Methods for improving the yield of capsaicin catalyzed by n-acyltransferase, and mutants, genes and applications thereof

Abstract

This invention relates to the field of agricultural biotechnology, specifically to a method for increasing the yield of capsaicin synthesized by N-acyltransferase, its mutant F45I, the gene, and its applications. The invention provides multiple advantageous mutation sites for the key enzyme N-acyltransferase that catalyzes this step, and through combined mutations, obtains a combined mutant with a more significant yield increase, substantially enhancing capsaicin production.

Description

Technical Field

[0001] This invention relates to the field of agricultural biotechnology, specifically to a method for increasing the yield of capsaicin synthesized by N-acyltransferase, its mutant F45I, gene, and applications. Background Technology

[0002] Capsaicin is a class of amide compounds synthesized from medium- and long-chain fatty acids (C9-C11) and vanillin amine. Natural capsaicin is primarily derived from chili peppers. Due to its antioxidant and antibacterial properties, capsaicin is commonly used as a food preservative. It also possesses broad-spectrum antibacterial and bactericidal functions, making it an excellent alternative to antibiotics in feed additives. Microbial biosynthesis offers unique advantages in increasing yield, reducing pollution, and promoting sustainable production.

[0003] CN116837016A discloses a method for constructing a recombinant Escherichia coli engineered strain for producing capsaicin vanillonamide, as well as the recombinant strain and its application. Using nonanoic acid and vanillinamine as substrates, the engineered Escherichia coli strain for producing capsaicin vanillonamide synthesizes vanillonamide through cell transformation. The engineered strain expresses exogenous gene encoding the vanillonamide synthase system, which includes an N-acyltransferase gene and a CoA ligase gene.

[0004] In the synthesis of capsaicin, N-acyltransferase is the key enzyme limiting its yield increase. It catalyzes the formation of an acyl bond between fatty acids in the form of coenzyme A and vanillinamine, thereby generating capsaicin-like substances. Figure 1 Therefore, N-acyltransferases can be genetically engineered to increase capsaicin production. Summary of the Invention

[0005] One of the objectives of this invention is to provide a method for increasing the yield of capsaicin synthesized by N-acyltransferase.

[0006] Another aspect of the present invention is to provide a high-activity mutant of N-acyltransferase.

[0007] Another object of the present invention is to provide a gene encoding the above-mentioned high-activity mutant of N-acyltransferase.

[0008] Another object of the present invention is to provide the application of the above-mentioned high-activity mutant of N-acyltransferase.

[0009] The method according to the present invention for increasing the yield of capsaicin synthesized by N-acyltransferase includes the step of mutating the N-acyltransferase with the amino acid sequence shown in SEQ ID NO: 33 as follows:

[0010] Single point mutation: F45I;

[0011] Two-point mutations: N12T F45, H39S F45I, Y42H F45I, F45I N112K, F45I Q238E;

[0012] Three-point protrusions: N12T H39S F45I, N12T Y42H F45I, N12T F45I N112K, N12T F45IQ238E, H39S Y42H F45I, H39S F45I N112K, H39S F45I Q238E, Y42H F45I N112K, Y42HF45I Q238E, F45I N112K Q238E;

[0013] Four-point mutations: N12T H39S Y42H F45I, N12T H39S F45I N112K, N12T H39S F45IQ238E, N12T Y42H F45I N112K, N12T Y42H F45I Q238E, N12T F45I N112K Q238E, H39SY42H F45I N112K, H39S Y42H F45I Q238E, H39S F45I N112K Q238E, Y42H F45I N112KQ238E;

[0014] Five-point mutations: N12T H39S Y42H F45I N112K, N12T H39S Y42H F45I Q238E, N12TH39S F45I N112K Q238E, N12T Y42H F45I N112K Q238E, H39S Y42H F45I N112K Q238E; or

[0015] Six-point mutation: N12T H39S Y42H F45I N112K Q238E.

[0016] According to the present invention, a series of proteins with enhanced catalytic efficiency were obtained by combining single-point and multi-point mutants of CaNAT derived from chili pepper, the sequences of which are shown in SEQ ID NO: 1 to SEQ ID NO: 32.

[0017] Furthermore, based on the amino acid sequence of the leucine dehydrogenase mutant provided above, those skilled in the art can obtain the sequence of its encoding gene. Due to codon degeneracy, there is more than one gene sequence encoding the above amino acid sequence, and all genes capable of encoding the above leucine dehydrogenase mutant are within the protection scope of this invention.

[0018] According to the technical solution of this application, a recombinant microorganism is obtained by introducing the coding sequence of a protein, such as the amino acid sequence shown in SEQ ID NO: 1 to SEQ ID NO: 32, into a target microorganism; the introduction may be by integrating a foreign gene into the host chromosome, or by expressing it outside the chromosome by a plasmid.

[0019] This invention provides for any of the following applications of the N-acyltransferase mutant or the gene encoding the N-acyltransferase mutant:

[0020] Fermentation to produce capsaicin-like substances;

[0021] Whole-cell catalytic synthesis of capsaicin-like substances;

[0022] Construct engineered bacteria for synthesizing capsaicin.

