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

Abstract

The invention discloses a mutant of fatty acid photo-decarboxylase McFAP and application thereof, wherein the amino acid sequence of the mutant of fatty acid photo-decarboxylase McFAP is shown as SEQ ID NO. 4. The mutant of the McFAP enriches the variety of FAP, fills the blank that the existing FAP cannot catalyze decarboxylation of saturated straight-chain fatty acid with 6-12 carbon atoms, has wider decarboxylation substrate spectrum and better decarboxylation effect, enables efficient and sustainable biosynthesis of fuel to be possible, and has wide industrial application prospect.

Description

Mutant of fatty acid light decarboxylase McFAP and application thereof

Technical Field

The invention belongs to the technical field of enzyme engineering, in particular to a fatty acid photodecarboxylation enzymeMcMutant of FAP and application thereof.

Background

Currently, biotechnology is continuously moving from medicine, agriculture, food to industrial fields (such as chemical industry, materials and energy sources). Gasoline, diesel, plastics, rubber, fiber and many large numbers of traditional petrochemical products are continually being replaced by industrial bio-manufactured products from renewable raw materials. The chemical industrial process with high temperature, high pressure and high pollution is continuously transferred to the biological processing process with mild condition, cleanness and environmental protection.

The exploration and practice of producing renewable biofuels and chemicals in an environmentally friendly manner has received great attention. The biomass is used as the raw material, the microorganism or enzyme is utilized to produce the fuel, the development and utilization of renewable biomass resources for directional conversion to produce the biogas and the liquid fuel are very wide in development prospect, and the technology becomes an important task of scientific research of researchers in related fields of various countries.

Enzymes found to date to be useful in the decarboxylation of fatty acids are mainly amino acid decarboxylases, haloperoxidases and fatty acid decarboxylations to terminal olefmic enzymes (OleT) _JE ) Etc., but most enzymes are not efficient in catalysis and require the addition of expensive cofactors and/or oxidants that tend to break unsaturated bonds of the lipid, a significant disadvantage in terms of application research. Therefore, the search for efficient novel decarboxylase is the key to breaking the bottleneck of biofuel production research.

Fatty acid light decarboxylase (fatty acid photodecarboxylase, FAP, EC 4.1.1.106) belongs to the family of glucose-methanol-choline (GMC) oxidoreductases, an optical driving enzyme that converts fatty acids to alk (en) enes using only blue light without the addition of expensive cofactors. The fatty acid can form various alkane (alkene) hydrocarbons by only removing one carboxyl, and is almost perfectly matched with the components of gasoline and diesel oil obtained by processing petroleum crude oil. The photocatalysis energy consumption is low, the process is clean, the on-off state of the reaction is convenient to regulate and control, the biological enzyme has strong catalysis specificity and mild conditions. Therefore, the optical driving enzyme FAP which combines the advantages of the two and meets the green development expectations becomes an emerging research hot spot. With the demand and preference of green energy, the preparation of alkane (alkene) hydrocarbon by decarboxylation of fatty acid becomes a key research route for developing biofuel, and has wide application prospect in the green chemical manufacturing process of biofuel.

Compared with the prior art, the FAP directly utilizes the light energy, is more energy-saving and environment-friendly, is simple and convenient, does not introduce double bonds at the tail end of a carbon chain, and is the product, namely the required alkane. FAP is taken as an optical driving enzyme for converting fatty acid into alkane (alkene) hydrocarbon by using blue light, the reaction condition for the decarboxylation reaction of fatty acid is mild, the conversion rate is high (more than 90 percent), the combustion heat value of the product is higher than that of ester fuel molecules, the byproduct is only carbon dioxide, and only light energy is needed in the process, so that the significance of environmental protection is very great, and the FAP represents a brand-new application field.

However, only the currently reported light decarboxylasesCvFAP、CrFAP、EsiFAP、GsuFAP、NgaFAP, in which only relatively intensive studies are madeCvFAP one, the rest only express the verified decarboxylation activityCvFAP and other currently reported photo-decarboxylases show obvious preference for saturated fatty acids with the carbon number of 16-22, and the catalytic decarboxylation activity of short and medium chain saturated fatty acids with the carbon number of less than 12 is obviously reduced (when the reaction is 14-h,CvFAP catalyzes lauric acid decarboxylation in 11% yield, and n-caproic acid decarboxylation by decoy molecule means 12 h only produced 1.6 mM product, an algal photoenzyme converts fattyacids to hydrocarbons and Hydrocarbon synthesis via photoenzymatic decarboxylation of carboxylic acids. The main components of the gasoline are aliphatic hydrocarbon and naphthene with 5-12 carbon atoms, so that the development and search of the photo-decarboxylase capable of catalyzing the decarboxylation of short and medium chain fatty acids have wide prospects in the aspects of enriching the types of the photo-decarboxylase and expanding the green energy development path.

Disclosure of Invention

Based on this, it is an object of the present invention to provide a fatty acid photodecarboxylaseMcFAP mutant capable of catalyzing fatty acid with 6-12 carbon atoms to removeAnd (3) a carboxylic acid.

The specific technical scheme for realizing the aim of the invention comprises the following steps:

fatty acid light decarboxylaseMcMutant of FAP, said fatty acid light decarboxylaseMcThe amino acid sequence of the FAP mutant is shown as SEQ ID NO. 4.

The invention also provides the fatty acid photo-decarboxylaseMcThe coding gene of the FAP mutant has the nucleotide sequence shown in SEQ ID NO. 3.

