A fatty acid elongase mutant g11783-f233a and its use in preparing c20:1 delta 11

Abstract

The application discloses a fatty acid elongase mutant g11783-F233A and application thereof in preparation of C20:1 Δ11 The application discloses a fatty acid elongase mutant g11783-F233A and application thereof in preparation of C20:1 Δ11 The application also provides related nucleic acid molecules, a recombinant expression vector, a recombinant saccharomyces cerevisiae and a C20:1 Δ11 The application also provides related nucleic acid molecules, a recombinant expression vector, a recombinant saccharomyces cerevisiae and a C20:1 Δ11 The application also provides related nucleic acid molecules, a recombinant expression vector, a recombinant saccharomyces cerevisiae and a C20:1

Description

Technical Field

[0001] This invention relates to the fields of enzyme engineering and fatty acid metabolism engineering, and more specifically, to a fatty acid elongation enzyme mutant g11783-F233A and its application in the preparation of C20:1 Δ11 Applications in [the field]. Background Technology

[0002] cis-11-eicosenoic acid (C20:1) Δ11 Gonadotropin, also known as omega-9 monounsaturated fatty acid, is an ω-9 long-chain monounsaturated fatty acid. Studies have shown that its C20:1 ratio... Δ11 It can reduce reactive oxygen species levels and inhibit the expression of inflammation-related enzymes and pro-inflammatory factors, showing potential to alleviate oxidative stress and inflammatory responses. Furthermore, C20:1 Δ11 It can improve the stability of emulsion systems and mixed micelles, promote the release and micellization of fat-soluble nutrients such as lutein during digestion, thereby improving their bioavailability. Based on the above characteristics, C20:1 Δ11 It can be used as a high-value functional lipid raw material for the development of functional foods, nutritional preparations, and fat-soluble active substance delivery systems. Therefore, achieving a C20:1 ratio is crucial. Δ11 The stable and controllable bioprocessing of this material has significant research value and potential application value.

[0003] Currently, C20:1 Δ11 It can be obtained mainly through natural oil separation, chemical synthesis, or biosynthesis. However, the C20:1 ratio in natural oils... Δ11 Biosynthesis typically occurs alongside various other fatty acids, and its content and composition are easily affected by the source of raw materials, making subsequent separation and purification processes complex. Chemical synthesis usually involves multiple steps and may face challenges in controlling reaction selectivity. In contrast, biosynthesis using microbial cell factories offers advantages such as mild reaction conditions, controllable production processes, and ease of large-scale cultivation, achieving a C20:1 ratio. Δ11 This provides a new technical approach for the stable preparation of [the substance / method / property].

[0004] In biosynthesis, C20:1 Δ11 From C18:1 Δ9 It is formed by adding two carbon atoms to the carboxyl terminus. This carbon chain elongation process is catalyzed by a fatty acid elongation system located in the endoplasmic reticulum membrane, in which fatty acid elongases play a key role in substrate recognition and product chain length determination. Previous studies have shown that some plant-derived fatty acid elongases can catalyze C18:1... Δ9 Extend the generation of C20:1 Δ11 For example, Arabidopsis-derived elongases, in addition to recognizing C18:1... Δ9In addition to substrates, it can catalyze the elongation of various saturated and unsaturated fatty acid substrates, and can further increase the C20:1 ratio. Δ11 Extend the generation of C22:1 Δ13 When the elongation enzyme derived from *Capsella bursa-pastoris* is expressed in *Saccharomyces cerevisiae*, it can catalyze C18:1... Δ9 Extend the generation of C20:1 Δ11 In addition, it can recognize other monounsaturated fatty acid substrates and form C20:1 Δ13 Other elongation products, including those from C20:1 fatty acid substrates. This demonstrates that existing elongation enzymes may exhibit catalytic activity towards a variety of fatty acid substrates, leading to the formation of different elongation products. When used in C20:1... Δ11 In directed biosynthesis, there is still room for further optimization in substrate selectivity and product specificity. Therefore, it is necessary to develop methods that can effectively recognize C18:1 Δ9 And catalyzes its elongation to generate C20:1 Δ11 A novel fatty acid elongation enzyme to enrich C20:1 Δ11 Enzyme elements required for bioprocessing, and provide the technological basis for their targeted biosynthesis. Summary of the Invention

