparation of genes for expression of cis / trans fatty acid isomerase

[0036] Total genomic DNA was isolated from Pseudomonas putida KT2440 (ATCC 47054, obtained from https: / / www.atcc.org / ). A gene presumed to encode cis / trans fatty acid isomerase (SEQ ID NO: 1, Uniprot ID: Q88KB4) was amplified using overhang PCR. The forward and reverse primer sequences used are shown in Table 1, and the amplified gene product was 2,274 bp in size.

[0037] Sequence number: Forward primer: CAGCCGGGCGATGGCCATGCAGGCCCCCCAGTCGAGSeq number 7 Reverse primer: GTGGTGGTGGTGGTGGTGCTCGAGGTTCTCGTAGCGGTTCGSeq number 8

[0038]

[0039] 1-2. Preparation of plasmids for expression of recombinant cis / trans fatty acid isomerase

[0040] A plasmid for expressing a recombinant cis / trans fatty acid isomerase was constructed using the pET26b(+) plasmid. At this time, the pelB signal sequence and histidine fusion protein sequence (HHHHHH) inherent in the pET26b(+) plasmid were constructed so that the cis / trans fatty acid isomerase could be smoothly expressed in the intermembrane space in an E. coli strain and also be smoothly separated and purified. That is, the cis / trans fatty acid isomerase prepared in Example 1-1 was inserted using the Nco1 (pelB site) and Xho1 (histidine fusion protein site) restriction enzyme sites within the pET26b(+) plasmid.

[0041] Specifically, after preparing the pET26b(+) plasmid, the plasmid was cleaved through Nco1 and Xho1 restriction enzyme treatment, and the prepared recombinant cis / trans fatty acid isomerase expression gene was in-fusion cloned (Fig. 1). The size of the recombinant pET26b(+) plasmid into which the recombinant cis / trans fatty acid isomerase gene was successfully inserted was 7,538 bp (Fig. 2a).

[0042] Thereafter, in order to mass-produce the recombinant pET26b(+) plasmid, Escherichia coliDH5α strain was transformed by heat treatment at 42°C for 30 seconds, and a transformant was obtained by utilizing kanamycin antibiotic resistance. The recombinant plasmid extracted from the resulting transformant was named pET26b(+)-CTI.

[0043]

[0044] [Example 2: Production of E. coli expressing recombinant cis / trans fatty acid isomerase]

[0045] 2-1. Production of E. coli expressing recombinant cis / trans fatty acid isomerase

[0046] The pET26b(+)-CTI plasmid prepared in Example 1 was transformed into Escherichia coliBL21(DE3) strain by heat treatment at 42°C for 30 seconds, and a transformant was obtained by utilizing kanamycin antibiotic resistance.

[0047] Meanwhile, when the transformant was cultured to express cis / trans fatty acid isomerase, it was confirmed that the cis / trans fatty acid isomerase did not achieve cytochrome c maturation under aerobic culture conditions and was not expressed in an active form.

[0048]

[0049] 2-2. Production of E. coli expressing active recombinant cis / trans fatty acid isomerase using double transformation

[0050] The above transformant was double-transformed with the pEC86 plasmid (The National Culture Collection of Switzerland, CCOS 891, b in Fig. 2) containing the cytochrome c maturation protein complex gene, and transformed again so that cis / trans fatty acid isomerase could be expressed in an active form.

[0051] Specifically, to further introduce the pEC86 plasmid and achieve double transformation, the transformant of Example 2-1 was treated with 0.1 M calcium chloride (CaCl2) to transform into a soluble cell, and then the pEC86 plasmid was transformed into the soluble cell by heat treatment at 42°C for 30 seconds, and a double transformant was obtained using kanamycin and chloramphenicol antibiotic resistance. Thereafter, the plasmid extract extracted from the double transformant was treated with Nco1 and Xho1 restriction enzymes, and as a result, bands appeared at 7.5 kb (confirming the pET26b(+)-CTI plasmid) and 11.7 kb (confirming the pEC86 plasmid), confirming that the two plasmids were introduced and that E. coli was completely double-transformed (Fig. 3).

[0052]

[0053] [Example 3: Production of recombinant cis / trans fatty acid isomerase]

[0054] First, the double-transformant E. coli clone prepared through the above Example 2-2 was taken, and 7.5 mL of the culture solution grown in Luria-Bertani liquid medium containing 20 μg / mL kanamycin and 50 μg / mL chloramphenicol antibiotics was inoculated into 750 mL of the medium, and cultured at 37°C and 200 rpm until the OD600 value was between 0.6 and 0.7. After storing the culture solution on ice for 30 minutes, 0.005 mM IPTG and 0.5 mM 5-aminolevulinic acid were added to the stored culture solution, and cultured at 20°C and 200 rpm for 20 hours. Thereafter, the cells were harvested using a centrifuge (4000 g, 20 minutes, 4°C).

[0055] The obtained cells were dispersed in 15 mL of 20 mM Tris-HCl (pH 7.5) buffer containing 10 mM imidazole and 200 mM sodium chloride, and then the cells were completely disrupted using an ultrasonicator and the solubilized protein fraction was recovered using a centrifuge (13000 g, 20 min, 4°C). The solubilized protein fraction was filtered and concentrated, and then separated using a high-speed protein liquid chromatography equipped with a HisTrap HP Ni-NTA affinity chromatography column along an imidazole concentration gradient (Fig. 4a). Subsequently, each separated fraction was confirmed through SDS-PAGE analysis, and it was confirmed that the fractions (5-9) eluted at 227.14-277.80 mM imidazole concentration were fractions containing the recombinant cis / trans fatty acid isomerase (Fig. 4b). Afterwards, the fraction eluted at the above 227.14 to 277.80 mM imidazole concentration was filtered using 50 mM potassium phosphate buffer (pH 7.5), concentrated, and confirmed through SDS-PAGE analysis. As a result, a clear band appeared at 86 kDa, confirming that the recombinant cis / trans fatty acid isomerase was produced intact (Fig. 4c).