The invention also provides the fatty acid photo-decarboxylaseMcMutant of FAP and application of coding gene thereof in catalyzing fatty acid decarboxylation.

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 of these embodiments, the fatty acid is a saturated linear fatty acid.

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

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

The invention also provides a fatty acid light decarboxylaseMcThe preparation method of the FAP mutant comprises the steps of expressing and purifying the recombinant engineering strain.

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

The invention also provides a method for catalyzing the decarboxylation of the fatty acid, which uses the fatty acid photo-decarboxylaseMcWhole cells of the mutant of FAP undergo a catalytic reaction.

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

in the present invention, the inventors constructed a fatty acid photo-decarboxylase by deletion mutation based on their own years of experienceMcThe FAP mutant has good decarboxylation effect on the straight-chain fatty acid with 6-18 carbon atoms,especially, the decarboxylation effect on the medium-chain saturated straight-chain fatty acid with 6-12 carbon atoms is very excellent (the decarboxylation effect on C8:0 can reach more than 90% in 30 min), the inventionMcThe FAP mutant enriches the variety of FAP, fills the blank that the existing FAP cannot catalyze decarboxylation of saturated straight-chain fatty acid with 6-12 carbon atoms, has wider decarboxylation substrate spectrum and better decarboxylation effect, enables efficient and sustainable biosynthesis of fuel to be possible, and has wide industrial application prospect.

Drawings

FIG. 1 is a diagram of embodiment 1 of the present inventionMcFAP@E. coliAnd (3) verifying the reaction result by photo-enzymatic decarboxylation.

FIG. 2 is a schematic diagram of embodiment 2 of the present inventionMcSDS-PAGE protein map of FAP-S; wherein M is a protein marker;1.McFAP-S total bacteria; 2.McFAP-S supernatant; 3.McFAP-S precipitation; 4.McFAP-S crude enzyme; 5.McFAP-S permeate; 6.0.5 M imidazole elutionMcFAP-S pure enzyme.

FIG. 3 is a diagram of embodiment 3 of the present inventionMcReaction time profile of FAP-S catalyzed decarboxylation of n-octanoic acid.

FIG. 4 is a diagram of example 3 of the present inventionMcThe effect of the amount of FAP-S enzyme on the efficiency of catalyzing decarboxylation of n-octanoic acid is shown in the graph.

FIG. 5 is a graph showing the concentration of n-octanoic acid as a substrate in example 3 of the present inventionMcFAP-S catalytic decarboxylation efficiency affects the results.

FIG. 6 is a graph showing the reaction temperature versus the reaction temperature in example 3 of the present inventionMcFAP-S catalyzed n-octanoic acid decarboxylation efficiency affects the results.

FIG. 7 shows the pH of the reaction in example 3 of the present inventionMcFAP-S catalyzes the decarboxylation efficiency of n-octanoic acid affecting the results.

FIG. 8 is a pair of the present invention in example 3McStorage stability study of FAP-S in dark at 4 ℃.

FIG. 9 is a pair of the present invention in example 3McFAP-S pH tolerance study.

FIG. 10 is a pair of the embodiment 3 of the present inventionMcThe light inactivation factor of FAP-S was examined.

FIG. 11 is a diagram of example 4 of the present inventionMcFAP@E. coliAnd (3) withMcFAP-S@E. coliSubstrate development research diagram for catalyzing saturated fatty acids with different chain lengths。

FIG. 12 is a diagram of example 5 of the present inventionMcFAP@E. coliAnd (3) withCvFAP@E. coliThe decarboxylation efficiency of the catalytic palmitic acid was compared with the experimental results.

Detailed Description

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

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and/or" as used herein includes any and all combinations of one or more of the associated listed items.

In the present invention, the gene is derived from a gene library by sequence analysisMicractinium conductrixIs a light decarboxylase of (2)McThe FAP gene sequence (nucleotide sequence is SEQ ID NO. 1) is obtained by gene synthesis, and the gene is expressed by taking escherichia coli BL21 (DE 3) as a host to obtainMcWhole cells of FAP (designated asMcFAP@E. coliThe same applies below), photo decarboxylaseMcFAP is successfully expressed and purified (the amino acid sequence is SEQ ID NO. 2), and the basic enzymatic properties of the FAP are simultaneously explored,McFAP is blue light catalyzed photo-decarboxylase, has very good universality on chain length of a fatty acid substrate for catalyzing decarboxylation, and has good decarboxylation effect on straight-chain saturated fatty acid with 6-18 carbon atoms.

SEQ ID NO.1 (coding gene of fatty acid photo-decarboxylase McFAP, 3438 bp):