[0005] In view of this, the purpose of this invention is to provide a fatty acid elongation enzyme mutant g11783-F233A, its encoded nucleic acid molecule, recombinant expression vector, recombinant host cell, and applications, so as to obtain a product capable of catalyzing C18:1 Δ9 Extend the generation of C20:1 Δ11 The fatty acid elongation enzyme element is C20:1. Δ11 It provides the technological foundation for biosynthesis.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] A fatty acid elongase mutant g11783-F233A is obtained by replacing phenylalanine at position 233 of wild-type fatty acid elongase g11783 with alanine.

[0008] Furthermore, the amino acid sequence of the wild-type fatty acid elongation enzyme g11783 is shown in SEQ ID NO:1, and the amino acid sequence of the fatty acid elongation enzyme mutant g11783-F233A is shown in SEQ ID NO:2.

[0009] The present invention also provides a nucleic acid molecule encoding the above-mentioned fatty acid elongase mutant g11783-F233A, the nucleotide sequence of which is shown in SEQ ID NO:3.

[0010] The present invention also provides a recombinant expression vector containing the above-mentioned nucleic acid molecule, wherein the nucleic acid molecule is operatively linked to a promoter capable of driving its expression in a host cell.

[0011] The present invention also provides a recombinant host cell containing the above-mentioned nucleic acid molecule or the above-mentioned recombinant expression vector, and capable of expressing the fatty acid elongase mutant g11783-F233A.

[0012] Furthermore, the recombinant host cell is recombinant Saccharomyces cerevisiae.

[0013] This invention also provides the above-mentioned fatty acid elongation enzyme mutant g11783-F233A, a nucleic acid molecule encoding the mutant g11783-F233A, a recombinant expression vector containing the nucleic acid molecule, or a recombinant host cell in the biosynthesis of C20:1 Δ11 Applications in [the field].

[0014] This invention also provides a biosynthetic C20:1 Δ11 The method includes culturing recombinant host cells capable of expressing the fatty acid elongase mutant g11783-F233A, and adding C18:1 to the culture system. Δ9 This allows the fatty acid elongation enzyme mutant g11783-F233A to catalyze C18:1 Δ9 A carbon chain elongation reaction occurs, producing C20:1 Δ11 .

[0015] Further, the method includes: seeding the recombinant host cells into SC-Ura liquid culture medium containing galactose, and adding C18:1... Δ9 , so that the C18:1 Δ9 The final concentration in the culture system was 230-270 μmol / L, and the culture was carried out in the dark with shaking at 20±1℃ and 200-250 r / min for 45-50 h.

[0016] As can be seen from the above technical solution, compared with the prior art, the present invention has the following beneficial effects:

[0017] 1. This invention obtains the fatty acid elongation enzyme mutant g11783-F233A by replacing phenylalanine at position 233 of the wild-type fatty acid elongation enzyme g11783 with alanine. The wild-type fatty acid elongation enzyme g11783 is C18:1 Δ9 No quantifiable elongation products were detected when it was used as a substrate, while the fatty acid elongase mutant g11783-F233A was able to catalyze C18:1 Δ9 Extend the generation of C20:1 Δ11The substrate conversion rate reached 17.20%, thus achieving a high conversion efficiency for C18:1. Δ9 The activity of the extended activity.

[0018] 2. The substrate selectivity of the fatty acid elongation enzyme mutant g11783-F233A described in this invention differs from that of the wild-type fatty acid elongation enzyme g11783. The wild-type fatty acid elongation enzyme g11783 can catalyze C18:3 Δ9,12,15 An elongation reaction occurred, while the fatty acid elongase mutant g11783-F233A did not produce a quantifiable elongation product for this substrate, and a catalytic C18:1 elongation reaction was obtained. Δ9 Extend the generation of C20:1 Δ11 The ability of the 233rd amino acid substitution to alter the substrate selectivity of fatty acid elongases indicates that the substitution at position 233 can alter the substrate selectivity of fatty acid elongases.

