CN114854726B - Mutant of fatty acid light decarboxylase McFAP and application thereof - Google Patents

Abstract

The invention discloses a mutant of fatty acid photodecarboxylase McFAP and its application. The amino acid sequence of the mutant of fatty acid photodecarboxylase McFAP is shown in SEQ ID NO. 4. The McFAP mutant of the present invention enriches the types of FAP and fills the gap that existing FAP cannot catalyze decarboxylation of saturated straight-chain fatty acids with 6 to 12 carbon atoms. The decarboxylation substrate spectrum is significantly wider and the decarboxylation effect is better. It makes efficient and sustainable biosynthesis of fuel possible and has broad industrial application prospects.

Description

Mutants of fatty acid photodecarboxylase McFAP and their applications

Technical field

The invention belongs to the technical field of enzyme engineering. Specifically, the invention relates to a mutant of fatty acid photodecarboxylase Mc FAP and its application.

Background technique

Currently, biotechnology continues to shift from medicine, agriculture, and food to industrial fields (such as chemicals, materials, and energy). Gasoline, diesel, plastics, rubber, fibers and many bulk traditional petrochemical products are increasingly being replaced by industrial biomanufactured products from renewable feedstocks. High-temperature, high-pressure, and high-pollution chemical industrial processes are also constantly shifting to mild, clean and environmentally friendly biological processing processes.

The exploration and practice of producing renewable biofuels and chemicals in an environmentally friendly manner has received great attention. Using biomass as raw material and using microorganisms or enzymes to produce fuels has very broad development prospects. The development and utilization of renewable biomass resources directed conversion technology to produce biogas and liquid fuels has become an important scientific research topic for researchers in related fields in various countries. Task.

The enzymes discovered so far that can be used for fatty acid decarboxylation are mainly decarboxylase enzymes such as amino acid decarboxylase, haloperoxidase and fatty acid decarboxylation synthesis terminal olefinase (OleT _JE ). However, the catalytic efficiency of most enzymes is not high and requires The addition of additional expensive cofactors and/or oxidants that easily destroy the unsaturated bonds of oils is a significant shortcoming in applied research. Therefore, finding efficient new decarboxylase enzymes is the key to breaking through the bottleneck in biofuel preparation research.

Fatty acid photodecarboxylase (FAP, EC 4.1.1.106) belongs to the glucose-methanol-choline (GMC) oxidoreductase family. It is a kind of enzyme that can convert fatty acids into alkanes using only blue light without adding expensive cofactors. Light-driven enzyme of (alkene) hydrocarbons. Fatty acids only need to remove one carboxyl group to form various alkanes (olefins), which are almost perfectly matched with the composition of gasoline and diesel oil obtained by processing petroleum crude oil. Photocatalysis has low energy consumption, clean process, and is easy to control the switching state of the reaction. Biological enzymes have strong catalytic specificity and mild conditions. Therefore, the light-driven enzyme FAP, which combines the advantages of both and meets the expectations of green development, has become an emerging research hotspot. With the demand and preference for green energy, the preparation of alkanes (olefins) through decarboxylation of fatty acids has become a key research route for the development of biofuels, which has broad application prospects in the green chemical manufacturing process of biofuels.

In comparison, FAP directly uses light energy, which is more energy-saving, environmentally friendly, simple and convenient, and does not introduce double bonds at the end of the carbon chain. The product is the required alkane. FAP is a light-driven enzyme that uses blue light to convert fatty acids into alkanes (alkenes). The reaction conditions for decarboxylation of fatty acids are mild, the conversion rate is high (up to more than 90%), and the combustion heat value of the product is higher than that of esters. The fuel molecule and the by-product are only carbon dioxide, and only light energy is used in the process. This is of great significance to environmental protection and represents a new application field.

However, the only photodecarboxylases that have been reported so far are Cv FAP, Cr FAP, Esi FAP, Gsu FAP, and Nga FAP. Among them, only Cv FAP has been relatively intensively studied. The rest only express and verify decarboxylation activity, and Cv FAP and others are currently reported The passed photodecarboxylase showed a clear preference for saturated fatty acids with carbon atoms of 16 to 22, and its catalytic decarboxylation activity for short and medium-chain saturated fatty acids with less than 12 carbon atoms was significantly reduced (at 14 h of reaction, Cv FAP catalyzed laurel The acid decarboxylation yield is 11%. The decarboxylation reaction of n-hexanoic acid is catalyzed by decoy molecule means to produce only 1.6 mM product for 12 hours. An algal photoenzyme convertsfattyacids to hydrocarbons and Hydrocarbon synthesis via photoenzymaticdecarboxylation of carboxylic acids). The main components of gasoline are aliphatic hydrocarbons and cycloalkanes with 5 to 12 carbon atoms. Therefore, exploring photodecarboxylase that can catalyze the decarboxylation of short and medium-chain fatty acids will not only enrich the types of photodecarboxylase, but also expand green energy. There are broad prospects for development approaches.

Contents of the invention

Based on this, one of the purposes of the present invention is to provide a mutant of fatty acid photodecarboxylase Mc FAP, which can catalyze the decarboxylation of fatty acids with 6 to 12 carbon atoms.

Specific technical solutions to achieve the above-mentioned invention objectives include the following:

A mutant of fatty acid photodecarboxylase Mc FAP, the amino acid sequence of the mutant of fatty acid photodecarboxylase Mc FAP is shown in SEQ ID NO. 4.

The present invention also provides a gene encoding a mutant of the fatty acid photodecarboxylase Mc FAP, and the nucleotide sequence is shown in SEQ ID NO. 3.

The present invention also provides the application of the mutant of the fatty acid photodecarboxylase Mc FAP and its encoding gene in catalyzing the decarboxylation of fatty acids.

In some embodiments, the fatty acid has 6 to 12 carbon atoms.

In some embodiments, the fatty acid has 7 to 8 carbon atoms.

In some embodiments, the fatty acid is a saturated linear fatty acid.

The invention also provides a recombinant expression vector inserted with the above encoding gene.

The invention also provides a recombinant engineering strain transformed with the above recombinant expression vector.

The present invention also provides a method for preparing a mutant of fatty acid photodecarboxylase Mc FAP, which is obtained by expressing and purifying the above recombinant engineering strain.

The present invention also provides the application of the above-mentioned recombinant expression vector or recombinant engineering strain in catalyzing fatty acid decarboxylation.

The present invention also provides a method for catalyzing the decarboxylation of fatty acids, which uses whole cells of a mutant of the fatty acid photodecarboxylase Mc FAP to perform a catalytic reaction.

Compared with the prior art, the present invention has the following beneficial effects:

In the present invention, based on many years of experience, the inventor constructed a mutant of fatty acid photodecarboxylase Mc FAP through deletion mutation, and found that it has a good decarboxylation effect on linear fatty acids with carbon atoms of 6 to 18, especially It has a very excellent decarboxylation effect on medium-chain saturated linear fatty acids with 6 to 12 carbon atoms (the decarboxylation effect on C8:0 can reach more than 90% within 30 minutes). The mutant of Mc FAP of the present invention enriches the properties of FAP. species, filling the gap that existing FAP cannot catalyze decarboxylation of saturated straight-chain fatty acids with carbon atoms of 6 to 12. The decarboxylation substrate spectrum is significantly wider and the decarboxylation effect is better, making efficient and sustainable biosynthesis of fuels possible. Broad industrial application prospects.

Description of drawings

Figure 1 shows the results of the photoenzyme decarboxylation verification reaction of Mc FAP@ E. coli in Example 1 of the present invention.

Figure 2 is the SDS-PAGE protein profile of Mc FAP-S in Example 2 of the present invention; wherein, M. protein marker; 1. Mc FAP-S total bacteria; 2. Mc FAP-S supernatant; 3. Mc FAP- S precipitation; 4. Mc FAP-S crude enzyme; 5. Mc FAP-S through solution; 6.0.5 M imidazole elution Mc FAP-S pure enzyme.

Figure 3 is a reaction time curve of Mc FAP-S catalyzing the decarboxylation of n-octanoic acid in Example 3 of the present invention.

Figure 4 is a graph showing the effect of Mc FAP-S enzyme addition on the catalytic decarboxylation efficiency of n-octanoic acid in Example 3 of the present invention.

Figure 5 is a graph showing the effect of the concentration of the substrate n-octanoic acid on the catalytic decarboxylation efficiency of Mc FAP-S in Example 3 of the present invention.

Figure 6 is a graph showing the effect of reaction temperature on Mc FAP-S catalyzed n-octanoic acid decarboxylation efficiency in Example 3 of the present invention.

Figure 7 is the result of the effect of reaction pH on Mc FAP-S catalyzed n-octanoic acid decarboxylation efficiency in Example 3 of the present invention.

Figure 8 is a graph showing the storage stability of Mc FAP-S under dark conditions at 4°C in Example 3 of the present invention.