ATGGCTGAAATGGCAGGTGGTGGTGAAGGTGATGGTATGCTGATGGGCGGCGCGGGTAGCGCAAACACTACCGACGCGTGTTATAGCGATCCGTCTAATCCGGATTGCGCAGCGTTTGAGCGCTCCGACGATGATTGGGCGGCGGACATCGAACTGCTGTGCTCTGCGATGCCGTTCATGCCGGGCTGCACCCTGGCGGAACAGTGCATGAATGGCACCGCCGCCGGTGAATATTGCGAAATGTCCAGTCTGGCTGGTAACATCTGTCTGGATATGCCGGGCATGAAAGGCTGTGAGGCATGGAACGCACTGTGTGGCGCGGCCAGCGCCGTTGAACAGTGTTCCTCTCCGGGCCCGGTTGTGGCACTCCCGACCACCGCGCTGGCCAAAGAAGGCCTGGAATCTCTGTGCTCTACCCATTGTATGGACGGTTGCCCAGACTGTGAAATGGGTAAACTGTGGAACACCTGCACCGACCCGCTGAGTGTTCTGGCGTGGATGTGCTACGCAATGCCGGACATGCCGGAATGTCTGGCTGCTCCGCAGGGCTCCGGCATGGTGGTGGCTTGCGGTGACGCTGAGGTTGCAGCTACCTTCCCGCTGGTGTGCGCGCAACCGCCGACCCCGGCGGCTAACTTTCAGCACCGCCTTCGTACCTGCCGTACCGCCGGCGTTGCGGCATCCGCATCCGGTTCTCCGGCAGTCACTATGGCTGGCCTGTCAACTGTTCTGGCAGTACTGGCACTGCTGCCATCCCCGGTTGCTATGGCCATGACTCCGATGCCGACCCCGGCGCTCGCTCCGGGCCCGGCGATCGACGATATCGGCGGTAACTGCCCGCTGCTGGGTCGCGGTAACATGGAAGCTCCGTGTTATAGCGACCCGAGCGCGGCAGCATGCGTTTCCTTTGAACGCAGCGATGCTGGCTGGGCGGATGACCTGAGTCAGCTGTGTTCTGCGATGCCGTATGCTGTTGGCTGCTGGCTGTGGCACTTGTGTAAAACCGGCGCAGCAAGCGGGACTTACTGTGCGCTGCCGTCCCTGACCGCGAACGTATGTGTTGACGCACCGCTGGTGAACGCTACATCAGCGCCGGGCTGCGAAGCGTGGGCCGCACTGTGCGGCGCCCAGGGTAGCGTCGTTGCGCAGTGCTCTGCGCCAGGCCCGCTGCCGGACATCATCAACACCCTGACCACCCGTGACGGCATCAACTCCCTCTGCGGTATGCATTACATGGATGGGTGTAACGAATGTACCCCTCACGAAGGTCCGGCAGTTCACGACTTCGCGGCCTGTGCTGATCCGGGTCCACTGCCGACTCTGGCCCACCAGTGTTACGCGATGCCTGAAATGGGTGAATGTACCCAGACTGGTATTACCGCAATGTGCAGCGGCGCTGAAGCTCGTGCGACCTTTCCGACCGTTTGCGTGGATCCACCTAACCCGACGACACTGGCGCCGGCGCCTGCCGTTTCTGCCTGCGATGTTGCGGCGGGTGCTGGCGCGCCACCAGCGGCGTCTGCCCGTCCGGCGTCGCACAGCCGCGCGTCACTGGTTGCCTCCCGTAGCGGCTTCTGCGCGCCTTCCCCGGCGCTGCGCTCTCAGCGCACCTCTACTGTCGCGCCGGCGCGCCGCGCCGCGTCGGCGCCGCGTGCGAGCGCAGTTGACGATATTCAACGTGCTCTGAGCACCGCTGGAAGCCCGGTATCCGGTAAACAGTACGATTACATCCTGGTGGGTGGCGGCACCGCGGCATGCGTTTTGGCTAACCGTTTAACCGCGGACGGTAGCAAACGTGTACTGGTGCTGGAAGCGGGTGCGGACAACGTGAGCCGCGATGTTAAAGTCCCGGCTGCGATCACCCGTTTGTTCCGTTCACCGTTGGATTGGAACTTGTTCAGCGAATTGCAGGAACAGCTGGCTGCACGTCAGATCTATATGGCTCGCGGCCGCTTGCTGGGTGGGTCTAGCGCGACCAATGCTACTCTTTACCACCGTGGCGCGGCGGCGGATTATGATGCGTGGGGCGTGCCGGGCTGGGGCGCAGCTGACGTGCTGCCATGGTTCGTTAAGGCCGAAACCAACGCGGAGTTTGCGGCGGGCAAATATCACGGCGCAGGTGGTAACATGCGCGTTGAGAATCCGCGCTACTCCAACCCGCAGCTGCACGGTGCTTTCTTTGCAGCTGCGCAGCAGATGGGTCTGCCGCAGAATACCGACTTCAACAATTGGGATCAGGATCATGCAGGCTTTGGCACTTTTCAGGTTATGCAGGAAAAAGGCACCCGCGCTGATATGTACCGCCAGTATCTTAAACCAGCTCTTGGTCGTCCGAACCTGCAGGTTCTGACCGGTGCGTCTGTGACCAAAGTTCATATCGATAAAGCTGGCGGTAAACCGCGTGCTCTGGGCGTAGAGTTTTCTCTGGATGGTCCGGCTGGTGAACGTATGGCAGCAGAGCTGGCGCCGGGCGGTGAAGTTCTCATGTGCGCTGGCGCCGTGCATAGCCCGCACATTCTGCAGCTGTCTGGCGTTGGTTCGGCGGCTACTCTGGCAGACCACGGCATCGCAGCAGTGGCAGATCTGCCAGGTGTTGGTGCGAACATGCAGGACCAGCCGGCCTGCCTGACAGCGGCTCCCCTGAAAGACAAATACGATGGCATTTCGCTGACCGATCATATCTATAATAGCAAAGGCCAGATTCGCAAACGCGCTATCGCGTCCTACCTGCTTCAGGGTAAAGGTGGTCTGACGTCAACTGGCTGCGACCGTGGCGCGTTTGTACGTACCGCAGGCCAGGCACTGCCGGACCTGCAGGTGCGTTTCGTGCCAGGCATGGCACTGGATGCAGATGGTGTGTCCACCTACGTCCGTTTCGCAAAATTTCAGTCTCAGGGCCTGAAATGGCCGTCTGGCATCACCGTACAGCTTATTGCGTGTCGCCCGCACAGCAAAGGTTCTGTTGGCCTGAAAAACGCGGACCCGTTCACCCCGCCGAAACTGCGTCCGGGCTACCTGACCGACAAAGCGGGTGCGGATCTGGCGACCCTGCGCTCTGGTGTTCATTGGGCCCGTGATCTGGCATCTAGCGGTCCGCTGAGCGAATTTCTTGAAGGCGAACTGTTTCCGGGTAGCCAAGTTGTTTCCGATGATGATATTGATTCTTACATTCGTCGTACCATTCACTCCAGCAACGCGATTGTGGGCACCTGTCGTATGGGCGCGGCGGGTGAAGCGGGTGTTGTTGTGGATAACCAGCTGCGCGTTCAGGGTGTTGATGGTCTGCGTGTTGTTGACGCGAGCGTAATGCCGCGTATCCCAGGTGGTCAGGTGGGTGCGCCGGTTGTGATGCTGGCCGAACGTGCAGCAGCGATGCTGACCGGTCAGGCAGCGCTGGCTGGTGCTAGCGCTGCAGCTCCGCCGACCCCGGTCGCGGCT