[0023] Advantages of the technical solution of the present invention:

[0024] In the biosynthesis of capsaicin, the synthesis of the fatty acid vanillinamine from the activated medium-chain fatty acid is a key step. This invention provides several advantageous mutation sites for N-acyltransferase, a key enzyme that catalyzes this step, and combines mutations to obtain a combined mutant with a more significant increase in final yield, thereby greatly improving the yield of capsaicin. Attached Figure Description

[0025] Figure 1 This demonstrates the reaction catalyzed by N-acyltransferase;

[0026] Figure 2 The substrate binding pocket and key sites of CaNAT are shown. Detailed Implementation

[0027] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0028] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0029] This invention provides a highly active N-acyltransferase mutant derived from the plant pepper. Capsicum annuum Using N-acyltransferase (CaNAT) as the parent, a single-point mutation of CaNAT was first performed. M175L Based on this, perform CaNATN12T CaNAT H39S CaNAT Y42H CaNAT F45I CaNAT N112K and CaNAT Q238E The two-point, three-point, four-point, five-point, six-point, and seven-point combination mutants all showed a certain degree of enhancement in enzyme activity.

[0030] The culture media involved in the following examples are as follows:

[0031] LB liquid medium: yeast extract 5 g / L, tryptone 10 g / L, sodium chloride 10 g / L; pH 7.0, sterilized at 121℃ for 21 min;

[0032] LB solid medium: Agar powder with a mass concentration of 20 g / L is added to the liquid medium.

[0033] In the following examples, vanillonamide was analyzed and identified by high performance liquid chromatography and mass spectrometry: 3.0 mL of the conversion solution was mixed with 3.0 mL of ethyl acetate, centrifuged at 4500 r / min for 5 min, and 1.5 mL of the supernatant organic phase was collected and dried by rotary drying for 1.5 h. The sample was then dissolved in 0.3 mL of methanol, centrifuged at 12,000 r / min for 10 min, and the product was identified by LC-MS and the yield was determined by HPLC. Vanillonamide samples were identified using an XDB-C18 column in a liquid chromatography-mass spectrometry (LC-MS) system. The sample was injected into the column, and column separation was performed using 70% mobile phase A (acetonitrile) and 30% mobile phase B (0.1% formic acid in water). The chromatographic sample was charged in the mass spectrometer under an ESI(+) ion source with a capillary voltage of 2.5 kV, nitrogen as the carrier gas at a flow rate of 12 L / min, and a drying temperature of 350 °C. After primary mass spectrometry detection and capture of the target precursor ion, the precursor ion was further fragmented by energy enhancement, and the fragment ions were analyzed by secondary mass spectrometry. The ion capture range was set to 200–900 m / z. High-performance liquid chromatography (HPLC) was performed using a UV detector (SPD-20A). The sample was injected into a ZORBAX EclipsePlus C18 column, and gradient column separation was carried out at a mobile phase flow rate of 1.0 mL / min. Mobile phase A was acetonitrile, and mobile phase B was 0.1% trifluoroacetic acid (TFA) in water. The mobile phase program consisted of three stages: first, acetonitrile was increased from an initial concentration of 5% to 80% over 10 min; then, acetonitrile was maintained at 80% concentration for 8 min; and finally, the concentration of acetonitrile was reduced back to 5% over 4 min and maintained for 3 min.

[0034] The sequences involved in the following embodiments are:

[0035] >SEQ ID NO: 1 CANAT F45I:

[0036] MASAISETITTNGPSENNNLTITGKIHTRVRLATKSDLHHIYQLIYQIHAYHNFTHLYKATESSLGDLLFKENPLPLFYGPSVLLLEVSPTPFTQPKNNKDEGFKPVLTTFNLKFPVVEGQVEEFQSKYDDGNDKRDVFIAGYAFFYANYSCFYDKPGFYFESLYFRESYRKLGMGRLLFGTVASIAANNGFVSVEGIVAVWNKKSYDFYIDMGVEIFDEFRYGKLHGENLQKYADKQKNEGGNC;

[0037] >SEQ ID NO:2CANAT N12T F45I:

[0038] MASAISETITTTGPSENNNLTITGKIHTRVRLATKSDLHHIYQLIYQIHAYHNFTHLYKATESSLGDLLFKENPLPLFYGPSVLLLEVSPTPFTQPKNNKDEGFKPVLTTFNLKFPVVEGQVEEFQSKYDDGNDKRDVFIAGYAFFYANYSCFYDKPGFYFESLYFRESYRKLGMGRLLFGTVASIAANNGFVSVEGIVAVWNKKSYDFYIDMGVEIFDEFRYGKLHGENLQKYADKQKNEGGNC;

[0039] >SEQ ID NO: 3 CANAT H39S F45I:

[0040] MASAISETITTNGPSENNNLTITGKIHTRVRLATKSDLSHIYQLIYQIHAYHNFTHLYKATESSLGDLLFKENPLPLFYGPSVLLLEVSPTPFTQPKNNKDEGFKPVLTTFNLKFPVVEGQVEEFQSKYDDGNDKRDVFIAGYAFFYANYSCFYDKPGFYFESLYFRESYRKLGMGRLLFGTVASIAANNGFVSVEGIVAVWNKKSYDFYIDMGVEIFDEFRYGKLHGENLQKYADKQKNEGGNC;

[0041] >SEQ ID NO: 4CANAT Y42H F45I:

[0042] MASAISETITTNGPSENNNLTITGKIHTRVRLATKSDLHHIHQLIYQIHAYHNFTHLYKATESSLGDLLFKENPLPLFYGPSVLLLEVSPTPFTQPKNNKDEGFKPVLTTFNLKFPVVEGQVEEFQSKYDDGNDKRDVFIAGYAFFYANYSCFYDKPGFYFESLYFRESYRKLGMGRLLFGTVASIAANNGFVSVEGIVAVWNKKSYDFYIDMGVEIFDEFRYGKLHGENLQKYADKQKNEGGNC;

[0043] >SEQ ID NO: 5 CANAT F45I N112K :

[0044] MASAISETITTNGPSENNNLTITGKIHTRVRLATKSDLHHIYQLIYQIHAYHNFTHLYKATESSLGDLLFKENPLPLFYGPSVLLLEVSPTPFTQPKNNKDEGFKPVLTTFKLKFPVVEGQVEEFQSKYDDGNDKRDVFIAGYAFFYANYSCFYDKPGFYFESLYFRESYRKLGMGRLLFGTVASIAANNGFVSVEGIVAVWNKKSYDFYIDMGVEIFDEFRYGKLHGENLQKYADKQKNEGGNC;

[0045] >SEQ ID NO: 6 CANAT F45I Q238E:

[0046] MASAISETITTNGPSENNNLTITGKIHTRVRLATKSDLHHIYQLIYQIHAYHNFTHLYKATESSLGDLLFKENPLPLFYGPSVLLLEVSPTPFTQPKNNKDEGFKPVLTTFNLKFPVVEGQVEEFQSKYDDGNDKRDVFIAGYAFFYANYSCFYDKPGFYFESLYFRESYRKLGMGRLLFGTVASIAANNGFVSVEGIVAVWNKKSYDFYIDMGVEIFDEFRYGKLHGENLQKYADKEKNEGGNC;

[0047] >SEQ ID NO: 7 CANAT N12T H39S F45I:

[0048] MASAISETITTTGPSENNNLTITGKIHTRVRLATKSDLSHIYQLIYQIHAYHNFTHLYKATESSLGDLLFKENPLPLFYGPSVLLLEVSPTPFTQPKNNKDEGFKPVLTTFNLKFPVVEGQVEEFQSKYDDGNDKRDVFIAGYAFFYANYSCFYDKPGFYFESLYFRESYRKLGMGRLLFGTVASIAANNGFVSVEGIVAVWNKKSYDFYIDMGVEIFDEFRYGKLHGENLQKYADKQKNEGGNC;

[0049] >SEQ ID NO: 8 CANAT N12T Y42H F45I:

[0050] MASAISETITTTGPSENNNLTITGKIHTRVRLATKSDLHHIHQLIYQIHAYHNFTHLYKATESSLGDLLFKENPLPLFYGPSVLLLEVSPTPFTQPKNNKDEGFKPVLTTFNLKFPVVEGQVEEFQSKYDDGNDKRDVFIAGYAFFYANYSCFYDKPGFYFESLYFRESYRKLGMGRLLFGTVASIAANNGFVSVEGIVAVWNKKSYDFYIDMGVEIFDEFRYGKLHGENLQKYADKQKNEGGNC;

[0051] >SEQ ID NO: 9 CANAT N12T F45I N112K:

[0052] MASAISETITTTGPSENNNLTITGKIHTRVRLATKSDLHHIYQLIYQIHAYHNFTHLYKATESSLGDLLFKENPLPLFYGPSVLLLEVSPTPFTQPKNNKDEGFKPVLTTFKLKFPVVEGQVEEFQSKYDDGNDKRDVFIAGYAFFYANYSCFYDKPGFYFESLYFRESYRKLGMGRLLFGTVASIAANNGFVSVEGIVAVWNKKSYDFYIDMGVEIFDEFRYGKLHGENLQKYADKQKNEGGNC;

[0053] >SEQ ID NO: 10 CANAT N12T F45I Q238E:

[0054] MASAISETITTTGPSENNNLTITGKIHTRVRLATKSDLHHIYQLIYQIHAYHNFTHLYKATESSLGDLLFKENPLPLFYGPSVLLLEVSPTPFTQPKNNKDEGFKPVLTTFNLKFPVVEGQVEEFQSKYDDGNDKRDVFIAGYAFFYANYSCFYDKPGFYFESLYFRESYRKLGMGRLLFGTVASIAANNGFVSVEGIVAVWNKKSYDFYIDMGVEIFDEFRYGKLHGENLQKYADKEKNEGGNC;

[0055] >SEQ ID NO: 11 CANAT H39S Y42H F45I:

[0056] MASAISETITTNGPSENNNLTITGKIHTRVRLATKSDLSHIHQLIYQIHAYHNFTHLYKATESSLGDLLFKENPLPLFYGPSVLLLEVSPTPFTQPKNNKDEGFKPVLTTFNLKFPVVEGQVEEFQSKYDDGNDKRDVFIAGYAFFYANYSCFYDKPGFYFESLYFRESYRKLGMGRLLFGTVASIAANNGFVSVEGIVAVWNKKSYDFYIDMGVEIFDEFRYGKLHGENLQKYADKQKNEGGNC;

[0057] >SEQ ID NO: 12 CANAT H39S F45I N112K:

[0058] MASAISETITTNGPSENNNLTITGKIHTRVRLATKSDLSHIYQLIYQIHAYHNFTHLYKATESSLGDLLFKENPLPLFYGPSVLLLEVSPTPFTQPKNNKDEGFKPVLTTFKLKFPVVEGQVEEFQSKYDDGNDKRDVFIAGYAFFYANYSCFYDKPGFYFESLYFRESYRKLGMGRLLFGTVASIAANNGFVSVEGIVAVWNKKSYDFYIDMGVEIFDEFRYGKLHGENLQKYADKQKNEGGNC;

[0059] >SEQ ID NO: 13 CANAT H39S F45I Q238E:

[0060] MASAISETITTNGPSENNNLTITGKIHTRVRLATKSDLSHIYQLIYQIHAYHNFTHLYKATESSLGDLLFKENPLPLFYGPSVLLLEVSPTPFTQPKNNKDEGFKPVLTTFNLKFPVVEGQVEEFQSKYDDGNDKRDVFIAGYAFFYANYSCFYDKPGFYFESLYFRESYRKLGMGRLLFGTVASIAANNGFVSVEGIVAVWNKKSYDFYIDMGVEIFDEFRYGKLHGENLQKYADKEKNEGGNC;

[0061] >SEQ ID NO: 14 CANAT Y42H F45I N112K:

[0062] MASAISETITTNGPSENNNLTITGKIHTRVRLATKSDLHHIHQLIYQIHAYHNFTHLYKATESSLGDLLFKENPLPLFYGPSVLLLEVSPTPFTQPKNNKDEGFKPVLTTFKLKFPVVEGQVEEFQSKYDDGNDKRDVFIAGYAFFYANYSCFYDKPGFYFESLYFRESYRKLGMGRLLFGTVASIAANNGFVSVEGIVAVWNKKSYDFYIDMGVEIFDEFRYGKLHGENLQKYADKQKNEGGNC;

[0063] >SEQ ID NO: 15 CANAT Y42H F45I Q238E:

[0064] MASAISETITTNGPSENNNLTITGKIHTRVRLATKSDLHHIHQLIYQIHAYHNFTHLYKATESSLGDLLFKENPLPLFYGPSVLLLEVSPTPFTQPKNNKDEGFKPVLTTFNLKFPVVEGQVEEFQSKYDDGNDKRDVFIAGYAFFYANYSCFYDKPGFYFESLYFRESYRKLGMGRLLFGTVASIAANNGFVSVEGIVAVWNKKSYDFYIDMGVEIFDEFRYGKLHGENLQKYADKEKNEGGNC;

[0065] >SEQ ID NO:16 CANAT F45I N112K Q238E:

[0066] MASAISETITTNGPSENNNLTITGKIHTRVRLATKSDLHHIYQLIYQIHAYHNFTHLYKATESSLGDLLFKENPLPLFYGPSVLLLEVSPTPFTQPKNNKDEGFKPVLTTFKLKFPVVEGQVEEFQSKYDDGNDKRDVFIAGYAFFYANYSCFYDKPGFYFESLYFRESYRKLGMGRLLFGTVASIAANNGFVSVEGIVAVWNKKSYDFYIDMGVEIFDEFRYGKLHGENLQKYADKEKNEGGNC;

[0067] >SEQ ID NO: 17 CANAT N12T H39S Y42H F45I:

[0068] MASAISETITTTGPSENNNLTITGKIHTRVRLATKSDLSHIHQLIYQIHAYHNFTHLYKATESSLGDLLFKENPLPLFYGPSVLLLEVSPTPFTQPKNNKDEGFKPVLTTFNLKFPVVEGQVEEFQSKYDDGNDKRDVFIAGYAFFYANYSCFYDKPGFYFESLYFRESYRKLGMGRLLFGTVASIAANNGFVSVEGIVAVWNKKSYDFYIDMGVEIFDEFRYGKLHGENLQKYADKQKNEGGNC;

[0069] >SEQ ID NO: 18 CANAT N12T H39S F45I N112K:

[0070] MASAISETITTTGPSENNNLTITGKIHTRVRLATKSDLSHIYQLIYQIHAYHNFTHLYKATESSLGDLLFKENPLPLFYGPSVLLLEVSPTPFTQPKNNKDEGFKPVLTTFKLKFPVVEGQVEEFQSKYDDGNDKRDVFIAGYAFFYANYSCFYDKPGFYFESLYFRESYRKLGMGRLLFGTVASIAANNGFVSVEGIVAVWNKKSYDFYIDMGVEIFDEFRYGKLHGENLQKYADKQKNEGGNC;