[0019] 3. The fatty acid elongase mutant g11783-F233A described in this invention involves only a single amino acid site substitution, has a well-defined structure, and encodes a nucleic acid that is easy to synthesize, clone, and heterologously express. It can be used as a C20:1... Δ11 Novel biosynthesized enzyme elements for constructing C20:1 Δ11 This provides a technological basis for microbial production systems and for further optimization through metabolic engineering. Attached Figure Description

[0020] Figure 1 This is a partial amino acid sequence alignment diagram of wild-type fatty acid elongase g11783 and the fatty acid elongase mutant g11783-F233A. The sequence shown represents amino acid residues 221-240, and the arrow indicates that amino acid 233 was mutated from phenylalanine F to alanine A.

[0021] Figure 2 Different recombinant brewer's yeasts were fed C18:3 Δ9,12,15 Or C18:1 Δ9 GC-MS total ion chromatogram after [previous treatment]; (A) Empty vector control Saccharomyces cerevisiae fed C18:3 Δ9,12,15 The results of subsequent tests; (B) Recombinant Saccharomyces cerevisiae expressing wild-type fatty acid elongation enzyme g11783 was fed to C18:3 Δ9,12,15 The results of subsequent tests; (C) Recombinant Saccharomyces cerevisiae expressing mutant fatty acid elongation enzyme g11783-F233A was fed with C18:3 Δ9,12,15 The test results afterward; (D) Empty vector control Saccharomyces cerevisiae fed C18:1 Δ9 The results of subsequent tests; (E) Recombinant Saccharomyces cerevisiae expressing wild-type fatty acid elongation enzyme g11783 fed with C18:1 Δ9 The results of subsequent tests; (F) Recombinant Saccharomyces cerevisiae expressing mutant fatty acid elongation enzyme g11783-F233A was fed with C18:1Δ9 The results of the subsequent tests. Detailed Implementation

[0022] The present invention will be further described in detail below with reference to specific embodiments, but the scope of protection of the present invention is not limited to the following embodiments.

[0023] Example 1: Identification of the F233 candidate modification site for fatty acid elongase g11783

[0024] The amino acid sequence of wild-type fatty acid elongation enzyme g11783 is shown in SEQ ID NO:1. Previous functional identification results indicate that wild-type fatty acid elongation enzyme g11783 can catalyze C18:3... Δ9,12,15 A carbon chain elongation reaction occurs, but with a C18:1 ratio. Δ9 When the substrate was used, no quantifiable extended product was detected.

[0025] To obtain a catalyst capable of catalyzing C18:1 Δ9 Extend the generation of C20:1 Δ11 A fatty acid elongase mutant was obtained, and candidate amino acid sites were screened for wild-type fatty acid elongase g11783. Phenylalanine at position 233 is located downstream of the conserved catalytic motif of HXXHH, and was therefore selected as a candidate modification site. Replacing phenylalanine at position 233 with alanine resulted in a mutant named g11783-F233A, whose amino acid sequence is shown in SEQ ID NO:2. Sequence alignment of wild-type fatty acid elongase g11783 and mutant g11783-F233A near amino acid position 233 is shown below. Figure 1 As shown.

[0026] Example 2: Site-directed mutagenesis construction of g11783-F233A

[0027] Using the wild-type recombinant expression plasmid pYES2-g11783, which was verified to be correct by sequencing, as a template, the fatty acid elongase mutant g11783-F233A was constructed by site-directed mutagenesis.

[0028] Site-directed mutagenesis primers were designed based on the codon corresponding to phenylalanine at position 233 in the wild-type g11783 coding sequence to replace the codon encoding phenylalanine with the codon encoding alanine. The site-directed mutagenesis primers used are as follows:

[0029] F: 5'-CATCATTTTGCAGTGCTGCTGTATGTTTGGCAGA-3'

[0030] R: 5'-GCAGCACTGCAAAATGATGATACCAATGCAGCA-3'

[0031] Site-directed mutagenesis was performed using a rapid site-directed mutagenesis kit (TIANGEN, KM101). Using pYES2-g11783 plasmid as a template (the wild-type fatty acid elongase g11783 gene was synthesized by a biotechnology company and constructed on pYES2, named pYES2-g11783), the site-directed mutagenesis reaction system was prepared according to the kit instructions. The total reaction volume was 50 μL, including 100 ng of pYES2-g11783 template, 2 μL of forward mutation primer, 2 μL of reverse mutation primer, 10 μL of reaction buffer, 1 μL of DNA polymerase, and nuclease-free water.