Figure 9 is a graph showing the pH tolerance of Mc FAP-S in Example 3 of the present invention.

Figure 10 is a graph showing the investigation of photo-inactivation factors of Mc FAP-S in Example 3 of the present invention.

Figure 11 is a diagram of substrate expansion research on Mc FAP@ E. coli and Mc FAP-S@ E. coli catalyzing saturated fatty acids with different chain lengths in Example 4 of the present invention .

Figure 12 is a comparative experimental result of the decarboxylation efficiency of palmitic acid catalyzed by Mc FAP@ E. coli and Cv FAP@ E. coli in Example 5 of the present invention.

Detailed ways

In order to facilitate an understanding of the invention, the invention will be described more fully below. The invention may be embodied in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that a thorough understanding of the present disclosure will be provided.

Unless otherwise defined, all technical and scientific terms used in the present invention have the same meanings commonly understood by those skilled in the technical field belonging to the present invention. The terms used in the description of the present invention are only for the purpose of describing specific embodiments and are not used to limit the present invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

In the present invention, the gene sequence of the photodecarboxylase Mc FAP derived from Micractinium conductrix is obtained through sequence analysis in the gene library (the nucleotide sequence is SEQ ID NO. 1), and the gene is obtained through gene synthesis and used in Escherichia coli BL21 (DE3) was used as the host for expression, and whole cells of Mc FAP were obtained (denoted as Mc FAP@ E. coli , the same below). The photodecarboxylase Mc FAP was successfully expressed and purified (the amino acid sequence is SEQ ID NO. 2). At the same time, its basic enzymatic properties were explored. Mc FAP is a blue light-catalyzed photodecarboxylase. It has very good universality for catalyzing the decarboxylation of fatty acid substrate chain lengths, and is suitable for linear chains with 6 to 18 carbon atoms. Saturated fatty acids all have good decarboxylation effects.

SEQ ID NO.1 (gene encoding fatty acid photodecarboxylase McFAP, 3438bp):

ATGGCTGAAATGGCAGGTGGTGGTGAAGGTGATGGTATGCTGATGGGCGGCGCGGGTAGCGCAAACACTACCGACGCGTGTTATAGCGATCCGTCTAATCCGGATTGCGCAGGTTTGAGCGCTCCGACGATGATTGGGCGGCGGACATCGAACTGCTGTGCTCTGCGATGCCGTTCATGCCGGGCTGCACCCTGGCGGAACAGTGCATGAATGGCACCGCCGCCGGTGAATATTGCGAAATGTCCAGTCT GGCTGGTAACATCTGTCTGGATATGCCGGGCATGAAAGGCTGTGAGGCATGGAACGCACTGTGTGGCGCGGCCAGCGCCGTTGAACAGTGTTCCTCTCCGGGCCCGGTTGTGGCACTCCCGACCACCGCGCTGGCCAAAGAAGGCCTGGAATCTCTGTGCTCTACCCATTGTATGGACGGTTGCCCAGACTGTGAAATGGGTAAACTGTGGAACACCTGCACCGACCCGCTGAGTGTTCTGGCGTGGATGTGCTA CGCAATGCCGGACATGCCGGAATGTCTGGCTGCTCCGCAGGGCTCCGGCATGGTGGTGGCTTGCGGTGACGCTGAGGTTGCAGCTACCTTCCCGCTGGTGTGCGGCCAACCGCCGACCCCGGCGGCTAACTTTCAGCACCGCCTTCGTACCTGCCGTACCGCCGGCGTTGCGGCATCCGCATCCGGTTTCTCCGGCAGTCACTATGGCTGGCCTGTCAACTGTTCTGGCAGTACTGGCACTGCTGCCATCCCCGGTT GCTATGGCCATGACTCCGATGCCGACCCCGGCGCTCGCTCCGGGCCCGGCGATCGACGATATCGGCGGTAACTGCCCGCTGCTGGGTCGCGGTAACATGGAAGCTCCGTGTTATAGCGACCCGAGCGCGGCAGCATGCGTTTCCTTTGAACGCAGCGATGCTGGCTGGGCGGATGACCTGAGTCAGCTGTGTTCTGCGATGCCGTATGCTGTTGGCTGCTGGCTGTGGCACTTGTGTAAAACCGGCGCAGCAA GCGGGACTTACTGTGCGCTGCCGTCCCTGACCGCGAACGTATGTGTTGACGCACCGCTGGTGAACGCTACATCAGCGCCGGGCTGCGAAGCGTGGGCCGCACTGTGCGGCGCCCAGGGTAGCGTCGTTGCGCAGTGCTCTGCGCCAGGCCCGCTGCCGGACATCATCAACACCCTGACCACCCGTGACGGCATCAACTCCCTCTGCGGTATGCATTACATGGATGGGTGTAACGAATGTACCCCTCACGAAGGTCCGGCAGTT CACGACTTCGCGGCCTGTGCTGATCCGGGTCCACTGCCGACTCTGGCCCACCAGTGTTACGCGATGCCTGAAATGGGTGAATGTACCCAGACTGGTATTACCGCAATGTGCAGCGGCGCTGAAGCTCGTGCGACCTTTCCGACCGTTTGCGTGGATCCACCTAACCCGACGACACTGGCGCCGGCGCCTGCCGTTTCTGCCTGCGATGTTGCGGCGGGTGCTGGCGCGCCACCAGCGGCGTCTGCCCGTCCGGCGTCGC ACAGCCGCGTCACTGGTTGCCTCCCGTAGCGGCTTCTGCGCGCCTTCCCCGGCGCTGCGCTCTCAGCGCACCTCTACTGTCGCGCCGGCGCGCCGCGCCGCGTCGGCGCCGCGTGCGAGCGCAGTTGACGATATTCAACGTGCTCTGAGCACCGCTGGAAGCCCGGTATCCGGTAAACAGTACGATTACATCCTGGTGGGTGGCGGCACCGCGGCATGCGTTTTGGCTAACCGTTTAACCGCGGACGGTAGCAAACG TGTACTGGTGCTGGAAGCGGGTGCGGACAACGTGAGCCGCGATGTTAAAGTCCCGGCTGCGATCACCCGTTTGTTCCGTTCACCGTTGGATTGGAACTTGTTCAGCGAATTGCAGGAACAGCTGGCTGCACGTCAGATCTATATGGCTCGCGGCCGCTTGCTGGGTGGGTCTAGCGCGACCAATGCTACTCTTTACCACCGTGGCGCGGCGGCGGATTATGATGCGTGGGGCGTGCCGGGCTGGGGCGCAGCTGA CGTGCTGCCATGGTTCGTTAAGGCCGAAACCAACGCGGAGTTTGCGGCGGGCAAATATCACGGCGCAGGTGGTAACATGCGCGTTGAGAATCCGCGCTACTCCAACCCGCAGCTGCACGGTGCTTTCTTTGCAGCTGCGCAGCAGATGGGTCTGCCGCAGAATACCGACTTCAACAATTGGGATCAGGATCATGCAGGCTTTGGCACTTTTCAGGTTATGCAGGAAAAAGGCACCCGCGCTGATATGTACCGCCAG TATCTTAAACCAGCTTCTTGGTCGTCCGAACCTGCAGGTTCTGACCGGTGCGTCTGTGACCAAAGTTCATATCGATAAAGCTGGCGGTAAACCGCGTGCTCTGGGCGTAGAGTTTTCTCTGGATGGTCCGGCTGGTGAACGTATGGCAGCAGAGCTGGCGCCGGGCGGTGAAGTTCTCATGTGCGCTGGCGCCGTGCATAGCCCGCACAATTCTGCAGCTGTCTGGCGTTGGTTCGGCGGCTACTCTGGCAGACCAC GGCATCGCAGCAGTGGCAGATCTGCCAGGTGTTGGTGCGAACATGCAGGACCAGCCGGCCTGCCTGACAGCGGCTCCCCTGAAAGACAAATACGATGGCATTTCGCTGACCGATCATATCTATAATAGCAAAGGCCAGATTCGCAAACGCGCTATCGCGTCCTACCTGCTTCAGGGTAAAGGTGGTCTGACGTCAACTGGCTGCGACCGTGGCGCGTTTGTACGTACCGCAGGCCAGGCACTGCCGGACCTGCAGGTG CGTTTCGTGCCAGGCATGGCACTGGATGCAGATGGTGTGTCCACCTACGTCCGTTTCGCAAAATTTCAGTCTCAGGGCCTGAAATGGCCGTCTGGCATCACCGTACAGCTTATTGCGTGTCGCCCGCACAGCAAAGGTTCTGTTGGCCTGAAAAACGCGGACCCGTTCACCCCGCCGAAACTGCGTCCGGGCTACCTGACCGACAAAGCGGGTGCGGATCTGGCGACCCTGCGCTCTGGTGTTCATTGGGCCCGTGATCTGG CATCTAGCGGTCCGCTGAGCGAATTTCTTGAAGGCGAACTGTTTCCGGGTAGCCAAGTTGTTTCCGATGATGATATTGATTCTTACATTCGTCGTACCATTCACTCCAGCAACGCGATTGTGGGCACCTGTCGTATGGGCGCGGCGGGTGAAGCGGGTTGTTGTGGATAACCAGCTGCGCGTTCAGGGTGTTGATGGTCTGCGTGTTGTTGACGCGAGCGTAATGCCGCGTATCCCAGGTGGTCAGGTGGGTGC GCCGGTTGTGATGCTGGCCGAACGTGCAGCAGCGATGCTGACCGGTCAGGCAGCGCTGGCTGGTGCTAGCGCTGCAGCTCCGCCGACCCCGGTCGCGGCT

