CN114854726A - Mutant of fatty acid light decarboxylase McFAP and application thereof - Google Patents

Abstract

The invention discloses a mutant of fatty acid light decarboxylase McFAP and application thereof, wherein the amino acid sequence of the mutant of the fatty acid light decarboxylase McFAP is shown as SEQ ID No. 4. The McFAP mutant enriches the variety of FAP, fills the blank that the existing FAP can not catalyze and decarboxylate the saturated straight chain fatty acid with 6-12 carbon atoms, has obviously wider decarboxylation substrate spectrum and better decarboxylation effect, enables the efficient 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, and particularly relates to a fatty acid light decarboxylase McFAP mutant and application thereof.

Background

Currently, biotechnology is continuously moving from medicine, agriculture, food to industrial fields (such as chemical industry, materials and energy). Gasoline, diesel, plastics, rubber, fiber, and many of the bulk of traditional petrochemical products are continually being replaced by industrial bio-manufactured products from renewable feedstocks. The chemical industrial process of 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 a raw material, the fuel is produced by utilizing microorganisms or enzymes, the development and the utilization of renewable biomass resources for directional conversion to produce the biogas and the liquid fuel have very wide development prospects, and the development and the utilization of the renewable biomass resources become important tasks of scientific research of research workers in related fields of various countries.

The enzymes which have been found to date to be useful for the decarboxylation of fatty acids are mainly amino acid decarboxylases, haloperoxidases and terminal olefin enzymes synthesized by the decarboxylation of fatty acids (OleT) _JE ) And the like, but most of the enzymes have low catalytic efficiency, and require additional addition of expensive cofactors and/or oxidants which are easy to damage unsaturated bonds of the grease, which is a significant disadvantage in application research. Therefore, the search for a novel decarboxylase with high efficiency is the key for breaking the bottleneck of the research of preparing the biofuel.

Fatty acid light decarboxylase (FAP, EC 4.1.1.106) belongs to glucose-methanol-choline (GMC) oxidoreductase family, and is a light-driven enzyme that can convert fatty acid into alkane (alkene) hydrocarbon by using only blue light without adding expensive cofactors. The fatty acid can form various kinds of alkane (alkene) hydrocarbon only by removing one carboxyl, and the alkane (alkene) hydrocarbon is almost perfectly matched with 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, the biological enzyme catalysis specificity is strong, and the condition is mild. Therefore, the light-driven enzyme FAP which combines the advantages of the two and meets the green development expectation becomes an emerging research hotspot. With the demand and preference of green energy, the preparation of alkane (alkene) hydrocarbon by decarboxylation of fatty acid becomes a key exploration 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, environment-friendly, simple and convenient, and does not introduce double bonds at the tail end of a carbon chain, and the product is the required alkane. FAP is used as a light-driven enzyme for converting fatty acid into alkane (alkene) hydrocarbon by using blue light, the reaction condition for the decarboxylation reaction of the fatty acid is mild, the conversion rate is high (up to more than 90%), 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 to be used in the process, so that the FAP has great significance for environmental protection and represents a brand-new application field.

However, only CvFAP, CrFAP, EsiFAP, GsuFAP and NgaFAP are reported as the currently reported optical decarboxylases, and only one kind of CvFAP is studied deeply, and the rest only expresses the verification decarboxylation activity, while the currently reported optical decarboxylases such as CvFAP and the like show obvious preference for the saturated fatty acid with the carbon number of 16-22 and have obviously reduced decarboxylation activity for the short and medium chain saturated fatty acid with the carbon number of less than 12 (in the reaction 14h, the yield of the CvFAP catalysis decarboxylation of lauric acid is 11%, and the decarboxylation reaction of n-hexanoic acid is catalyzed by decoy molecular means for 12h to generate only 1.6mM product, and organic phosphozyme convertes to fatty acids and Hydrocarbon synthesis via photochemical decarboxylation of carboxylic acids). The main components of the gasoline are aliphatic hydrocarbon and naphthenic hydrocarbon with 5-12 carbon atoms, so that the exploitation and exploration of the optical decarboxylase capable of catalyzing the decarboxylation of the short-chain and medium-chain fatty acids have wide prospects in the aspects of enriching the types of the optical decarboxylase and expanding the green energy development approach.