[0032] The site-directed mutagenesis PCR reaction procedure is as follows: First, pre-denaturation is performed at 95℃ for 2 min; then 18 cycles are performed, each cycle including denaturation at 94℃ for 2 s, annealing at 55℃ for 10 s and extension at 68℃ for 3 min; finally, extension is performed at 68℃ for 5 min.

[0033] After PCR amplification, the amplification products were digested and circularized according to the instructions of the site-directed mutagenesis kit to remove wild-type template plasmids that had not undergone mutation and to obtain plasmid products containing the F233A mutation.

[0034] The treated mutant product was added to *E. coli* DH5α competent cells, incubated on ice for 30 min, then heat-shocked at 42°C for 60 s, followed immediately by an ice incubation for 2 min. LB liquid medium was added, and the cells were incubated at 37°C and 200 rpm for 1 h to recover. The recovered bacterial culture was then spread onto LB solid medium containing 100 μg / mL ampicillin and incubated upside down at 37°C for 16 h.

[0035] Single clones were selected for colony PCR identification, and positive clones with expected amplification results were sent for sequencing. Sequencing results showed that the codon corresponding to phenylalanine at position 233 of g11783 had been replaced with the codon corresponding to alanine, and no other unexpected mutations were found in the target gene, thus obtaining the recombinant expression plasmid pYES2-g11783-F233A.

[0036] Positive clones with correct sequencing were selected for amplification culture, and the pYES2-g11783-F233A plasmid was extracted using a plasmid extraction kit for subsequent transformation of Saccharomyces cerevisiae. The encoding nucleotide sequence of g11783-F233A is shown in SEQ ID NO:3.

[0037] Example 3 Construction and Identification of Recombinant Saccharomyces cerevisiae

[0038] The wild-type recombinant expression plasmid pYES2-g11783, the mutant recombinant expression plasmid pYES2-g11783-F233A, and the empty pYES2 vector, which were verified by sequencing, were transformed into Saccharomyces cerevisiae INVSC1 competent cells using the PEG / LiAc method.

[0039] Saccharomyces cerevisiae competent cells were thoroughly mixed with 4 μg plasmid, 10 μg denatured salmon sperm DNA, and 500 μL PEG / LiAc transformation solution and incubated at 30°C for 30 min, followed by heat shock at 42°C for 15 min. After heat shock, the Saccharomyces cerevisiae cells were collected by centrifugation, resuspended, and plated on SD-Ura solid medium.

[0040] The conversion plates were incubated upside down at 30°C for 48 h until single colonies of Saccharomyces cerevisiae were formed.

[0041] Single colonies were picked from transformation plates of pYES2-g11783, pYES2-g11783-F233A, and pYES2 empty vectors, respectively, and colony PCR was performed using universal vector primers flanking the target gene. PCR products were detected by 1% agarose gel electrophoresis.

[0042] Strains in the pYES2-g11783 and pYES2-g11783-F233A transformants whose amplified band size matched the theoretical length of the target gene were identified as positive transformants. Based on this, recombinant *Saccharomyces cerevisiae* expressing the wild-type fatty acid elongation enzyme g11783 and recombinant *Saccharomyces cerevisiae* expressing the fatty acid elongation enzyme mutant g11783-F233A were obtained, respectively.

[0043] Control Saccharomyces cerevisiae containing the empty pYES2 vector.

[0044] The recombinant Saccharomyces cerevisiae was used for subsequent induction culture and fatty acid substrate feeding experiments.

[0045] Example 4: Induction culture and substrate feeding of recombinant Saccharomyces cerevisiae

[0046] Recombinant Saccharomyces cerevisiae g11783, recombinant Saccharomyces cerevisiae g11783-F233A, and empty vector control Saccharomyces cerevisiae, which were identified as positive by colony PCR, were inoculated into SD-Ura liquid medium and cultured overnight at 30℃ and 220 r / min with shaking to obtain seed culture.