SEQ ID NO.2 (amino acid sequence of fatty acid photodecarboxylase Mc FAP):

MAEMAGGGEGDGMLMGGAGSANTTDACYSDPSNPDCAAFERSDDDWAADIELLCSAMPFMPGCTLAEQCMNGTAAGEYCEMSSLAGNICLDMPGMKGCEAWNALCGAASAVEQCSSPGPVVALPTTALAKEGLESLCSTHCMDGCPDCEMGKLWNTCTDPLSVLAWMCYAMPDMPECLAAPQGSGMVVACGDAEVAATFPLVCAQPPTPAANFQHRLRTCRTAGVAASASGSPAVTMAG LSTVLAVLALLPSPVAMAMTPMPTPALAPGPAIDDIGGNCPLLGRGNMEAPCYSDPSAAACVSFERSDAGWADDLSQLCSAMPYAVGCWLWHLCKTGAASGTYCALPSLTANVCVDAPLVNATSAPGCEAWAALCGAQGSVVAQCSAPGPLPDIINTLTTRDGINSLCGMHYMDGCNECTPHEGPAVHDFAACADPGPLPTLAHQCYAMPEMGECTQTGITAMCSGAEARATFPTVCVDP PNPTTLAPAPAVSACDVAAGAGAPPAASARPASHSRASLVASRSGFCAPSPALRSQRTSTVAPARRAASAPRASAVDDIQRALSTAGSPVSGKQYDYILVGGGTAACVLANRLTADGSKRVLEAGADNVSRDVKVPAAITRLFRSPLDWNLFSELQEQLAARQIYMARGRLLGGSSATNATLYHRGAAADYDAWGVPGWGAADVLPWFVKAETNAEFAAGKYHGAGGNMR VENPRYSNPQLHGAFFAAAQQMGLPQNTDFNNWDQDHAGFGTFQVMQEKGTRADMYRQYLKPALGRPNLQVLTGASVTKVHIDKAGGKPRALGVEFSLDGPAGERMAAELAPGGEVLMCAGAVHSPHILQLSGVGSAATLADHGIAAVADLPGVGANMQDQPACLTAAPLKDKYDGISLTDHIYNSKGQIRKRAIASYLLQGKGGLTST GCDRGAFVRTAGQALPDLQVRFVPGMALDADGVSTYVRFAKFQSQGLKWPSGITVQLIACRPPHSKGSVGLKNADPFTPPKLRPGYLTDKAGADLATLRSGVHWARDLASSGPLSEFLEGELFPGSQVVSDDDDIDSYIRRTIHSSNAIVGTCRMGAAGEAGVVVDNQLRVQGVDGLRVVDASVMPRIPGGQVGAPVVMLAERAAAMLTGQAAL AGASAAAPPTPVAA

A mutant of fatty acid photodecarboxylase Mc FAP (hereinafter named Mc FAP-S) was obtained by truncating the N terminus of Mc FAP and performing deletion mutation. The Mc FAP mutant pure enzyme was purified through a nickel column (nucleotide sequence such as SEQ ID shown in NO.3, and the amino acid sequence is shown in SEQ ID NO.4). The catalytic effect of the Mc FAP mutant is greatly improved, and it can catalyze the decarboxylation of medium-chain fatty acids with carbon atoms of 6 to 12. It enriches the types of FAP and supplements the types of FAP enzymes that photocatalyze the decarboxylation of short-chain fatty acids.

SEQ ID NO.3 (gene encoding mutant of fatty acid photodecarboxylase Mc FAP):

CGTGCGAGCGCAGTTGACGATATTCAACGTGCTCTGAGCACCGCTGGAAGCCCGGTATCCGGTAAACAGTACGATTACATCCTGGTGGGTGGCGGCACCGCGGCATGCGTTTTGGCTAACCGTTTAACCGCGGACGGTAGCAAACGTGTACTGGTGCTGGAAGCGGGTGCGGACAACGTGAGCCGCGATGTTAAAGTCCCGGCTGCGATCACCCGTTTGTTCCGTTCACCGTTGGATTGGAACTTGTTCAGCGAATT GCAGGAACAGCTGGCTGCACGTCAGATCTATATGGCTCGCGGCCGCTTGCTGGGTGGGTCTAGCGCGACCAATGCTACTCTTTACCACCGTGGCGCGGCGGCGGATTATGATGCGTGGGGCGTGCCGGGCTGGGGCGCAGCTGACGTGCTGCCATGGTTCGTTAAGGCCGAAACCAACGCGGAGTTTGCGGCGGGCAAATATCACGGCGCAGGTGGTAACATGCGCGTTGAGAATCCGCGCTACTCCAACCCGCAG CTGCACGGTGCTTTCTTTGCAGCTGCGCAGCAGATGGGTCTGCCGCAGAATACCGACTTCAACAATTGGGATCAGGATCATGCAGGCTTTGGCACTTTTCAGGTTATGCAGGAAAAAGGCACCCGCGCTGATATGTACCGCCAGTATCTTAAACCAGCTTCTTGGTCGTCCGAACCTGCAGGTTCTGACCGGTGCGTCTGTGACCAAAGTTCATATCGATAAAGCTGGCGGTAAACCGCGTGCTCTGGGCGTAGA GTTTTCTCTGGATGGTCCGGCTGGTGAACGTATGGCAGCAGAGCTGGCGCGGCGGTGAAGTTCTCATGTGCGCTGGCCGTGCATAGCCCGCACATTCTGCAGCTGTCTGGCGTTGGTTCGGCGGCTACTCTGGCAGACCACGGCATCGCAGCAGTGGCAGATCTGCCAGGTGTTGGTGCGAACATGCAGGACCAGCCGGCCTGCCTGACAGCGGCTCCCCTGAAAGACAAATACGATGGCATTTCGCTGACCGA TCATATCTATAATAGCAAAGGCCAGATTCGCAAACGCGCTATCGCGTCCTACCTGCTTCAGGGTTAAAGGTGGTCTGACGTCAACTGGCTGCGACCGTGGCGCGTTTGTACGTACCGCAGGCCAGGCACTGCCGGACCTGCAGGTGCGTTTCGTGCCAGGCATGGCACTGGATGCAGATGGTGTGTCCACCTACGTCCGTTTCGCAAAATTTCAGTCTCAGGGCCTGAAATGGCCGTCTTGGCATCACCGTACAGCTT ATTGCGTGTCGCCCGCACAGCAAAGGTTCTGTTGGCCTGAAAAACGCGGACCCGTTCACCCCGCCGAAACTGCGTCCGGGCTACCTGACCGACAAAGCGGGTGCGGATCTGGCGACCCTGCGCTCTGGTGTTCATTGGGCCCGTGATCTGGCATCTAGCGGTCCGCTGAGCGAATTTCTTGAAGGCGAACTGTTTCCGGGTAGCCAAGTTGTTTCCGATGATGATATTGATTCTTACATTCGTCGTACCATTCACTCCAGCAA CGCGATTGTGGGCACCTGTCGTATGGGCGCGGCGGGTGAAGCGGGTTGTTGTGGATAACCAGCTGCGCGTTCAGGGTGTTGATGGTCTGCGTGTTGTTGACGCGAGCGTAATGCCGCGTATCCCAGGTGGTCAGGTGGGTGCCGGTTGTGATGCTGGCCGAACGTGCAGCAGCGATGCTGACCGGTCAGGCAGCGCTGGCTGGTGCTAGCGCTGCAGCTCCGCCGACCCCGGTCGCGGCT

SEQ ID NO.4 (amino acid sequence of mutant of fatty acid photodecarboxylase Mc FAP):