Disclosure of Invention

Based on the above, one of the objects of the present invention is to provide a mutant of fatty acid decarboxylase McFAP, which can catalyze decarboxylation of fatty acid with 6-12 carbon atoms.

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

a mutant of fatty acid light decarboxylase McFAP is disclosed, wherein the amino acid sequence of the mutant of fatty acid light decarboxylase McFAP is shown in SEQ ID NO. 4.

The invention also provides a coding gene of the fatty acid light decarboxylase McFAP mutant, and the nucleotide sequence is shown as SEQ ID NO. 3.

The invention also provides a mutant of the fatty acid light decarboxylase McFAP and application of a coding gene of the mutant in catalyzing the decarboxylation of the fatty acid.

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

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

In some of these embodiments, the fatty acid is a saturated straight chain 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 preparation method of the fatty acid light decarboxylase McFAP mutant, which is obtained by expressing and purifying the recombinant engineering strain.

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

The invention also provides a method for catalyzing the decarboxylation of the fatty acid, which is to use the whole cell of the mutant of the fatty acid light decarboxylase McFAP to carry out catalytic reaction.

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

in the invention, the inventor constructs a mutant of fatty acid light decarboxylase McFAP according to years of experience of the inventor by deletion mutation, and finds that the mutant has good decarboxylation effect on the straight chain fatty acid with the carbon atom number of 6-18, especially has excellent decarboxylation effect on the medium-chain saturated straight chain fatty acid with the carbon atom number of 6-12 (the decarboxylation effect on C8:0 can reach more than 90% in 30 min).

Drawings

Fig. 1 shows the results of the photochemical decarboxylation verification reaction of McFAP @ e.

FIG. 2 is an SDS-PAGE protein profile of McFAP-S in example 2 of the present invention; wherein, m. protein marker; McFAP-S total bacteria; McFAP-S supernatant; precipitation of McFAP-S; McFAP-S crude enzyme; McFAP-S pass-through; 6.0.5M imidazole elutes McFAP-S pure enzyme.

FIG. 3 is a graph of the reaction time for McFAP-S to catalyze the decarboxylation of n-octanoic acid in example 3 of the present invention.

FIG. 4 is a graph showing the effect of McFAP-S enzyme dosage on the efficiency of catalyzing the decarboxylation of n-octanoic acid in example 3 of the present invention.

FIG. 5 is a graph showing the effect of the concentration of the substrate, n-octanoic acid, on the efficiency of the catalytic decarboxylation by McFAP-S in example 3 of the present invention.

FIG. 6 is a graph showing the effect of reaction temperature on the efficiency of McFAP-S catalyzed decarboxylation of n-octanoic acid in example 3 of the present invention.

FIG. 7 shows the effect of reaction pH on the efficiency of McFAP-S catalyzed decarboxylation of n-octanoic acid in example 3 of the present invention.

FIG. 8 is a graph showing the examination of the storage stability of McFAP-S under dark condition at 4 ℃ in example 3 of the present invention.

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

FIG. 10 is a graph showing the consideration of the light inactivation factor of McFAP-S in example 3 of the present invention.

FIG. 11 is a graph showing the substrate extension studies of McFAP @ E.coli and McFAP-S @ E.coli in example 4 of the present invention in catalyzing saturated fatty acids of different chain lengths.

FIG. 12 shows the results of comparative experiments on the decarboxylation efficiency of palmitic acid catalyzed by McFAP @ E.coli and CvFAP @ E.coli in example 5 of the present invention.