SEQ ID NO.2 (fatty acid light decarboxylase)McAmino acid sequence of FAP):

MAEMAGGGEGDGMLMGGAGSANTTDACYSDPSNPDCAAFERSDDDWAADIELLCSAMPFMPGCTLAEQCMNGTAAGEYCEMSSLAGNICLDMPGMKGCEAWNALCGAASAVEQCSSPGPVVALPTTALAKEGLESLCSTHCMDGCPDCEMGKLWNTCTDPLSVLAWMCYAMPDMPECLAAPQGSGMVVACGDAEVAATFPLVCAQPPTPAANFQHRLRTCRTAGVAASASGSPAVTMAGLSTVLAVLALLPSPVAMAMTPMPTPALAPGPAIDDIGGNCPLLGRGNMEAPCYSDPSAAACVSFERSDAGWADDLSQLCSAMPYAVGCWLWHLCKTGAASGTYCALPSLTANVCVDAPLVNATSAPGCEAWAALCGAQGSVVAQCSAPGPLPDIINTLTTRDGINSLCGMHYMDGCNECTPHEGPAVHDFAACADPGPLPTLAHQCYAMPEMGECTQTGITAMCSGAEARATFPTVCVDPPNPTTLAPAPAVSACDVAAGAGAPPAASARPASHSRASLVASRSGFCAPSPALRSQRTSTVAPARRAASAPRASAVDDIQRALSTAGSPVSGKQYDYILVGGGTAACVLANRLTADGSKRVLVLEAGADNVSRDVKVPAAITRLFRSPLDWNLFSELQEQLAARQIYMARGRLLGGSSATNATLYHRGAAADYDAWGVPGWGAADVLPWFVKAETNAEFAAGKYHGAGGNMRVENPRYSNPQLHGAFFAAAQQMGLPQNTDFNNWDQDHAGFGTFQVMQEKGTRADMYRQYLKPALGRPNLQVLTGASVTKVHIDKAGGKPRALGVEFSLDGPAGERMAAELAPGGEVLMCAGAVHSPHILQLSGVGSAATLADHGIAAVADLPGVGANMQDQPACLTAAPLKDKYDGISLTDHIYNSKGQIRKRAIASYLLQGKGGLTSTGCDRGAFVRTAGQALPDLQVRFVPGMALDADGVSTYVRFAKFQSQGLKWPSGITVQLIACRPHSKGSVGLKNADPFTPPKLRPGYLTDKAGADLATLRSGVHWARDLASSGPLSEFLEGELFPGSQVVSDDDIDSYIRRTIHSSNAIVGTCRMGAAGEAGVVVDNQLRVQGVDGLRVVDASVMPRIPGGQVGAPVVMLAERAAAMLTGQAALAGASAAAPPTPVAA

by truncatingMcDeletion mutation of N-terminal of FAP to obtain fatty acid light decarboxylaseMcMutant of FAP (hereinafter referred to asMcFAP-S), and purifying by nickel column to obtainMcFAP mutant pure enzyme (nucleotide sequence is shown as SEQ ID NO.3, amino acid sequence is shown as SEQ ID NO. 4).McThe catalytic effect of the FAP mutant is greatly improved, and the FAP mutant can catalyze the decarboxylation of the medium-chain fatty acid with 6-12 carbon atoms. The FAP enzyme type of the photocatalytic decarboxylation short chain fatty acid is supplemented.