RASAVDDIQRALSTAGSPVSGKQYDYILVGGGTAACVLANRLTADGSKRVLVLEAGADNVSRDVKVPAAITRLFRSPLDWNLFSELQEQLAARQIYMARGRLLGGSSATNATLYHRGAAADYDAWGVPGWGAADVLPWFVKAETNAEFAAGKYHGAGGNMRVENPRYSNPQLHGAFFAAAQQMGLPQNTDFNNWDQDHAGFGTFQVMQEKGTRAD MYRQYLKPALGRPNLQVLTGASVTKVHIDKAGGKPRALGVEFSLDGPAGERMAAELAPGGEVLMCAGAVHSPHILQLSGVGSAATLADHGIAAVADLPGVGANMQDQPACLTAAPLKDKYDGISLTDHIYNSKGQIRKRAIASYLLQGKGGLTSTGCDRGAFVRTAGQALPDLQVRFVPGMALDADGVSTYVRFAKFQSQGLKWPSGI TVQLIACRPHSKGSVGLKNADPFTPPKLRPGYLTDKAGADLATLRSGVHWARDLASSGPLSEFLEGELFPGSQVVSDDDIDSYIRRTIHSSNAIVGTCRMGAAGEAGVVVDNQLRVQGVDGLRVVDASVMPRIPGGQVGAPVVMLAERAAAMLTGQAALAGASAAAPPTPVAA

In the following examples, the contents of fatty acids and alkanes before and after the decarboxylation reaction were detected by the Agilent 7890B gas chromatography system (Agilent Technologies, Palo Alto, CA, USA), and the chromatographic analysis column was KB-FFAP (30 m × 0.25 mm, 0.25 μm). , the specific chromatographic analysis method is: injection volume: 1 μL; injector temperature: 250°C; split ratio: 30:1; detector temperature: 280°C; temperature rise program: initial temperature is 110°C, maintained for 3.4 minutes, Then increase to 190°C at a rate of 25°C min ^-1 , hold for 2.1 min, then increase again to 230°C at a rate of 25°C min ^-1 , hold for 2 min, and finally increase to 250°C at a rate of 30°C min ^-1 , keep for 12 minutes. Each fatty acid and alkane standard was used to characterize the chromatographic peak time, and the above standards were used to prepare standard solutions with different concentrations, n-octanol was used as the internal standard, and the standard curve was obtained through gas phase detection for quantitative calculation.

In the following examples, plasmid pET28a- Mc FAP was synthesized by Sangon Bioengineering (Shanghai) Co., Ltd.; empty plasmid pET28a was stored in the applicant's laboratory; Escherichia coli BL21 (DE3) competent cells were purchased from Weidi Biotechnology Co., Ltd.; plasmid extraction kit, purchased from Sangon Bioengineering (Shanghai) Co., Ltd.; all other chemicals were purchased from Sigma-Aldrich, TCI or Aladdin Company, which have the highest purity and can be used without further purification.

The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

Example 1 Preparation of Mc FAP@ E. coli and verification of photoenzymatic decarboxylation of catalyzed fatty acids

The recombinant E.coli BL21 (DE3) strain containing plasmid pET28a- Mc FAP was cultured at 37°C in Terrific Broth (TB) containing 50 μg/mL kanamycin. When the OD ₆₀₀ reached 0.7 -0.8, 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) was added, and the cells were incubated at 17°C for 20 h. Collect the bacteria by centrifugation at 4000 rpm for 30 minutes at 4°C; wash with Tris-HCl buffer (50 mM, pH 8, containing 100mM NaCl), and centrifuge again (10000 rpm, 20min, 4°C); press the cell pellet according to 1: 2 (w/v) was suspended in the same buffer, added with 1 mM phenylmethylsulfonyl chloride (PMSF) and 5% glycerol (w/v), frozen in liquid nitrogen, and stored at -80°C until use.

In order to verify the catalytic decarboxylation effect of Mc FAP, E. coli cells containing the empty plasmid pET28a vector (denoted as empty WC) were prepared according to the same method.

Add 500 μL of Mc FAP@ E. coli with a wet mass concentration of 0.5 g/mL, 300 μL of 170 mM fatty acid DMSO solution, and 200 μL of Tris-HCl buffer (100mM, pH 8.0) into a 5 mL transparent reaction bottle , the total reaction volume is 1mL; then place it in a homemade photocatalytic reaction device (add a blue light irradiation device to the ordinary catalytic reaction device), and react under 500 rpm, 30°C, blue light (10 W, 220 V) irradiation 12 h; after the reaction, take the reaction mixture into a 2mL EP tube, add 1 mL of ethyl acetate solution of 25 mM n-octanol internal standard for extraction, that is, the extraction volume ratio is 1:1, and the extracted mixture is centrifuged at 11000 rpm for 4 After 1 min, take the upper organic phase and put it into a 2 mL chromatography bottle for GC analysis. The same reaction system was placed in the dark with other conditions unchanged to catalyze the decarboxylation of fatty acids.

Replace Mc FAP@ E. coli with empty WC and keep other conditions unchanged to catalyze the decarboxylation of fatty acids.

The experimental results are shown in Figure 1. As can be seen from Figure 1, empty WC has no decarboxylation effect on fatty acids, Mc FAP@ E. coli has no decarboxylation effect on fatty acids under blue light conditions, and Mc FAP@ E. coli has a decarboxylation effect on fatty acids under blue light conditions, thus verifying that Mc FAP@ E. coli catalyzes the decarboxylation reaction of fatty acid substrates under blue light conditions.

Example 2 Construction and purification of Mc FAP-S mutant

The full length of the Mc FAP gene is 3438 bp, with a total of 1146 amino acids. From the 551st amino acid from the N terminus to the C terminus, a total of 596 amino acids are the constructed mutant Mc FAP-S. The amino acid sequence is shown in SEQ ID NO.4 , the nucleotide sequence is shown in SEQ ID NO.3.

By designing primers (as shown in Table 1), using the Mc FAP gene as a template, the target gene (1788 bp) and pET28a vector (5362 bp) were obtained through PCR amplification (the system and procedures are shown in Table 2).

Table 1 Primers used to construct Mc FAP-S

Table 2 PCR reaction system and procedures

Recover the amplified target gene and vector according to Sangon SanPrep column PCR product gel recovery operation manual. Measure the DNA concentration of the target gene and vector obtained above and perform seamless cloning according to the requirements of the seamless cloning kit (reaction system and The procedure is shown in Table 2). After seamless cloning, the resulting plasmid was transformed and cultured, and the sample was sent for sequencing. The sequencing result was correctly the constructed deletion mutant Mc FAP-S.

The instruments, chromatography columns, and purification chromatography columns used for the purification of Mc FAP-S are: HisPrep ^TM FF16/10; HiPrep ^TM 26/10 salt exchange column. First use the loading buffer (50 mM Tris-HCl 300 mM NaCl 10mM imidazole 5% (v/v) glycerol pH 9) to equilibrate the chromatography column. After equilibrium, pump the crude enzyme solution into the column. After the loading is completed, use the loading buffer. liquid to equilibrium. Then use elution buffer (50mM Tris-HCl, 300mM NaCl, 500mM imidazole, 5% (v/v) glycerol, pH 9) for elution, collect peak samples, and detect by SDS-PAGE electrophoresis. The sample containing the peak corresponding to the target protein is salt-exchanged using a salt-exchange column. Add the protein to the salt-exchange column balanced with salt-exchange buffer (50 mM Tris-HCl 150 mM NaCl 5% (v/v) glycerol pH 9), then continue elution with the salt-exchange buffer, and collect the elution The protein is concentrated and aliquoted, pre-frozen in liquid nitrogen, and stored at -80°C for later use. The flow rate throughout the entire process is 5 mL min ^-1 .

The SDS-PAGE protein profile of Mc FAP-S after purification with a nickel column and elution with 0.5 M imidazole is shown in Figure 2. From Figure 2, it can be seen that Mc FAP-S has achieved good expression and purification effects.

Example 3 Characterization of enzymatic properties of Mc FAP-S

Definition of FAP enzyme activity: Under the conditions of 30°C, 500 rpm, blue light (10 W, 220 V), the amount of enzyme required to catalyze the reaction of 1 μmol n-octanoic acid within 1 minute to generate 1 μmol n-heptane is defined as 1 U.

1. Optimize reaction time

In this example, 12 reaction times are selected to optimize the Mc FAP-S catalyzed n-octanoic acid decarboxylation process (5min, 10min, 15min, 20min, 25min, 30min, 35min, 40min, 45min, 50min, 55min and 60min), in which 150 μL 140 mM n-octanoic acid DMSO solution (the concentration of n-octanoic acid in the reaction system is 20 mM, the DMSO addition amount is 15%), the enzyme addition amount is 40 μM, and Tris-HCl buffer (100 mM, pH 9.0) makes up the total reaction volume 1 mL into a 5 mL transparent reaction bottle. Then place it in a self-made photocatalytic reaction device and react for a certain period of time under 500 rpm, 30°C, blue light (10 W, 220 V); after the reaction is completed, take the reaction mixture into a 2 mL EP tube and add 1 mL of ethyl acetate solution of 25 mM n-octanol internal standard was used for extraction, that is, the extraction volume ratio was 1:1. After the extracted mixture was centrifuged at 11000 rpm for 4 min, the upper organic phase was taken and placed in a 2 mL chromatographic bottle for GC analysis.