[0047] Saccharomyces cerevisiae cells were collected from the seed culture by centrifugation, and the cells were transferred to SC-Ura liquid induction medium containing 2% galactose. The initial cell density OD of the culture system was adjusted. 600 It is 0.2.

[0048] C18:3Δ9,12,15 And C18:1 Δ9 Fatty acid feeding experiments were conducted using different fatty acid substrates. Different fatty acid substrates were added to independent culture systems, with a final concentration of 250 μmol / L in each system.

[0049] After adding the fatty acid substrate, the cells were cultured at 20℃ and 220 r / min in the dark with shaking for 48 h. After the culture was completed, the Saccharomyces cerevisiae cells from each group were collected for subsequent detection of fatty acid composition and substrate transformation.

[0050] Example 5: Yeast cell collection, fatty acid methyl esterification and extraction

[0051] The *Saccharomyces cerevisiae* culture, after being fed with fatty acid substrates, was centrifuged at 4℃ and 8000 r / min for 10 min. The supernatant was discarded, and the *Saccharomyces cerevisiae* cell pellet was collected. The *Saccharomyces cerevisiae* cells were washed twice with sterile water to remove residual culture medium and exogenous fatty acids adhering to the cell surface. After each wash, the cells were centrifuged at 4℃ and 8000 r / min for 10 min, and the cell pellet was collected. The washed *Saccharomyces cerevisiae* cells were then freeze-dried for 24 h to obtain lyophilized bacterial powder. 300 mg of the lyophilized bacterial powder was weighed and added to 1.0 mL of toluene, 2.0 mL of 1% sulfuric acid-methanol solution, and 700 μL of nonadecanoic acid internal standard solution. The mixture was reacted at 50℃ and 150 r / min in the dark for 16 h to convert the fatty acids in the sample into the corresponding fatty acid methyl esters. After the methyl esterification reaction was completed, the sample was cooled to room temperature, and 1 mL of sodium chloride solution and 1 mL of n-hexane were added. After thorough shaking and mixing, the mixture was centrifuged at 3000 r / min for 5 min to separate the aqueous and organic phases. The upper n-hexane phase was collected. 1 mL of n-hexane was added again to the remaining aqueous phase, and the extraction was repeated once more. The two n-hexane phases were combined. The combined n-hexane phase was dried using anhydrous sodium sulfate and filtered through a 0.22 μm organic phase filter to obtain the fatty acid methyl ester sample for subsequent GC-MS detection.

[0052] Example 6: GC-MS detection and substrate selectivity analysis

[0053] The fatty acid methyl ester samples prepared in Example 5 were analyzed using gas chromatography-mass spectrometry (GC-MS). The GC conditions were as follows: injection port temperature 250℃, injection volume 1 μL, split ratio 10:1, carrier gas high-purity helium, and carrier gas flow rate 1.0 mL / min. The column temperature program was set as follows: initial temperature 50℃, hold for 1 min; increase to 175℃ at 25℃ / min; then increase to 230℃ at 4℃ / min and hold for 5 min. Mass spectrometry was performed in electron impact ionization mode, with an ion source temperature of 230℃, an interface temperature of 250℃, and a scan range of m / z 50-550. The fatty acid components in the samples were analyzed based on the retention times and mass spectrometric characteristics of each fatty acid methyl ester peak.

[0054] With C18:3 Δ9,12,15 When used as a substrate, the corresponding C20:3 was not detected in the empty vector control *Saccharomyces cerevisiae* sample. Δ11,14,17 Extended product peaks; a significant C20:3 ratio was detected in recombinant Saccharomyces cerevisiae samples expressing wild-type fatty acid elongation enzyme g11783. Δ11,14,17 The extended product peak indicates that wild-type g11783 can catalyze C18:3 Δ9,12,15 Carbon chain elongation reaction occurred; no quantifiable C20:3 was detected in the recombinant Saccharomyces cerevisiae sample expressing the fatty acid elongase mutant g11783-F233A. Δ11,14,17 Extending the product peak ( Figure 2 AC).