SEQ ID NO.3 (fatty acid light decarboxylase)McCoding gene for mutant of FAP):

CGTGCGAGCGCAGTTGACGATATTCAACGTGCTCTGAGCACCGCTGGAAGCCCGGTATCCGGTAAACAGTACGATTACATCCTGGTGGGTGGCGGCACCGCGGCATGCGTTTTGGCTAACCGTTTAACCGCGGACGGTAGCAAACGTGTACTGGTGCTGGAAGCGGGTGCGGACAACGTGAGCCGCGATGTTAAAGTCCCGGCTGCGATCACCCGTTTGTTCCGTTCACCGTTGGATTGGAACTTGTTCAGCGAATTGCAGGAACAGCTGGCTGCACGTCAGATCTATATGGCTCGCGGCCGCTTGCTGGGTGGGTCTAGCGCGACCAATGCTACTCTTTACCACCGTGGCGCGGCGGCGGATTATGATGCGTGGGGCGTGCCGGGCTGGGGCGCAGCTGACGTGCTGCCATGGTTCGTTAAGGCCGAAACCAACGCGGAGTTTGCGGCGGGCAAATATCACGGCGCAGGTGGTAACATGCGCGTTGAGAATCCGCGCTACTCCAACCCGCAGCTGCACGGTGCTTTCTTTGCAGCTGCGCAGCAGATGGGTCTGCCGCAGAATACCGACTTCAACAATTGGGATCAGGATCATGCAGGCTTTGGCACTTTTCAGGTTATGCAGGAAAAAGGCACCCGCGCTGATATGTACCGCCAGTATCTTAAACCAGCTCTTGGTCGTCCGAACCTGCAGGTTCTGACCGGTGCGTCTGTGACCAAAGTTCATATCGATAAAGCTGGCGGTAAACCGCGTGCTCTGGGCGTAGAGTTTTCTCTGGATGGTCCGGCTGGTGAACGTATGGCAGCAGAGCTGGCGCCGGGCGGTGAAGTTCTCATGTGCGCTGGCGCCGTGCATAGCCCGCACATTCTGCAGCTGTCTGGCGTTGGTTCGGCGGCTACTCTGGCAGACCACGGCATCGCAGCAGTGGCAGATCTGCCAGGTGTTGGTGCGAACATGCAGGACCAGCCGGCCTGCCTGACAGCGGCTCCCCTGAAAGACAAATACGATGGCATTTCGCTGACCGATCATATCTATAATAGCAAAGGCCAGATTCGCAAACGCGCTATCGCGTCCTACCTGCTTCAGGGTAAAGGTGGTCTGACGTCAACTGGCTGCGACCGTGGCGCGTTTGTACGTACCGCAGGCCAGGCACTGCCGGACCTGCAGGTGCGTTTCGTGCCAGGCATGGCACTGGATGCAGATGGTGTGTCCACCTACGTCCGTTTCGCAAAATTTCAGTCTCAGGGCCTGAAATGGCCGTCTGGCATCACCGTACAGCTTATTGCGTGTCGCCCGCACAGCAAAGGTTCTGTTGGCCTGAAAAACGCGGACCCGTTCACCCCGCCGAAACTGCGTCCGGGCTACCTGACCGACAAAGCGGGTGCGGATCTGGCGACCCTGCGCTCTGGTGTTCATTGGGCCCGTGATCTGGCATCTAGCGGTCCGCTGAGCGAATTTCTTGAAGGCGAACTGTTTCCGGGTAGCCAAGTTGTTTCCGATGATGATATTGATTCTTACATTCGTCGTACCATTCACTCCAGCAACGCGATTGTGGGCACCTGTCGTATGGGCGCGGCGGGTGAAGCGGGTGTTGTTGTGGATAACCAGCTGCGCGTTCAGGGTGTTGATGGTCTGCGTGTTGTTGACGCGAGCGTAATGCCGCGTATCCCAGGTGGTCAGGTGGGTGCGCCGGTTGTGATGCTGGCCGAACGTGCAGCAGCGATGCTGACCGGTCAGGCAGCGCTGGCTGGTGCTAGCGCTGCAGCTCCGCCGACCCCGGTCGCGGCT

SEQ ID NO.4 (fatty acid light decarboxylase)McAmino acid sequence of mutant of FAP):

RASAVDDIQRALSTAGSPVSGKQYDYILVGGGTAACVLANRLTADGSKRVLVLEAGADNVSRDVKVPAAITRLFRSPLDWNLFSELQEQLAARQIYMARGRLLGGSSATNATLYHRGAAADYDAWGVPGWGAADVLPWFVKAETNAEFAAGKYHGAGGNMRVENPRYSNPQLHGAFFAAAQQMGLPQNTDFNNWDQDHAGFGTFQVMQEKGTRADMYRQYLKPALGRPNLQVLTGASVTKVHIDKAGGKPRALGVEFSLDGPAGERMAAELAPGGEVLMCAGAVHSPHILQLSGVGSAATLADHGIAAVADLPGVGANMQDQPACLTAAPLKDKYDGISLTDHIYNSKGQIRKRAIASYLLQGKGGLTSTGCDRGAFVRTAGQALPDLQVRFVPGMALDADGVSTYVRFAKFQSQGLKWPSGITVQLIACRPHSKGSVGLKNADPFTPPKLRPGYLTDKAGADLATLRSGVHWARDLASSGPLSEFLEGELFPGSQVVSDDDIDSYIRRTIHSSNAIVGTCRMGAAGEAGVVVDNQLRVQGVDGLRVVDASVMPRIPGGQVGAPVVMLAERAAAMLTGQAALAGASAAAPPTPVAA

in the following examples, the content of fatty acids and alkanes before and after decarboxylation was measured by Agilent 7890B gas chromatography system (Agilent Technologies, palo Alto, calif., USA), chromatographic column KB-FFAP (30 m ×0.25mm,0.25 μm), the specific chromatographic method is: sample injection volume: 1. mu L; injector temperature: 250 ℃; split ratio: 30:1; detector temperature: 280 ℃; the temperature-raising program is as follows: the initial temperature is 110℃for 3.4 min, followed by 25℃for min ^-1 Is of the speed of (1)The temperature is raised to 190 ℃ and kept for 2.1 min, and then the temperature is kept at 25 ℃ for min again ^-1 Is heated to 230 ℃ for 2 min and finally is heated to 30 ℃ for min ^-1 The rate of (2) was raised to 250℃and held for 12 min. The method comprises the steps of carrying out qualitative analysis on chromatographic peak time by adopting each fatty acid and alkane standard substance, preparing standard solutions with different concentrations by adopting the standard substances, taking n-octanol as an internal standard, and obtaining a standard curve through gas phase detection for quantitative calculation.