The results are shown in Figure 3. Mc FAP-S catalyzed the decarboxylation of n-octanoic acid quickly within 5 minutes. After 5 minutes, the conversion rate increased steadily, and the conversion rate reached 95% in 30 minutes.

2. Optimize the amount of reaction enzyme

In this example, 4 enzyme dosages are selected to optimize the Mc FAP-S catalyzed n-octanoic acid decarboxylation process (the reaction enzyme dosages are final concentrations of 6 μM, 12 μM, 24 μM, and 36 μM respectively). Other conditions and treatment methods remain unchanged, and the steps are the same as above. .

The results are shown in Figure 4. As the amount of enzyme in the reaction system increases, the reaction conversion rate increases.

3. Optimize reaction substrate concentration

In this example, 5 substrate concentrations are selected to optimize the Mc FAP-S catalyzed n-octanoic acid decarboxylation process (the reaction substrate concentrations are 10mM, 20mM, 30mM, 40mM, and 50mM respectively). Other conditions and treatment methods remain unchanged, and the steps are the same as above. .

The results are shown in Figure 5. As the substrate concentration in the reaction system increases, the reaction conversion rate decreases, but the actual production rate remains unchanged.

4. Optimize the reaction temperature

In this example, 5 reaction temperatures are selected to optimize the Mc FAP-S catalyzed n-octanoic acid decarboxylation process (reaction temperatures are 20°C, 30°C, 40°C, 45°C, and 50°C respectively). Other conditions and treatment methods remain unchanged, and the steps Same as above.

The results are shown in Figure 6. The optimal temperature for Mc FAP-S to catalyze the decarboxylation of n-octanoic acid is 40°C. The relative enzyme activity is more than 80% between 30°C and 45°C. The relative enzyme activity decreases rapidly when the temperature is higher than 45°C.

5. Optimize reaction pH

In this example, 5 pH values were selected to optimize the Mc FAP-S catalyzed n-octanoic acid decarboxylation process (the pH values of the reaction system were 6, 7, 8, 9, and 10 respectively). The other conditions and treatment methods remained unchanged, and the steps were the same as above.

The results are shown in Figure 7. The optimal temperature for Mc FAP-S to catalyze the decarboxylation of n-octanoic acid is 8~9, but the conversion rate is more than 80% in the pH range of 6 to 10, that is, Mc FAP-S catalyzes the decarboxylation of n-octanoic acid. It has good catalytic effect in the pH range of 6~10.

6. Examine storage stability

In this example, the storage stability of Mc FAP-S catalyzing the decarboxylation of n-octanoic acid was selected to be examined under dark conditions at 4°C, and 12 storage times were selected to examine the residual enzyme activity of Mc FAP-S catalyzing the decarboxylation of n-octanoic acid (the storage time is 10 min, 30 min, 1 h, 3 h, 6 h, 12 h, 1 d, 2 d, 3 d, 5 d, 7 d, 10 d), other conditions and treatment methods remain unchanged, and the steps are the same as above.

The results are shown in Figure 8. After Mc FAP-S was stored under dark conditions at 4°C for 10 days, the residual activity of catalyzing the decarboxylation of n-octanoic acid was still more than 70%.

7. Optimize pH tolerance

In this example, 5 pH values are selected to incubate the Mc FAP-S enzyme solution (the pH of the incubation system is 6, 7, 8, 9, and 10 respectively). The incubation conditions are 4°C and light-proof conditions, and other conditions and treatment methods remain unchanged. The steps are the same as above.

The results are shown in Figure 9. Under dark conditions at 4°C, the residual activity of Mc FAP-S in catalyzing the decarboxylation of n-octanoic acid after incubation for 5 days in the pH range of 6~10 is still more than 50%. At the same time, within the range of pH 6~10, pH had no significant effect on the enzyme activity of Mc FAP-S, and the residual activity after incubation at pH 6~8 was slightly better than that at pH 9~10.

8. Examine the factors of light inactivation

This example chooses to examine the storage stability of Mc FAP-S's enzyme activity that catalyzes the decarboxylation of n-octanoic acid when stored at room temperature under different lighting conditions. Five different lighting environments are selected to incubate the Mc FAP-S pure enzyme solution (blue light irradiation, sunlight irradiation, dark Storage, red light irradiation, and system containing 5% DMSO and 10 mM n-octanoic acid were incubated with red light irradiation), and 5 storage times were selected to examine the residual enzyme activity of Mc FAP-S catalyzing the decarboxylation of n-octanoic acid (the storage time was 10 min, 30 min, 1 h, 2 h, 3 h), other conditions and treatment methods remain unchanged, and the steps are the same as above.

The results are shown in Figure 10. Under normal temperature conditions, blue light irradiation has the greatest impact on the pure enzyme activity of Mc FAP-S. The residual enzyme activity after 10 minutes is less than 10%; after 3 hours of sunlight irradiation, the residual enzyme activity of Mc FAP-S is less than 50%. %; Mc FAP-S under dark conditions and red light irradiation conditions, the residual enzyme activity is still above 90% after 3 hours; while adding 10 mM n-octanoic acid substrate and incubating with 5% DMSO, Mc FAP-S, After 3 hours of sunlight irradiation, the residual enzyme activity was still more than 90%, that is, the addition of substrate and co-incubation significantly inhibited the photoinactivation of Mc FAP-S. Based on the above reasons, in the following examples, the whole-cell catalytic form was selected to conduct substrate expansion research experiments on catalyzing saturated fatty acids with different chain lengths.

Example 4 Research on substrate expansion of Mc FAP@ E. coli and Mc FAP-S@ E. coli catalyzed fatty acids

Different fatty acid substrates (saturated linear fatty acids with 6 to 18 carbon atoms) were selected for photoenzyme-catalyzed deacidification. The reaction time was 30 minutes. Other relevant reaction conditions were carried out according to the photoenzyme decarboxylation verification experiment in Example 1.

The results are shown in Figure 11. Both Mc FAP@ E. coli and Mc FAP-S@ E. coli can catalyze the decarboxylation effect of C6:0~C18:0 fatty acids. With the same amount of whole cells added, Mc FAP-S The efficiency of @ E. coli in catalyzing the decarboxylation of C7:0~C12:0 fatty acids is significantly higher than that of Mc FAP@ E. coli in catalyzing the decarboxylation of C7:0~C12:0 fatty acids.

Example 5 Comparison of Mc FAP@ E. coli and Cv FAP@ E. coli catalytic decarboxylation efficiency of palmitate

In this example, 6 reaction times were selected to compare the decarboxylation efficiency of Mc FAP@ E. coli and Cv FAP@ E. coli (preparation method is the same as Mc FAP@ E. coli ) in catalyzing palmitic acid (1, 2, 3, 4, 5 and 6h), the addition amount of Mc FAP@ E. coli and Cv FAP@ E. coli was 0.25 g/mL, and other relevant reaction conditions were carried out according to the photoenzyme decarboxylation verification experiment of Example 1.

The results are shown in Figure 12. After Mc FAP@ E. coli catalyzes the decarboxylation reaction of palmitic acid for 6 hours, the catalytic conversion rate of palmitic acid decarboxylation can reach more than 90%. Under the same reaction conditions, Cv FAP @E. coli reacted for 6 hours to generate 30 mM product, with a conversion rate of only about 60%. It can be seen that with the same amount of whole cells added, the decarboxylation efficiency of Mc FAP@ E. coli in catalyzing palmitic acid is significantly higher than that of Cv FAP @E. coli .

The technical features of the above-described embodiments can be combined in any way. To simplify the description, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, All should be considered to be within the scope of this manual.

The above-mentioned embodiments only express several implementation modes of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the invention. It should be noted that, for those of ordinary skill in the art, several modifications and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the scope of protection of the patent of the present invention should be determined by the appended claims.

sequence list

<110> South China University of Technology, Guangdong Youzi Biomanufacturing Research Institute Co., Ltd.