[0055] With C18:1 Δ9 When used as a substrate, no quantifiable C20:1 was detected in either the empty vector control *Saccharomyces cerevisiae* or the recombinant *Saccharomyces cerevisiae* expressing wild-type fatty acid elongation enzyme g11783. Δ11 The product peak was elongated; a significant C20:1 ratio was detected in the recombinant Saccharomyces cerevisiae sample expressing the fatty acid elongase mutant g11783-F233A. Δ11 The product peak was prolonged. The results indicate that g11783-F233A can catalyze C18:1 Δ9 A carbon chain elongation reaction occurs, producing C20:1 Δ11 ( Figure 2 DF).

[0056] The formula for calculating substrate conversion rate is as follows:

[0057] Substrate conversion rate (%) = (Amount of product) / (Amount of remaining substrate + Amount of product) × 100%

[0058] Table 1 shows the transformation results of wild-type fatty acid elongase g11783 and its mutant g11783-F233A on different fatty acid substrates. Fatty acid elongase g11783 exhibits a C18:3 conversion ratio. Δ9,12,15When used as a substrate, it can catalyze a carbon chain elongation reaction and generate C20:3. Δ11,14,17 The substrate conversion rate was 27.31%; the fatty acid elongase mutant g11783-F233A exhibited a C18:1 ratio. Δ9 When used as a substrate, it can catalyze a carbon chain elongation reaction to generate C20:1. Δ11 The substrate conversion rate was 17.20%.

[0059] Table 1. Transformation results of different fatty acid substrates by wild-type fatty acid elongase g11783 and fatty acid elongase mutant g11783-F233A.

[0060]

[0061] Note: ND indicates that no quantifiable corresponding elongation product was detected.

[0062] The above results indicate that after replacing phenylalanine at position 233 of wild-type fatty acid elongase g11783 with alanine, the resulting mutant g11783-F233A acquired the ability to catalyze the elongation of C18:1Δ9 to C20:1Δ11 and exhibited substrate selectivity different from that of wild-type g11783, indicating that the F233A substitution altered the substrate selectivity of g11783.

Claims

1. A fatty acid elongation enzyme mutant g11783-F233A, characterized in that, The fatty acid elongase mutant was obtained by replacing phenylalanine at position 233 of the wild-type fatty acid elongase g11783 shown in SEQ ID NO:1 with alanine.

2. The fatty acid elongation enzyme mutant g11783-F233A according to claim 1, characterized in that, It has catalytic C18:1 Δ9 A carbon chain elongation reaction occurs, producing C20:1 Δ11 The activity.

3. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the fatty acid elongase mutant g11783-F233A as described in claim 1.

4. The nucleic acid molecule according to claim 3, characterized in that, The nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO:

3.

5. A recombinant expression vector, characterized in that, It contains the nucleic acid molecule as described in claim 3 or 4.

6. A recombinant host cell, characterized in that, It contains the nucleic acid molecule as described in claim 3 or 4, or the recombinant expression vector as described in claim 5, and is capable of expressing the fatty acid elongase mutant g11783-F233A as described in claim 1.

7. The recombinant host cell according to claim 6, characterized in that, The host cell is Saccharomyces cerevisiae.

8. The fatty acid elongation enzyme mutant g11783-F233A according to claim 1, the nucleic acid molecule according to claim 3 or 4, the recombinant expression vector according to claim 5, or the recombinant host cell according to claim 6 in the preparation of C20:1 Δ11 Applications in [the context of the text].

9. A method for preparing C20:1 Δ11 The method is characterized by, Recombinant host cells expressing the fatty acid elongase mutant g11783-F233A were cultured, and C18:1 was added to the culture system. Δ9 , so that the C18:1 Δ9 Under the catalysis of the fatty acid elongation enzyme mutant g11783-F233A, a carbon chain elongation reaction occurs, producing C20:1 Δ11 .

10. The preparation method according to claim 9, characterized in that, The recombinant host cells were seeded into SC-Ura liquid medium containing galactose, and C18:1 was added. Δ9 , so that the C18:1 Δ9 The final concentration in the culture system was 230-270 μmol / L, and the culture was carried out in the dark with shaking at 20±1℃ and 200-250 r / min for 45-50 h.

Images