[0071] >SEQ ID NO:19 CANAT N12T H39S F45I Q238E:

[0072] MASAISETITTTGPSENNNLTITGKIHTRVLATKSDLSHIYQLIYQIHAYHNFTHLYKATESSLGDLLFKENPLFYGPSVLLLEVSPTPFTQPKNNKDEGFKPVLTTFNLKFPVVEGQVE EFQSKYDDGNDKRDVFIAGYAFFYAFFYANYSCFYDKPGFYFESLYFRESYRKLGMGRLLFGTVASIAANNGFVSVEGIVAVWNKKSYDFYIDMGVEIFDEFRYGKLHGENLQKYADKEKNEGGNC;

[0073] >SEQ ID NO: 20 WING N12T Y42H F45I N112K:

[0074] MASAISETITTTGPSENNNLTITGKIHTRVRLATKSDLHHIHQLIYQIHAYHNFTHLYKATESSLGDLLFKENPLFYGPSVLLEVSPTPFTQNKNKDEGFKPVLTTFKLKFPVVEGQVE EFQSKYDDGNDKRDVFIAGYAFFYAFFYANYSCFYDKPGFYFESLYFRESYRKLGMGRLLFGTVASIAANNGFVSVEGIVAVWNKKSYDFYIDMGVEIFDEFRYGKLHGENLQKYADKQKNEGGNC;

[0075] >SEQ ID NO: 21 WING N12T Y42H F45I Q238E:

[0076] MASAISETITTTGPSENNNLTITGKIHTRVRLATKSDLHHIHQLIYQIHAYHNFTHLYKATESSLGDLLFKENPLPLFYGPSVLLLEVSPTPFTQPKNNKDEGFKPVLTTFNLKFPVVEGQVEEFQSKYDDGNDKRDVFIAGYAFFYANYSCFYDKPGFYFESLYFRESYRKLGMGRLLFGTVASIAANNGFVSVEGIVAVWNKKSYDFYIDMGVEIFDEFRYGKLHGENLQKYADKEKNEGGNC;

[0077] >SEQ ID NO: 22 CANAT N12T F45I N112K Q238E:

[0078] MASAISETITTTGPSENNNLTITGKIHTRVRLATKSDLHHIYQLIYQIHAYHNFTHLYKATESSLGDLLFKENPLPLFYGPSVLLLEVSPTPFTQPKNNKDEGFKPVLTTFKLKFPVVEGQVEEFQSKYDDGNDKRDVFIAGYAFFYANYSCFYDKPGFYFESLYFRESYRKLGMGRLLFGTVASIAANNGFVSVEGIVAVWNKKSYDFYIDMGVEIFDEFRYGKLHGENLQKYADKEKNEGGNC;

[0079] >SEQ ID NO: 23 CANAT H39S Y42H F45I N112K:

[0080] MASAISETITTNGPSENNNLTITGKIHTRVRLATKSDLSHIHQLIYQIHAYHNFTHLYKATESSLGDLLFKENPLPLFYGPSVLLLEVSPTPFTQPKNNKDEGFKPVLTTFKLKFPVVEGQVEEFQSKYDDGNDKRDVFIAGYAFFYANYSCFYDKPGFYFESLYFRESYRKLGMGRLLFGTVASIAANNGFVSVEGIVAVWNKKSYDFYIDMGVEIFDEFRYGKLHGENLQKYADKQKNEGGNC;

[0081] >SEQ ID NO: 24 CANAT H39S Y42H F45I Q238E:

[0082] MASAISETITTNGPSENNNLTITGKIHTRVRLATKSDLSHIHQLIYQIHAYHNFTHLYKATESSLGDLLFKENPLPLFYGPSVLLLEVSPTPFTQPKNNKDEGFKPVLTTFNLKFPVVEGQVEEFQSKYDDGNDKRDVFIAGYAFFYANYSCFYDKPGFYFESLYFRESYRKLGMGRLLFGTVASIAANNGFVSVEGIVAVWNKKSYDFYIDMGVEIFDEFRYGKLHGENLQKYADKEKNEGGNC;

[0083] >SEQ ID NO:25 CANAT H39S F45I N112K Q238E:

[0084] MASAISETITTNGPSENNNLTITGKIHTRVRLATKSDLSHIYQLIYQIHAYHNFTHLYKATESSLGDLLFKENPLPLFYGPSVLLLEVSPTPFTQPKNNKDEGFKPVLTTFKLKFPVVEGQVEEFQSKYDDGNDKRDVFIAGYAFFYANYSCFYDKPGFYFESLYFRESYRKLGMGRLLFGTVASIAANNGFVSVEGIVAVWNKKSYDFYIDMGVEIFDEFRYGKLHGENLQKYADKEKNEGGNC;

[0085] >SEQ ID NO: 26 CANAT Y42H F45I N112K Q238E:

[0086] MASAISETITTNGPSENNNLTITGKIHTRVRLATKSDLHHIHQLIYQIHAYHNFTHLYKATESSLGDLLFKENPLPLFYGPSVLLLEVSPTPFTQPKNNKDEGFKPVLTTFKLKFPVVEGQVEEFQSKYDDGNDKRDVFIAGYAFFYANYSCFYDKPGFYFESLYFRESYRKLGMGRLLFGTVASIAANNGFVSVEGIVAVWNKKSYDFYIDMGVEIFDEFRYGKLHGENLQKYADKEKNEGGNC;

[0087] >SEQ ID NO: 27 CANAT N12T H39S Y42H F45I N112K:

[0088] MASAISETITTTGPSENNNLTITGKIHTRVRLATKSDLSHIHQLIYQIHAYHNFTHLYKATESSLGDLLFKENPLFYGPSVLLLEVSPTPFTQPKNNKDEGFKPVLTTFKLKFPVVEGQVE EFQSKYDDGNDKRDVFIAGYAFFYAFFYANYSCFYDKPGFYFESLYFRESYRKLGMGRLLFGTVASIAANNGFVSVEGIVAVWNKKSYDFYIDMGVEIFDEFRYGKLHGENLQKYADKQKNEGGNC;

[0089] >SEQ ID NO: 28 WING N12T H39S Y42H F45I Q238E:

[0090] MASAISETITTTGPSENNNLTITGKIHTRVRLATKSDLSHIHQLIYQIHAYHNFTHLYKATESSLGDLLFKENPLFYGPSVLLLEVSPTPFTQPKNNKDEGFKPVLTTFNLKFPVVEGQVE EFQSKYDDGNDKRDVFIAGYAFFYAFFYANYSCFYDKPGFYFESLYFRESYRKLGMGRLLFGTVASIAANNGFVSVEGIVAVWNKKSYDFYIDMGVEIFDEFRYGKLHGENLQKYADKEKNEGGNC;

[0091] >SEQ ID NO: 29 CANAT N12T H39S F45I N112K Q238E:

[0092] MASAISETITTTGPSENNNLTITGKIHTRVRLATKSDLSHIYQLIYQIHAYHNFTHLYKATESSLGDLLFKENPLPLFYGPSVLLLEVSPTPFTQPKNNKDEGFKPVLTTFKLKFPVVEGQVEEFQSKYDDGNDKRDVFIAGYAFFYANYSCFYDKPGFYFESLYFRESYRKLGMGRLLFGTVASIAANNGFVSVEGIVAVWNKKSYDFYIDMGVEIFDEFRYGKLHGENLQKYADKEKNEGGNC;

[0093] >SEQ ID NO: 30 CANAT N12T Y42H F45I N112K Q238E:

[0094] MASAISETITTTGPSENNNLTITGKIHTRVRLATKSDLHHIHQLIYQIHAYHNFTHLYKATESSLGDLLFKENPLPLFYGPSVLLLEVSPTPFTQPKNNKDEGFKPVLTTFKLKFPVVEGQVEEFQSKYDDGNDKRDVFIAGYAFFYANYSCFYDKPGFYFESLYFRESYRKLGMGRLLFGTVASIAANNGFVSVEGIVAVWNKKSYDFYIDMGVEIFDEFRYGKLHGENLQKYADKEKNEGGNC;

[0095] >SEQ ID NO: 31 CANAT H39S Y42H F45I N112K Q238E:

[0096] MASAISETITTNGPSENNNLTITGKIHTRVRLATKSDLSHIHQLIYQIHAYHNFTHLYKATESSLGDLLFKENPLPLFYGPSVLLLEVSPTPFTQPKNNKDEGFKPVLTTFKLKFPVVEGQVEEFQSKYDDGNDKRDVFIAGYAFFYANYSCFYDKPGFYFESLYFRESYRKLGMGRLLFGTVASIAANNGFVSVEGIVAVWNKKSYDFYIDMGVEIFDEFRYGKLHGENLQKYADKEKNEGGNC;

[0097] >SEQ ID NO: 32 CANAT N12T H39S Y42H F45I N112K Q238E:

[0098] MASAISETITTTGPSENNNLTITGKIHTRVRLATKSDLSHIHQLIYQIHAYHNFTHLYKATESSLGDLLFKENPLPLFYGPSVLLLEVSPTPFTQPKNNKDEGFKPVLTTFKLKFPVVEGQVE EFQSKYDDGNDKRDVFIAGYAFFYANYSCFYDKPGFYFESLYFRESYRKLGMGRLLFGTVASIAANNGFVSVEGIVAVWNKKSYDFYIDMGVEIFDEFRYGKLHGENLQKYADKEKNEGGNC;

[0099] SEQ ID NO: 33CANAT:

[0100] MASAISETITTNGPSENNNLTITGKIHTRVRLATKSDLHHIYQLFYQIHAYHNFTHLYKATESSLGDLLFKENPLPLFYGPSVLLLEVSPTPFTQPKNNKDEGFKPVLTTFNLKFPVVEGQVE EFQSKYDDGNDKRDVFIAGYAFFYANYSCFYDKPGFYFESLYFRESYRKLGMGRLLFGTVASIAANNGFVSVEGIVAVWNKKSYDFYIDMGVEIFDEFRYGKLHGENLQKYADKQKNEGGNC. Example 1:

[0101] 1.1 Effect of single-point mutants on enzyme activity in capsaicin production

[0102] To rapidly characterize the catalytic performance of N-acyltransferases, plasmid pCDF-CaNAT-PhCL was transformed into E. coli EC6 (BL21(DE3) with fadE, fadR, acrR, crp, dppA, and yeaR genes knocked out) (refer to CN116837016A). The effect of CaNAT mutation on capsaicin yield was evaluated using a whole-cell catalytic method.