In the following examples, plasmid pET28a-McFAP was synthesized by the division of bioengineering (Shanghai); empty plasmid pET28a is stored for the applicant laboratory; coli BL21 (DE 3) competent cells, purchased from Biotech Inc. ]; plasmid extraction kit, purchased from the division of bioengineering (Shanghai); all other chemicals were purchased from Sigma-Aldrich, TCI or ala Ding Gongsi, which were the highest purity and used without further purification.

The invention is described in detail below with reference to the drawings and the specific embodiments.

Example 1McFAP@E. coliIs verified by photo-enzymatic decarboxylation of catalytic fatty acids

Plasmid pET28a-McRecombination of FAPE.coliBL21 (DE 3) strain was cultured at 37℃in super Broth (Terrific Broth, TB) containing 50. Mu.g/mL kanamycin, when OD ₆₀₀ To 0.7-0.8, 0.5 mM isopropyl β -D-1-thiogalactoside (IPTG) was added and the cells were incubated at 17 ℃ for 20 h. Centrifuging at 4000 rpm at 4deg.C for 30min to obtain bacteria; washed with Tris-HCl buffer (50 mM, pH 8, containing 100mM NaCl) and centrifuged again (10000 rpm,20min,4 ℃); cell pellet was prepared as 1:2 (w/v) are suspended in the same buffer, 1 mM phenylmethylflavanthracene chloride (PMSF) and 5% glycerol (w/v) are added, frozen in liquid nitrogen and stored at-80 ℃ for later use.

To verifyMcCatalytic decarboxylation of FAP escherichia coli cells containing empty plasmid pET28a vector (designated empty WC) were prepared in the same way.

500 mu L of the mixture was subjected to wet weight concentration of 0.5. 0.5 g/mLMcFAP@E. coli300 mu L of 170 mM fatty acid DMSO solution, 200 mu L of Tris-HCl buffer solution100mM, pH 8.0) was added to a 5 mL clear reaction flask with a total reaction volume of 1mL; then placing the mixture in a self-made photocatalytic reaction device (a blue light irradiation device is added on a common catalytic reaction device), and reacting at 500 rpm and 30 ℃ under the irradiation of blue light (10W, 220V) for 12 h; after the reaction, the reaction mixture was taken in a 2 mL EP tube, and 1mL of a 25mM ethyl acetate solution as an internal standard of n-octanol was added for extraction, i.e., the extraction volume ratio was 1:1, the extracted mixture was centrifuged at 11000 rpm for 4 min, and the upper organic phase was taken for GC analysis in a 2 mL chromatographic bottle. The same reaction system was placed in the dark, with the other conditions unchanged, to catalyze the decarboxylation of fatty acids.

Will beMcFAP@E. coliThe other conditions were unchanged, and the fatty acid decarboxylation was catalyzed, changing to empty WC.

The experimental results are shown in FIG. 1. As can be seen from fig. 1, empty WC has no decarboxylation effect on fatty acids,McFAP@E. colino decarboxylation effect on fatty acid under the condition of no blue light,McFAP@E. colihas decarboxylation effect on fatty acid under blue light condition, thereby verifyingMcFAP@E. coliThe decarboxylation of fatty acid substrates is catalyzed under blue light conditions.

Example 2McConstruction and purification of FAP-S mutants

McThe FAP gene has total length of 3438bp, total 1146 amino acids, total 596 amino acids from 551 th amino acid at N end to C end as constructed mutantMcFAP-S with amino acid sequence shown in SEQ ID NO.4 and nucleotide sequence shown in SEQ ID NO. 3.

By designing primers (as shown in Table 1) toMcThe FAP gene was used as a template, and the target gene (1788 bp) and pET28a vector (5362 bp) were obtained by PCR amplification (the system and procedure are shown in Table 2).

Table 1 for constructionMcPrimers for FAP-S

TABLE 2PCR reaction System and program

Recovering amplified target gene and carrier according to the manual of SanPrep column PCR product gel recovery, measuring DNA concentration of the target gene and carrier, seamless cloning according to the requirement of seamless cloning kit (reaction system and program are shown in Table 2), transforming and culturing the plasmid, sample feeding and sequencing, and obtaining correct sequencing resultMcFAP-S。

McThe apparatus, chromatographic column and purification chromatographic column for FAP-S purification and sample loading and sample collection are as follows: his Prep ^TM FF16/10；HiPrep ^TM 26/10 salt-changing column. The column was equilibrated with loading buffer (50 mM Tris-HCl 300 mM NaCl 10mM imidazole 5% (v/v) glycerol pH 9), after equilibration, the crude enzyme was pumped in and after loading was completed the column was equilibrated with loading buffer. Then eluted with elution buffer (50 mM Tris-HCl 300 mM NaCl 500 mM imidazole 5% (v/v) glycerol pH 9) and peak samples were collected and detected by SDS-PAGE electrophoresis. And (3) carrying out salt exchange on the sample received by the peak corresponding to the target protein by using a salt exchange column. Adding the protein into a salt exchange column balanced by salt exchange buffer solution (50 mM Tris-HCl 150 mM NaCl 5% (v/v) glycerol pH 9), continuing eluting by using the salt exchange buffer solution, collecting the eluted protein, concentrating, packaging, pre-freezing with liquid nitrogen, and preserving at-80 ℃ for later use. The whole flow velocity is 5 mL min ^-1 。

McThe SDS-PAGE protein map of FAP-S after purification by nickel column and elution by 0.5. 0.5M imidazole is shown in FIG. 2, and as can be seen from FIG. 2,Mcthe FAP-S obtains better expression and purification effects.