[0056]

[0057] [Example 4: Activity analysis of recombinant cis / trans fatty acid isomerase]

[0058] 4-1. Induction of enzyme reaction

[0059] To confirm whether the cis / trans fatty acid isomerase produced through the above Example 3 exhibits full activity, an enzymatic reaction was performed on monounsaturated fatty acids (oleic acid, palmitoleic acid, cis-vaccenic acid). The enzymatic reaction system was prepared based on reversed micelles. 0.09 mL of the recombinant enzyme solution was added to an isooctane solution containing 4.41 mL of 113.38 mM sodium 1,4-bis(2-ethylhexoxy)-1,4-dioxobutane-2-sulfonate, and the mixture was vigorously mixed for more than 1 minute to prepare a stable reversed micelle reaction system. The monounsaturated fatty acid substrate solution was prepared by dissolving each substance in isooctane to a concentration of 1 mM. The enzyme reaction was initiated by adding 0.5 mL of the substrate solution to the reversed micelle reaction system and was performed at 37°C and 800 rpm for 30 minutes.

[0060]

[0061] 4-2. Analysis of enzyme reaction products

[0062] To analyze the cis / trans composition of unsaturated fatty acids after the enzymatic reaction, the unsaturated fatty acids in the reaction solution were converted to methyl esters. First, only the isooctane solvent in 0.3 mL of the reaction solution was completely vaporized using a nitrogen generator, and the dried reaction solution was completely dissolved in 0.5 mL of a mixed solution of toluene and methanol (toluene: methanol = 52:48, v / v). 0.5 mL of a methylation reagent (a strong acidic BF3-methanol solution) was added to the completely dissolved reaction solution, and the methylation reaction was performed at 37°C for 20 minutes. 1 mL of hexane was mixed with the obtained supernatant to extract the synthesized unsaturated fatty acid methyl ester, and impurities were washed using 1 mL of distilled water.

[0063] The above cis / trans unsaturated fatty acid methyl ester extract was filtered through a 0.45 μm PTFE filter, and then separated using gas chromatography equipped with a DB-FastFAME column in which a cyanopropyl functional group was modified and the substance was packed as a stationary phase, and detected using a flame ionization detector (Fig. 5).

[0064] Afterwards, from the results of the above Fig. 5, the conversion rate was calculated through the concentration of trans-unsaturated fatty acids compared to the concentration of total unsaturated fatty acids, and it was confirmed that the conversion rates from cis-unsaturated fatty acids (oleic acid, palmitoleic acid, cis-vaccenic acid) to trans-unsaturated fatty acids (elaidic acid, palmitelidic acid, vaccenic acid) were 17.99%, 47.65%, and 28.72%, respectively, as a result of reacting cis / trans fatty acid isomerase for 30 minutes. The above results indicate that the activity was performed regardless of the length or double bond position of the unsaturated fatty acid. In addition, it means that cis / trans fatty acid isomerase was fully expressed in an active form.

[0065]

[0066] [Example 5: Structural analysis of recombinant cis / trans fatty acid isomerase]

[0067] In this example, in order to structurally confirm the expression pattern of the recombinant cis / trans fatty acid isomerase produced through double transformation, the protein structure of the recombinant cis / trans fatty acid isomerase produced through Example 3 was predicted.

[0068] First, the amino acid sequence of SEQ ID NO: 6, the entire sequence of the recombinant cis / trans fatty acid isomerase, was analyzed using SignalP (version 6.0). As a result, the pelB signal sequence (SEQ ID NO: 4) inserted at the N-terminus was accurately predicted to be a signal sequence domain, and it was predicted to be cleaved between the 22nd and 23rd amino acids and matured upon delivery to the intermembrane space (Fig. 6).

[0069] Furthermore, the protein tertiary structure was predicted using deep learning-based RoseTTAFold. Specifically, information corresponding to the remaining sequence, excluding the signal sequence (amino acids 1-22) of the amino acid sequence represented by SEQ ID NO: 6, was analyzed using deep learning-based RoseTTAFold. As a result, a tertiary structure model was predicted with an average estimated reliability of 0.80 (maximum 1.00) and an atomic distance error ranging from a minimum of 0.64 A to a maximum of 10.56 A (Fig. 7).

[0070] Through this, it was confirmed that the recombinant cis / trans fatty acid isomerase is an active site consisting of a cytochrome c domain consisting of six short alpha helices from the 33rd to the 190th amino acid after cytochrome c maturation, and that the remaining site is a structural domain that is not directly related to the enzyme activity.

Claims

1. A method for producing cis / trans fatty acid isomerase, characterized by culturing double-transformed Escherichia coli to express cis / trans fatty acid isomerase and a cytochrome c maturation system.

2. In paragraph 1, The above cis / trans fatty acid isomerase is, A method for producing a cis / trans fatty acid isomerase, characterized in that the N-terminal signal sequence is replaced with a pelB signal sequence consisting of the amino acid sequence of sequence number 4 and expressed.

3. In paragraph 1, The above cis / trans fatty acid isomerase production method is, A method for producing cis / trans fatty acid isomerase, characterized by transforming E. coli using a pEC86 plasmid (CCOS 891) in which a cytochrome c maturation system is introduced.

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