[0103] The dominant mutants of this enzyme were designed, and four single-point mutation sites (G66A, Q126K, F45I, V201D) were selected. The four single-point mutants were constructed on plasmids to replace the original CaNAT sites. The mutants were transformed into EC6 strain, incubated, and then plated on solid LB plates containing 50 µg / mL streptomycin and cultured overnight at 37°C. Single colonies on the plates were selected for colony PCR detection, and positive transformants were selected.

[0104] Seed culture was prepared by inoculating the culture into LB medium containing 50 µg / mL streptomycin and incubating overnight in a shaker (37°C, 200 rpm). The next day, the culture was transferred to fresh LB medium containing 50 µg / mL streptomycin and cultured at 37°C, 200 rpm to determine the bacterial density (OD). 600 When the pH approached 0.8, IPTG was added to a final concentration of 0.5 mM, followed by induction culture at 4°C and 200 r / min for 16 h. The induced bacterial cells were collected by centrifugation at 5000 r / min for 5 min, washed with an equal volume of water, and then centrifuged again to collect the cells, repeating this process once more. The bacterial cells were resuspended in 0.1 M KH₂PO₄-K₂HPO₄ buffer (pH 6.0) containing 20 g / L glucose. The bacterial density (OD₂) was determined. 600 The concentration was adjusted to approximately 5.0. The cell transformation system contained 2 g / L sodium nonanoate, 2 g / L vanillinamine, 0.5 g / L Tween-80, and 10 mL / L trace elements. The cell transformation solution volume was 15 mL, placed in a 100 mL Erlenmeyer flask, and the transformation reaction was carried out in a constant temperature shaker at 30℃ (250 r / min). The yield of vanillinamine was determined after 24 h of transformation.

[0105] The capsaicin yield of the control strain CaNATwt was 151.8 mg / L, while that of the mutant CaNAT was... F45I (SEQ ID NO:1) had a capsaicin yield of 218.6 mg / L, while the other three mutants, CaNAT, had a different yield. G66A CaNAT Q126K and CaNAT V201D All of these will lead to a decrease in production.

[0106] like Figure 2 As shown, gray represents the surface of the substrate channel, black represents the substrate acyl-CoA, and black spheres represent the F45 site. F45I optimizes the kinetics of substrate entry and product release by removing the steric hindrance at the substrate channel entrance.

[0107] 1.2 Effect of double-point mutants on enzyme activity in capsaicin production

[0108] Similar to the method in 1.1, a two-point combination mutant was constructed, and its catalytic and capsaicin yield was tested.

[0109] The capsaicin yields of the two-point combination mutant and the engineered strain with increased yields were as follows:

[0110] CaNAT N12T F45I , SEQ ID NO: 2, 316.5 mg / L;

[0111] CaNAT H39S F45I , SEQ ID NO: 3, 336.5 mg / L;

[0112] CaNAT Y42H F45I , SEQ ID NO: 4, 325.4 mg / L;

[0113] CaNAT F45I N112K , SEQ ID NO: 5, 297.3mg / L;

[0114] CaNAT F45I Q238E , SEQ ID NO: 6, 306.5mg / L.

[0115] 1.3 Effect of three-point mutants on enzyme activity in capsaicin production

[0116] Similar to the method in 1.1, a three-point combination mutant was constructed, and its catalytic and capsaicin yield was tested.

[0117] In the control group (CaNATwt), the capsaicin yield was 151.8 mg / L. The capsaicin yields of the three-point combination mutant and the engineered strain were respectively:

[0118] CaNAT N12T H39S F45I , SEQ ID NO: 7, 418.0mg / L;

[0119] CaNAT N12T Y42H F45I , SEQ ID NO: 8, 375.6mg / L;

[0120] CaNAT N12T F45I N112K , SEQ ID NO: 9, 386.2 mg / L;

[0121] CaNAT N12T F45I Q238E , SEQ ID NO: 10, 422.8 mg / L;

[0122] CaNAT H39S Y42H F45I , SEQ ID NO: 11, 384.7 mg / L;

[0123] CaNATH39S F45I N112K , SEQ ID NO: 12, 411.6 mg / L;

[0124] CaNAT H39S F45I Q238E , SEQ ID NO: 13, 413.1 mg / L;

[0125] CaNAT Y42H F45I N112K , SEQ ID NO: 14, 402.1mg / L;

[0126] CaNAT Y42H F45I Q238E , SEQ ID NO: 15, 387.5mg / L;

[0127] CaNAT F45I N112K Q238E , SEQ ID NO: 16, 402.4 mg / L.