Example 3McCharacterization of the enzymatic Properties of FAP-S

FAP enzyme activity definition: under the conditions of 30 ℃, 500 rpm and blue light (10W, 220V) irradiation, the enzyme amount required for the catalytic reaction of 1. Mu. Mol of n-octanoic acid to 1. Mu. Mol of n-heptane within 1 min was defined as 1U.

1. Optimizing the reaction time

In this example, 12 reaction time pairs were selectedMcOptimization of FAP-S catalyzed n-octanoic acid decarboxylation process(5 min, 10min, 15min, 20min, 25min, 30min, 35min, 40min, 45min, 50min, 55min and 60 min), wherein 150. Mu.L of 140-mM-octanoic acid DMSO solution (20 mM of octanoic acid concentration, 15% of DMSO addition in the reaction system) was added, the enzyme addition was 40. Mu.M, and Tris-HCl buffer (100 mM, pH 9.0) was added to make up the total reaction volume of 1mL, and the mixture was added to a 5-mL transparent reaction flask. Then placing the mixture in a self-made photocatalytic reaction device, and reacting for a certain time under the irradiation of blue light (10W, 220V) at 500 rpm and 30 ℃; after the reaction, the reaction mixture was taken in a 2 mL EP tube, and 1mL of an ethyl acetate solution of 25mM n-octanol internal standard was added for extraction, i.e., the extraction volume ratio was 1:1, the extracted mixture was centrifuged at 11000 rpm for 4 min, and the upper organic phase was taken for GC analysis in a 2 mL chromatographic bottle.

The results are shown in figure 3 of the drawings,McFAP-S catalyzes the decarboxylation of n-octanoic acid to react rapidly within 5min, the conversion rate is steadily increased after 5min, and the conversion rate can reach 95% after 30 min.

2. Optimizing the amount of the added reaction enzyme

In this example, 4 enzyme addition pairs were selectedMcThe FAP-S catalyzed n-octanoic acid decarboxylation process was optimized (final concentrations of 6. Mu.M, 12. Mu.M, 24. Mu.M and 36. Mu.M, respectively) with the other conditions and treatments unchanged, as described above.

As a result, as shown in FIG. 4, the reaction conversion increased with the increase in the amount of enzyme added to the reaction system.

3. Optimizing the concentration of the reaction substrate

This example selects 5 substrate concentration pairsMcFAP-S catalyzed n-octanoic acid decarboxylation was optimized (reaction substrate concentrations 10mM, 20mM, 30mM, 40mM, 50mM, respectively) with the other conditions and treatments unchanged, as described above.

As a result, as shown in FIG. 5, the reaction conversion rate was decreased with an increase in the substrate concentration in the reaction system, but the actual production rate was unchanged.

4. Optimizing the reaction temperature

In this example, 5 pairs of reaction temperatures were selectedMcThe FAP-S catalyzed decarboxylation process of the n-octanoic acid is optimized (the reaction temperature is respectively 20 ℃,30 ℃,40 ℃, 45℃,50 ℃ and other conditions and treatment modes are unchanged, and the steps are the same.

The results are shown in figure 6 of the drawings,Mcthe optimal temperature for the FAP-S to catalyze the decarboxylation of the n-octanoic acid is 40 ℃, the relative enzyme activity is over 80 percent at the temperature of 30-45 ℃, and the relative enzyme activity is rapidly reduced at the temperature of higher than 45 ℃.

5. Optimizing the reaction pH

This example selects 5 pH pairsMcThe FAP-S catalyzed n-octanoic acid decarboxylation process is optimized (the pH of the reaction system is 6, 7, 8, 9 and 10 respectively), other conditions and treatment modes are unchanged, and the steps are the same.

The results are shown in figure 7 of the drawings,Mcthe optimal temperature of the FAP-S catalyzed decarboxylation of the n-octanoic acid is 8-9, but the conversion rate is more than 80% in the pH range of 6-10, namelyMcThe FAP-S catalysis of decarboxylation of the n-octanoic acid has a good catalytic effect within the pH range of 6-10.

6. Examination of storage stability

Selection and investigation of the present embodimentMcThe FAP-S catalyzed n-octanoic acid decarboxylation enzyme activity has storage stability under dark condition at 4deg.C, and 12 storage times are selected for investigationMcThe residual enzyme activity (retention time is 10min,30 min,1 h,3 h,6 h,12 h,1 d,2 d,3 d,5 d,7 d,10d) of the FAP-S catalyzed n-octanoic acid decarboxylation is unchanged, and other conditions and treatment modes are the same as the steps.

The results are shown in figure 8 of the drawings,McFAP-S is stored for 10d under dark condition at 4 ℃, and the residual activity of catalyzing decarboxylation of n-octanoic acid is still more than 70%.