<120> Mutants of fatty acid photodecarboxylase McFAP and their applications

<130> 1

<160> 8

<170> SIPOSequenceListing 1.0

<210> 1

<211> 3438

<212> DNA

<213> Artificial Sequence

<400> 1

atggctgaaa tggcaggtgg tggtgaaggt gatggtatgc tgatgggcgg cgcgggtagc 60

gcaaacacta ccgacgcgtg ttatagcgat ccgtctaatc cggattgcgc agcgtttgag 120

cgctccgacg atgattgggc ggcggacatc gaactgctgt gctctgcgat gccgttcatg 180

ccgggctgca ccctggcgga acagtgcatg aatggcaccg ccgccggtga atattgcgaa 240

atgtccagtc tggctggtaa catctgtctg gatatgccgg gcatgaaagg ctgtgaggca 300

tggaacgcac tgtgtggcgc ggccagcgcc gttgaacagt gttcctctcc gggcccggtt 360

gtggcactcc cgaccaccgc gctggccaaa gaaggcctgg aatctctgtg ctctacccat 420

tgtatggacg gttgcccaga ctgtgaaatg ggtaaactgt ggaacacctg caccgacccg 480

ctgagtgttc tggcgtggat gtgctacgca atgccggaca tgccggaatg tctggctgct 540

ccgcagggct ccggcatggt ggtggcttgc ggtgacgctg aggttgcagc taccttcccg 600

ctggtgtgcg cgcaaccgcc gaccccggcg gctaactttc agcaccgcct tcgtacctgc 660

cgtaccgccg gcgttgcggc atccgcatcc ggttctccgg cagtcactat ggctggcctg 720

tcaactgttc tggcagtact ggcactgctg ccatccccgg ttgctatggc catgactccg 780

atgccgaccc cggcgctcgc tccgggcccg gcgatcgacg atatcggcgg taactgcccg 840

ctgctgggtc gcggtaacat ggaagctccg tgttatagcg acccgagcgc ggcagcatgc 900

gtttcctttg aacgcagcga tgctggctgg gcggatgacc tgagtcagct gtgttctgcg 960

atgccgtatg ctgttggctg ctggctgtgg cacttgtgta aaaccggcgc agcaagcggg 1020

acttactgtg cgctgccgtc cctgaccgcg aacgtatgtg ttgacgcacc gctggtgaac 1080

gctacatcag cgccgggctg cgaagcgtgg gccgcactgt gcggcgccca gggtagcgtc 1140

gttgcgcagt gctctgcgcc aggcccgctg ccggacatca tcaacaccct gaccacccgt 1200

gacggcatca actccctctg cggtatgcat tacatggatg ggtgtaacga atgtacccct 1260

cacgaaggtc cggcagttca cgacttcgcg gcctgtgctg atccgggtcc actgccgact 1320

ctggcccacc agtgttacgc gatgcctgaa atgggtgaat gtacccagac tggtattacc 1380

gcaatgtgca gcggcgctga agctcgtgcg acctttccga ccgtttgcgt ggatccacct 1440

aacccgacga cactggcgcc ggcgcctgcc gtttctgcct gcgatgttgc ggcgggtgct 1500

ggcgcgccac cagcggcgtc tgcccgtccg gcgtcgcaca gccgcgcgtc actggttgcc 1560

tcccgtagcg gcttctgcgc gccttccccg gcgctgcgct ctcagcgcac ctctactgtc 1620

gcgccggcgc gccgcgccgc gtcggcgccg cgtgcgagcg cagttgacga tattcaacgt 1680

gctctgagca ccgctggaag cccggtatcc ggtaaacagt acgattacat cctggtgggt 1740

ggcggcaccg cggcatgcgt tttggctaac cgtttaaccg cggacggtag caaacgtgta 1800

ctggtgctgg aagcgggtgc ggacaacgtg agccgcgatg ttaaagtccc ggctgcgatc 1860

acccgtttgt tccgttcacc gttggattgg aacttgttca gcgaattgca ggaacagctg 1920

gctgcacgtc agatctatat ggctcgcggc cgcttgctgg gtgggtctag cgcgaccaat 1980

gctactcttt accaccgtgg cgcggcggcg gattatgatg cgtggggcgt gccgggctgg 2040

ggcgcagctg acgtgctgcc atggttcgtt aaggccgaaa ccaacgcgga gtttgcggcg 2100

ggcaaatatc acggcgcagg tggtaacatg cgcgttgaga atccgcgcta ctccaacccg 2160

cagctgcacg gtgctttctt tgcagctgcg cagcagatgg gtctgccgca gaataccgac 2220

ttcaacaatt gggatcagga tcatgcaggc tttggcactt ttcaggttat gcaggaaaaa 2280

ggcacccgcg ctgatatgta ccgccagtat cttaaaccag ctcttggtcg tccgaacctg 2340

caggttctga ccggtgcgtc tgtgaccaaa gttcatatcg ataaagctgg cggtaaaccg 2400

cgtgctctgg gcgtagagtt ttctctggat ggtccggctg gtgaacgtat ggcagcagag 2460

ctggcgccgg gcggtgaagt tctcatgtgc gctggcgccg tgcatagccc gcacattctg 2520

cagctgtctg gcgttggttc ggcggctact ctggcagacc acggcatcgc agcagtggca 2580

gatctgccag gtgttggtgc gaacatgcag gaccagccgg cctgcctgac agcggctccc 2640

ctgaaagaca aatacgatgg catttcgctg accgatcata tctataatag caaaggccag 2700

attcgcaaac gcgctatcgc gtcctacctg cttcagggta aaggtggtct gacgtcaact 2760

ggctgcgacc gtggcgcgtt tgtacgtacc gcaggccagg cactgccgga cctgcaggtg 2820

cgtttcgtgc caggcatggc actggatgca gatggtgtgt ccacctacgt ccgtttcgca 2880

aaatttcagt ctcagggcct gaaatggccg tctggcatca ccgtacagct tattgcgtgt 2940

cgcccgcaca gcaaaggttc tgttggcctg aaaaacgcgg acccgttcac cccgccgaaa 3000

ctgcgtccgg gctacctgac cgacaaagcg ggtgcggatc tggcgaccct gcgctctggt 3060

gttcattggg cccgtgatct ggcatctagc ggtccgctga gcgaatttct tgaaggcgaa 3120

ctgtttccgg gtagccaagt tgtttccgat gatgatattg attcttacat tcgtcgtacc 3180

attcactcca gcaacgcgat tgtgggcacc tgtcgtatgg gcgcggcggg tgaagcgggt 3240

gttgttgtgg ataaccagct gcgcgttcag ggtgttgatg gtctgcgtgt tgttgacgcg 3300

agcgtaatgc cgcgtatccc aggtggtcag gtgggtgcgc cggttgtgat gctggccgaa 3360

cgtgcagcag cgatgctgac cggtcaggca gcgctggctg gtgctagcgc tgcagctccg 3420

ccgaccccgg tcgcggct 3438

<210> 2

<211> 1146

<212> PRT

<213> Artificial Sequence

<400> 2

Met Ala Glu Met Ala Gly Gly Gly Glu Gly Asp Gly Met Leu Met Gly

1 5 10 15

Gly Ala Gly Ser Ala Asn Thr Thr Asp Ala Cys Tyr Ser Asp Pro Ser

20 25 30

Asn Pro Asp Cys Ala Ala Phe Glu Arg Ser Asp Asp Asp Trp Ala Ala

35 40 45

Asp Ile Glu Leu Leu Cys Ser Ala Met Pro Phe Met Pro Gly Cys Thr

50 55 60

Leu Ala Glu Gln Cys Met Asn Gly Thr Ala Ala Gly Glu Tyr Cys Glu

65 70 75 80

Met Ser Ser Leu Ala Gly Asn Ile Cys Leu Asp Met Pro Gly Met Lys

85 90 95

Gly Cys Glu Ala Trp Asn Ala Leu Cys Gly Ala Ala Ser Ala Val Glu

100 105 110

Gln Cys Ser Ser Pro Gly Pro Val Val Ala Leu Pro Thr Thr Ala Leu

115 120 125

Ala Lys Glu Gly Leu Glu Ser Leu Cys Ser Thr His Cys Met Asp Gly

130 135 140

Cys Pro Asp Cys Glu Met Gly Lys Leu Trp Asn Thr Cys Thr Asp Pro

145 150 155 160

Leu Ser Val Leu Ala Trp Met Cys Tyr Ala Met Pro Asp Met Pro Glu

165 170 175

Cys Leu Ala Ala Pro Gln Gly Ser Gly Met Val Val Ala Cys Gly Asp

180 185 190

Ala Glu Val Ala Ala Thr Phe Pro Leu Val Cys Ala Gln Pro Pro Thr

195 200 205

Pro Ala Ala Asn Phe Gln His Arg Leu Arg Thr Cys Arg Thr Ala Gly

210 215 220

Val Ala Ala Ser Ala Ser Gly Ser Pro Ala Val Thr Met Ala Gly Leu

225 230 235 240

Ser Thr Val Leu Ala Val Leu Ala Leu Leu Pro Ser Pro Val Ala Met

245 250 255

Ala Met Thr Pro Met Pro Thr Pro Ala Leu Ala Pro Gly Pro Ala Ile

260 265 270

Asp Asp Ile Gly Gly Asn Cys Pro Leu Leu Gly Arg Gly Asn Met Glu