[0128] 1.4 Effect of the four-point mutant on the enzyme activity of capsaicin production

[0129] As in Example 1, a four-point combination mutant was constructed and its catalytic and capsaicin yield was tested.

[0130] The capsaicin yields of the four-point combination mutant and the engineered strain were as follows:

[0131] CaNAT N12T H39S Y42H F45I , SEQ ID NO: 17, 489.3mg / L;

[0132] CaNAT N12T H39S F45I N112K , SEQ ID NO: 18, 478.3mg / L;

[0133] CaNAT N12T H39S F45I Q238E , SEQ ID NO: 19, 484.0mg / L;

[0134] CaNAT N12T Y42H F45I N112K , SEQ ID NO: 20, 478.4mg / L;

[0135] CaNAT N12T Y42H F45I Q238E SEQ ID NO: 21 479.7 mg / L;

[0136] CaNAT N12T F45I N112K Q238E , SEQ ID NO: 22, 504.5mg / L;

[0137] CaNAT H39S Y42H F45I N112K , SEQ ID NO: 23, 505.2mg / L;

[0138] CaNAT H39S Y42H F45I Q238E, SEQ ID NO: 24, 472.3mg / L;

[0139] CaNAT H39S F45I N112K Q238E , SEQ ID NO: 25, 483.3mg / L;

[0140] CaNAT Y42H F45I N112K Q238E , SEQ ID NO: 26, 465.6mg / L.

[0141] 1.5 Effect of the five-point mutant on the enzyme activity of capsaicin production

[0142] Similar to the method in 1.1, a five-point combination mutant was constructed, and its catalytic and capsaicin yield was tested.

[0143] The capsaicin yields of the five-point combination mutants and engineered strains were as follows:

[0144] CaNAT N12T H39S Y42H F45I N112K , SEQ ID NO: 27, 555.9mg / L;

[0145] CaNAT N12T H39S Y42H F45I Q238E , SEQ ID NO: 28, 540.8mg / L;

[0146] CaNAT N12T H39S F45I N112K Q238E , SEQ ID NO: 29, 553.4mg / L;

[0147] CaNAT N12T Y42H F45I N112K Q238E , SEQ ID NO: 30, 533.1mg / L;

[0148] CaNAT H39S Y42H F45I N112K Q238E , SEQ ID NO: 31, 562.4mg / L.

[0149] 1.6 Effect of the six-point mutant on the enzyme activity of capsaicin production

[0150] Similar to the method in 1.1, the six-point combination mutant was constructed and its catalytic and capsaicin yield was tested.

[0151] The capsaicin yields of the six-point combination mutants and engineered strains were as follows:

[0152] CaNAT N12T H39S Y42H F45I N112K Q238E , SEQ ID NO: 32, 616.2mg / L.

[0153] The above embodiments are only used to understand the technical aspects of this application and do not limit the scope of protection of this application.

Claims

1. A method for increasing the yield of capsaicin synthesized by N-acyltransferase, characterized in that, The method includes the following steps of mutating an N-acyltransferase with an amino acid sequence as shown in SEQ ID NO:33: Single point mutation: F45I; Two-point mutations: N12T F45, H39S F45I, Y42H F45I, F45I N112K or F45I Q238E; Three-point protrusion: N12T H39S F45I, N12T Y42H F45I, N12T F45I N112K, N12T F45I Q238E, H39S Y42H F45I, H39S F45I N112K, H39S F45I Q238E, Y42H F45I N112K, Y42H F45IQ238E or F45I N112K Q238E; Four-point mutations: N12T H39S Y42H F45I, N12T H39S F45I N112K, N12T H39S F45I Q238E, N12T Y42H F45I N112K, N12T Y42H F45I Q238E, N12T F45I N112K Q238E, H39S Y42HF45I N112K, H39S Y42H F45I Q238E, H39S F45I N112K Q238E, or Y42H F45I N112KQ238E; Five-point mutations: N12T H39S Y42H F45I N112K, N12T H39S Y42H F45I Q238E, N12T H39SF45I N112K Q238E, N12T Y42H F45I N112K Q238E or H39S Y42H F45I N112K Q238E; or Six-point mutation: N12T H39S Y42H F45I N112K Q238E.

2. A high-activity mutant of N-acyltransferase, characterized in that, The amino acid sequence of the N-acyltransferase high-activity mutant is shown in any one of SEQ ID NO: 1 to SEQ ID NO:

32.

3. N-acyltransferase gene, characterized in that, The N-acyltransferase gene encodes the high-activity mutant of the N-acyltransferase as described in claim 2.

4. A recombinant expression vector comprising the N-acyltransferase gene as described in claim 3.

5. A recombinant expression strain comprising the N-acyltransferase gene as described in claim 3.

6. The application of the N-acyltransferase high-activity mutant described in claim 2 in catalyzing the synthesis of capsaicin.

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