7. Optimizing pH tolerance

This example selects 5 pH pairsMcThe FAP-S enzyme solution is incubated (the pH of an incubation system is 6, 7, 8, 9 and 10 respectively), the incubation condition is 4 ℃ light-shielding condition, other conditions and treatment modes are unchanged, and the steps are the same.

The results are shown in FIG. 9, in the dark at 4 ℃,Mcthe residual activity of FAP-S for catalyzing decarboxylation of n-octanoic acid after incubation of 5 d within the pH range of 6-10 is still more than 50%. Meanwhile, the pH is in the range of 6-10, the pH is relative toMcThe enzyme activity of FAP-S has no obvious influence, and the residual activity after incubation at pH 6-8 is slightly better than that at pH 9-10.

8. Investigation of the light inactivation factor

Selection and investigation of the present embodimentMcThe FAP-S catalyzed n-octanoic acid decarboxylation enzyme activity has the storage stability of normal temperature preservation under different illumination conditions, and 5 different illumination environments are selected for incubationMcFAP-S pure enzyme solution (blue light irradiation, sunlight irradiation, dark preservation, red light irradiation, and red light irradiation co-incubation of system containing 5% DMSO 10mM n-octanoic acid) and selecting 5 preservation times for investigationMcThe residual enzyme activity (retention time is 10min,30 min,1 h,2 h,3 h) of the FAP-S catalyzed n-octanoic acid decarboxylation is unchanged, and other conditions and treatment modes are the same as the steps.

As a result, as shown in FIG. 10, under normal temperature conditions, the blue light was irradiated toMcThe effect of the activity of the pure enzyme activity of FAP-S is the greatest, and the residual enzyme activity is less than 10% after 10 min; after sunlight irradiates 3 hMcThe residual enzyme activity of FAP-S is less than 50%;Mcunder dark condition and red light irradiation condition, the residual enzyme activity of FAP-S after 3 h is still more than 90%; while under the condition of adding 10mM n-octanoic acid substrate and incubating with 5% DMSOMcThe residual enzyme activity of FAP-S after sunlight irradiation of 3 h is still more than 90%, namely the co-incubation of the added substrate obviously inhibitsMcLight inactivation of FAP-S. For the reasons described above, a whole cell catalytic format was chosen in the examples below for the substrate development study catalyzing saturated fatty acids of different chain lengths.

Example 4McFAP@E. coliAnd (3) withMcFAP-S@E. coliSubstrate development studies to catalyze fatty acids

Different fatty acid substrates (saturated straight-chain fatty acid with 6-18 carbon atoms) are selected for photocatalytic deacidification, the reaction time is 30min, and other relevant reaction conditions are carried out according to the photo-enzymatic decarboxylation verification experiment of the embodiment 1.

The results are shown in figure 11 of the drawings,McFAP@E. coliand (3) withMcFAP-S@E. coliCan catalyze the decarboxylation effect of the fatty acid with the ratio of C6:0 to C18:0, and under the condition of the same total cell addition,McFAP-S@E. colithe decarboxylation efficiency of the fatty acid with the catalytic ratio of C7:0 to C12:0 is obviously higher than that of the fatty acid with the catalytic ratio of C7:0 to C12:0McFAP@E. coliCatalytic C7:0-C12:0 fatty acid decarboxylation efficiency.

Example 5McFAP@E. coliAnd (3) withCvFAP@E. coliCatalytic softeningComparison of the decarboxylation efficiency of fatty acids

This example selects 6 reaction times to compareMcFAP@E. coliAnd (3) withCvFAP@E. coli(same preparation method)McFAP@E. coli) The decarboxylation efficiency of the catalytic palmitic acid (1, 2, 3, 4, 5 and 6 h),McFAP@E. coliand (3) withCvFAP@E. coliThe addition was 0.25 g/mL and other relevant reaction conditions were performed as per the photo-enzymatic decarboxylation validation experiment of example 1.

The results are shown in figure 12 of the drawings,McFAP@E. coliafter the reaction of catalyzing the palmitic acid to decarboxylate 6h, the conversion rate of catalyzing the palmitic acid to decarboxylate can reach more than 90 percent. Under the same reaction condition, the reaction mixture is prepared,CvFAP@E. coliafter reaction 6h, a product of 30mM is produced, and the conversion rate is only about 60%. From this, it was found that, when the total cell amount was the same,McFAP@E. colithe decarboxylation efficiency of the catalytic palmitic acid is significantly higher thanCvFAP@E. coli。

The technical features of the above-described embodiments may be arbitrarily combined, and all possible combinations of the technical features in the above-described embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description.

The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.

Sequence listing

<110> university of North China university of Mitsui, guangdong enzyme Biomanufacturing institute of England

<120> mutant of fatty acid light decarboxylase McFAP and application thereof

<130> 1

<160> 8

<170> SIPOSequenceListing 1.0

<210> 1

<211> 3438

<212> DNA

<213> Artificial sequence (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 (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 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 (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 (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 (Artificial Sequence)

<400> 5

atgggtcgcg gatccgaatt ccgtgcgagc gcagttgacg at 42

<210> 6

<211> 39

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 6

tgcggccgca agcttgtcga cagccgcgac cggggtcgg 39

<210> 7

<211> 27

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 7

gaattcggat ccgcgaccca tttgctg 27

<210> 8

<211> 23

<212> DNA

<213> Artificial sequence (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.