275 280 285

Ala Pro Cys Tyr Ser Asp Pro Ser Ala Ala Ala Cys Val Ser Phe Glu

290 295 300

Arg Ser Asp Ala Gly Trp Ala Asp Asp Leu Ser Gln Leu Cys Ser Ala

305 310 315 320

Met Pro Tyr Ala Val Gly Cys Trp Leu Trp His Leu Cys Lys Thr Gly

325 330 335

Ala Ala Ser Gly Thr Tyr Cys Ala Leu Pro Ser Leu Thr Ala Asn Val

340 345 350

Cys Val Asp Ala Pro Leu Val Asn Ala Thr Ser Ala Pro Gly Cys Glu

355 360 365

Ala Trp Ala Ala Leu Cys Gly Ala Gln Gly Ser Val Val Ala Gln Cys

370 375 380

Ser Ala Pro Gly Pro Leu Pro Asp Ile Ile Asn Thr Leu Thr Thr Arg

385 390 395 400

Asp Gly Ile Asn Ser Leu Cys Gly Met His Tyr Met Asp Gly Cys Asn

405 410 415

Glu Cys Thr Pro His Glu Gly Pro Ala Val His Asp Phe Ala Ala Cys

420 425 430

Ala Asp Pro Gly Pro Leu Pro Thr Leu Ala His Gln Cys Tyr Ala Met

435 440 445

Pro Glu Met Gly Glu Cys Thr Gln Thr Gly Ile Thr Ala Met Cys Ser

450 455 460

Gly Ala Glu Ala Arg Ala Thr Phe Pro Thr Val Cys Val Asp Pro Pro

465 470 475 480

Asn Pro Thr Thr Leu Ala Pro Ala Pro Ala Val Ser Ala Cys Asp Val

485 490 495

Ala Ala Gly Ala Gly Ala Pro Pro Ala Ala Ser Ala Arg Pro Ala Ser

500 505 510

His Ser Arg Ala Ser Leu Val Ala Ser Arg Ser Gly Phe Cys Ala Pro

515 520 525

Ser Pro Ala Leu Arg Ser Gln Arg Thr Ser Ser Thr Val Ala Pro Ala Arg

530 535 540

Arg Ala Ala Ser Ala Pro Arg Ala Ser Ala Val Asp Asp Ile Gln Arg

545 550 555 560

Ala Leu Ser Thr Ala Gly Ser Pro Val Ser Gly Lys Gln Tyr Asp Tyr

565 570 575

Ile Leu Val Gly Gly Gly Thr Ala Ala Cys Val Leu Ala Asn Arg Leu

580 585 590

Thr Ala Asp Gly Ser Lys Arg Val Leu Val Leu Glu Ala Gly Ala Asp

595 600 605

Asn Val Ser Arg Asp Val Lys Val Pro Ala Ala Ile Thr Arg Leu Phe

610 615 620

Arg Ser Pro Leu Asp Trp Asn Leu Phe Ser Glu Leu Gln Glu Gln Leu

625 630 635 640

Ala Ala Arg Gln Ile Tyr Met Ala Arg Gly Arg Leu Leu Gly Gly Ser

645 650 655

Ser Ala Thr Asn Ala Thr Leu Tyr His Arg Gly Ala Ala Ala Asp Tyr

660 665 670

Asp Ala Trp Gly Val Pro Gly Trp Gly Ala Ala Asp Val Leu Pro Trp

675 680 685

Phe Val Lys Ala Glu Thr Asn Ala Glu Phe Ala Ala Gly Lys Tyr His

690 695 700

Gly Ala Gly Gly Asn Met Arg Val Glu Asn Pro Arg Tyr Ser Asn Pro

705 710 715 720

Gln Leu His Gly Ala Phe Phe Ala Ala Ala Gln Gln Met Gly Leu Pro

725 730 735

Gln Asn Thr Asp Phe Asn Asn Trp Asp Gln Asp His Ala Gly Phe Gly

740 745 750

Thr Phe Gln Val Met Gln Glu Lys Gly Thr Arg Ala Asp Met Tyr Arg

755 760 765

Gln Tyr Leu Lys Pro Ala Leu Gly Arg Pro Asn Leu Gln Val Leu Thr

770 775 780

Gly Ala Ser Val Thr Lys Val His Ile Asp Lys Ala Gly Gly Lys Pro

785 790 795 800

Arg Ala Leu Gly Val Glu Phe Ser Leu Asp Gly Pro Ala Gly Glu Arg

805 810 815

Met Ala Ala Glu Leu Ala Pro Gly Gly Glu Val Leu Met Cys Ala Gly

820 825 830

Ala Val His Ser Pro His Ile Leu Gln Leu Ser Gly Val Gly Ser Ala

835 840 845

Ala Thr Leu Ala Asp His Gly Ile Ala Ala Val Ala Asp Leu Pro Gly

850 855 860

Val Gly Ala Asn Met Gln Asp Gln Pro Ala Cys Leu Thr Ala Ala Pro

865 870 875 880

Leu Lys Asp Lys Tyr Asp Gly Ile Ser Leu Thr Asp His Ile Tyr Asn

885 890 895

Ser Lys Gly Gln Ile Arg Lys Arg Ala Ile Ala Ser Tyr Leu Leu Gln

900 905 910

Gly Lys Gly Gly Leu Thr Ser Thr Gly Cys Asp Arg Gly Ala Phe Val

915 920 925

Arg Thr Ala Gly Gln Ala Leu Pro Asp Leu Gln Val Arg Phe Val Pro

930 935 940

Gly Met Ala Leu Asp Ala Asp Gly Val Ser Thr Tyr Val Arg Phe Ala

945 950 955 960

Lys Phe Gln Ser Gln Gly Leu Lys Trp Pro Ser Gly Ile Thr Val Gln

965 970 975

Leu Ile Ala Cys Arg Pro His Ser Lys Gly Ser Val Gly Leu Lys Asn

980 985 990

Ala Asp Pro Phe Thr Pro Pro Lys Leu Arg Pro Gly Tyr Leu Thr Asp

995 1000 1005

Lys Ala Gly Ala Asp Leu Ala Thr Leu Arg Ser Gly Val His Trp Ala

1010 1015 1020

Arg Asp Leu Ala Ser Ser Gly Pro Leu Ser Glu Phe Leu Glu Gly Glu

1025 1030 1035 1040

Leu Phe Pro Gly Ser Gln Val Val Ser Asp Asp Asp Ile Asp Ser Tyr

1045 1050 1055

Ile Arg Arg Thr Ile His Ser Ser Asn Ala Ile Val Gly Thr Cys Arg

1060 1065 1070

Met Gly Ala Ala Gly Glu Ala Gly Val Val Val Asp Asn Gln Leu Arg

1075 1080 1085

Val Gln Gly Val Asp Gly Leu Arg Val Val Asp Ala Ser Val Met Pro

1090 1095 1100

Arg Ile Pro Gly Gly Gln Val Gly Ala Pro Val Val Met Leu Ala Glu

1105 1110 1115 1120

Arg Ala Ala Ala Met Leu Thr Gly Gln Ala Ala Leu Ala Gly Ala Ser

1125 1130 1135

Ala Ala Ala Pro Pro Thr Pro Val Ala Ala

1140 1145

<210> 3

<211> 1788

<212> DNA

<213> Artificial Sequence

<400> 3

cgtgcgagcg cagttgacga tattcaacgt gctctgagca ccgctggaag cccggtatcc 60

ggtaaacagt acgattacat cctggtgggt ggcggcaccg cggcatgcgt tttggctaac 120

cgtttaaccg cggacggtag caaacgtgta ctggtgctgg aagcgggtgc ggacaacgtg 180

agccgcgatg ttaaagtccc ggctgcgatc acccgtttgt tccgttcacc gttggattgg 240

aacttgttca gcgaattgca ggaacagctg gctgcacgtc agatctatat ggctcgcggc 300

cgcttgctgg gtgggtctag cgcgaccaat gctactcttt accaccgtgg cgcggcggcg 360

gattatgatg cgtggggcgt gccgggctgg ggcgcagctg acgtgctgcc atggttcgtt 420

aaggccgaaa ccaacgcgga gtttgcggcg ggcaaatatc acggcgcagg tggtaacatg 480

cgcgttgaga atccgcgcta ctccaacccg cagctgcacg gtgctttctt tgcagctgcg 540

cagcagatgg gtctgccgca gaataccgac ttcaacaatt gggatcagga tcatgcaggc 600

tttggcactt ttcaggttat gcaggaaaaa ggcacccgcg ctgatatgta ccgccagtat 660

cttaaaccag ctcttggtcg tccgaacctg caggttctga ccggtgcgtc tgtgaccaaa 720

gttcatatcg ataaagctgg cggtaaaccg cgtgctctgg gcgtagagtt ttctctggat 780

ggtccggctg gtgaacgtat ggcagcagag ctggcgccgg gcggtgaagt tctcatgtgc 840

gctggcgccg tgcatagccc gcacattctg cagctgtctg gcgttggttc ggcggctact 900

ctggcagacc acggcatcgc agcagtggca gatctgccag gtgttggtgc gaacatgcag 960

gaccagccgg cctgcctgac agcggctccc ctgaaagaca aatacgatgg catttcgctg 1020

accgatcata tctataatag caaaggccag attcgcaaac gcgctatcgc gtcctacctg 1080

cttcagggta aaggtggtct gacgtcaact ggctgcgacc gtggcgcgtt tgtacgtacc 1140

gcaggccagg cactgccgga cctgcaggtg cgtttcgtgc caggcatggc actggatgca 1200

gatggtgtgt ccacctacgt ccgtttcgca aaatttcagt ctcagggcct gaaatggccg 1260

tctggcatca ccgtacagct tattgcgtgt cgcccgcaca gcaaaggttc tgttggcctg 1320

aaaaacgcgg acccgttcac cccgccgaaa ctgcgtccgg gctacctgac cgacaaagcg 1380

ggtgcggatc tggcgaccct gcgctctggt gttcattggg cccgtgatct ggcatctagc 1440

ggtccgctga gcgaatttct tgaaggcgaa ctgtttccgg gtagccaagt tgtttccgat 1500

gatgatattg attcttacat tcgtcgtacc attcactcca gcaacgcgat tgtgggcacc 1560

tgtcgtatgg gcgcggcggg tgaagcgggt gttgttgtgg ataaccagct gcgcgttcag 1620

ggtgttgatg gtctgcgtgt tgttgacgcg agcgtaatgc cgcgtatccc aggtggtcag 1680

gtgggtgcgc cggttgtgat gctggccgaa cgtgcagcag cgatgctgac cggtcaggca 1740

gcgctggctg gtgctagcgc tgcagctccg ccgaccccgg tcgcggct 1788

<210> 4

<211> 596

<212> PRT

<213> Artificial Sequence

<400> 4

Arg Ala Ser Ala Val Asp Asp Ile Gln Arg Ala Leu Ser Thr Ala Gly

1 5 10 15

Ser Pro Val Ser Gly Lys Gln Tyr Asp Tyr Ile Leu Val Gly Gly Gly

20 25 30

Thr Ala Ala Cys Val Leu Ala Asn Arg Leu Thr Ala Asp Gly Ser Lys

35 40 45

Arg Val Leu Val Leu Glu Ala Gly Ala Asp Asn Val Ser Arg Asp Val

50 55 60

Lys Val Pro Ala Ala Ile Thr Arg Leu Phe Arg Ser Pro Leu Asp Trp

65 70 75 80

Asn Leu Phe Ser Glu Leu Gln Glu Gln Leu Ala Ala Arg Gln Ile Tyr

85 90 95

Met Ala Arg Gly Arg Leu Leu Gly Gly Ser Ser Ala Thr Asn Ala Thr

100 105 110

Leu Tyr His Arg Gly Ala Ala Ala Asp Tyr Asp Ala Trp Gly Val Pro

115 120 125

Gly Trp Gly Ala Ala Asp Val Leu Pro Trp Phe Val Lys Ala Glu Thr

130 135 140

Asn Ala Glu Phe Ala Ala Gly Lys Tyr His Gly Ala Gly Gly Asn Met

145 150 155 160

Arg Val Glu Asn Pro Arg Tyr Ser Asn Pro Gln Leu His Gly Ala Phe

165 170 175

Phe Ala Ala Ala Gln Gln Met Gly Leu Pro Gln Asn Thr Asp Phe Asn

180 185 190

Asn Trp Asp Gln Asp His Ala Gly Phe Gly Thr Phe Gln Val Met Gln

195 200 205

Glu Lys Gly Thr Arg Ala Asp Met Tyr Arg Gln Tyr Leu Lys Pro Ala

210 215 220

Leu Gly Arg Pro Asn Leu Gln Val Leu Thr Gly Ala Ser Val Thr Lys

225 230 235 240

Val His Ile Asp Lys Ala Gly Gly Lys Pro Arg Ala Leu Gly Val Glu

245 250 255

Phe Ser Leu Asp Gly Pro Ala Gly Glu Arg Met Ala Ala Glu Leu Ala

260 265 270

Pro Gly Gly Glu Val Leu Met Cys Ala Gly Ala Val His Ser Pro His

275 280 285

Ile Leu Gln Leu Ser Gly Val Gly Ser Ala Ala Thr Leu Ala Asp His

290 295 300

Gly Ile Ala Ala Val Ala Asp Leu Pro Gly Val Gly Ala Asn Met Gln

305 310 315 320

Asp Gln Pro Ala Cys Leu Thr Ala Ala Pro Leu Lys Asp Lys Tyr Asp

325 330 335

Gly Ile Ser Leu Thr Asp His Ile Tyr Asn Ser Lys Gly Gln Ile Arg

340 345 350

Lys Arg Ala Ile Ala Ser Tyr Leu Leu Gln Gly Lys Gly Gly Leu Thr

355 360 365

Ser Thr Gly Cys Asp Arg Gly Ala Phe Val Arg Thr Ala Gly Gln Ala

370 375 380

Leu Pro Asp Leu Gln Val Arg Phe Val Pro Gly Met Ala Leu Asp Ala

385 390 395 400

Asp Gly Val Ser Thr Tyr Val Arg Phe Ala Lys Phe Gln Ser Gln Gly

405 410 415

Leu Lys Trp Pro Ser Gly Ile Thr Val Gln Leu Ile Ala Cys Arg Pro

420 425 430

His Ser Lys Gly Ser Val Gly Leu Lys Asn Ala Asp Pro Phe Thr Pro

435 440 445

Pro Lys Leu Arg Pro Gly Tyr Leu Thr Asp Lys Ala Gly Ala Asp Leu

450 455 460

Ala Thr Leu Arg Ser Gly Val His Trp Ala Arg Asp Leu Ala Ser Ser

465 470 475 480

Gly Pro Leu Ser Glu Phe Leu Glu Gly Glu Leu Phe Pro Gly Ser Gln

485 490 495

Val Val Ser Asp Asp Asp Ile Asp Ser Tyr Ile Arg Arg Thr Ile His

500 505 510

Ser Ser Asn Ala Ile Val Gly Thr Cys Arg Met Gly Ala Ala Gly Glu

515 520 525

Ala Gly Val Val Val Asp Asn Gln Leu Arg Val Gln Gly Val Asp Gly

530 535 540

Leu Arg Val Val Asp Ala Ser Val Met Pro Arg Ile Pro Gly Gly Gln

545 550 555 560

Val Gly Ala Pro Val Val Met Leu Ala Glu Arg Ala Ala Ala Met Leu

565 570 575

Thr Gly Gln Ala Ala Leu Ala Gly Ala Ser Ala Ala Ala Pro Pro Thr

580 585 590

Pro Val Ala Ala

595

<210> 5

<211> 42

<212> DNA

<213> Artificial Sequence

<400> 5

atgggtcgcg gatccgaatt ccgtgcgagc gcagttgacg at 42

<210> 6

<211> 39

<212> DNA

<213> Artificial Sequence

<400> 6

tgcggccgca agcttgtcga cagccgcgac cggggtcgg 39

<210> 7

<211> 27

<212> DNA

<213> Artificial Sequence

<400> 7

gaattcggat ccgcgaccca tttgctg 27

<210> 8

<211> 23

<212> DNA

<213> Artificial Sequence

<400> 8

gtcgacaagc ttgcggccgc act 23

Claims (5)

1. Fatty acid light decarboxylaseMcUse of mutant of FAP for catalyzing decarboxylation of fatty acid, said fatty acid light decarboxylaseMcThe amino acid sequence of the FAP mutant is shown as SEQ ID NO.4, and the fatty acid is saturated straight-chain fatty acid with 7-8 carbon atoms.

2. Fatty acid light decarboxylaseMcThe application of the coding gene of the FAP mutant in catalyzing the decarboxylation of fatty acid is shown in SEQ ID NO.3, wherein the fatty acid is saturated straight-chain fatty acid with 7-8 carbon atoms.

3. Inserted with fatty acid light decarboxylaseMcThe application of a recombinant expression vector of a coding gene of a FAP mutant in catalyzing decarboxylation of fatty acid, wherein the nucleotide sequence of the coding gene is shown as SEQ ID NO.3, and the fatty acid is saturated straight-chain fatty acid with 7-8 carbon atoms.

4. Application of recombinant engineering strain transferred into recombinant expression vector with inserted fatty acid light decarboxylase in catalyzing fatty acid decarboxylationMcFAP mutant coding gene, nucleotide sequence of the coding gene is shown as SEQ ID NO.3, and the fat isThe acid is a saturated straight-chain fatty acid with 7-8 carbon atoms.

5. A method for catalyzing decarboxylation of fatty acid, which is characterized in that a fatty acid photo-decarboxylase is usedMcPerforming catalytic reaction on whole cells of the FAP mutant, wherein the fatty acid is saturated straight-chain fatty acid with 7-8 carbon atoms; the fatty acid light decarboxylaseMcThe amino acid sequence of the FAP mutant is shown as SEQ ID NO. 4.