Detailed Description

In order that the invention may be more fully understood, reference will now be made to the following description. The present 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. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

In the invention, a gene sequence (nucleotide sequence is SEQ ID NO.1) of light decarboxylase McFAP from Micretinium product is obtained by performing sequence analysis in a gene bank, the gene is obtained by gene synthesis, escherichia coli BL21(DE3) is used as a host for expression to obtain a whole cell (marked as McFAP @ E. coli, the same below) of the McFAP, the light decarboxylase McFAP is successfully expressed and purified (amino acid sequence is SEQ ID NO.2), and basic enzymological properties of the light decarboxylase McFAP are researched, wherein the McFAP is a blue light catalyzed light decarboxylase, has very good universality on substrate chain length of fatty acid for catalyzing decarboxylation, and has good decarboxylation effect on linear chain saturated fatty acid with 6-18 carbon atoms.

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

ATGGCTGAAATGGCAGGTGGTGGTGAAGGTGATGGTATGCTGATGGGCG GCGCGGGTAGCGCAAACACTACCGACGCGTGTTATAGCGATCCGTCTAAT CCGGATTGCGCAGCGTTTGAGCGCTCCGACGATGATTGGGCGGCGGACAT CGAACTGCTGTGCTCTGCGATGCCGTTCATGCCGGGCTGCACCCTGGCGG AACAGTGCATGAATGGCACCGCCGCCGGTGAATATTGCGAAATGTCCAGT CTGGCTGGTAACATCTGTCTGGATATGCCGGGCATGAAAGGCTGTGAGGC ATGGAACGCACTGTGTGGCGCGGCCAGCGCCGTTGAACAGTGTTCCTCTC CGGGCCCGGTTGTGGCACTCCCGACCACCGCGCTGGCCAAAGAAGGCCTG GAATCTCTGTGCTCTACCCATTGTATGGACGGTTGCCCAGACTGTGAAAT GGGTAAACTGTGGAACACCTGCACCGACCCGCTGAGTGTTCTGGCGTGGA TGTGCTACGCAATGCCGGACATGCCGGAATGTCTGGCTGCTCCGCAGGGC TCCGGCATGGTGGTGGCTTGCGGTGACGCTGAGGTTGCAGCTACCTTCCC GCTGGTGTGCGCGCAACCGCCGACCCCGGCGGCTAACTTTCAGCACCGCC TTCGTACCTGCCGTACCGCCGGCGTTGCGGCATCCGCATCCGGTTCTCCGG CAGTCACTATGGCTGGCCTGTCAACTGTTCTGGCAGTACTGGCACTGCTG CCATCCCCGGTTGCTATGGCCATGACTCCGATGCCGACCCCGGCGCTCGC TCCGGGCCCGGCGATCGACGATATCGGCGGTAACTGCCCGCTGCTGGGTC GCGGTAACATGGAAGCTCCGTGTTATAGCGACCCGAGCGCGGCAGCATG CGTTTCCTTTGAACGCAGCGATGCTGGCTGGGCGGATGACCTGAGTCAGC TGTGTTCTGCGATGCCGTATGCTGTTGGCTGCTGGCTGTGGCACTTGTGTA AAACCGGCGCAGCAAGCGGGACTTACTGTGCGCTGCCGTCCCTGACCGCG AACGTATGTGTTGACGCACCGCTGGTGAACGCTACATCAGCGCCGGGCTG CGAAGCGTGGGCCGCACTGTGCGGCGCCCAGGGTAGCGTCGTTGCGCAGT GCTCTGCGCCAGGCCCGCTGCCGGACATCATCAACACCCTGACCACCCGT GACGGCATCAACTCCCTCTGCGGTATGCATTACATGGATGGGTGTAACGA ATGTACCCCTCACGAAGGTCCGGCAGTTCACGACTTCGCGGCCTGTGCTG ATCCGGGTCCACTGCCGACTCTGGCCCACCAGTGTTACGCGATGCCTGAA ATGGGTGAATGTACCCAGACTGGTATTACCGCAATGTGCAGCGGCGCTGA AGCTCGTGCGACCTTTCCGACCGTTTGCGTGGATCCACCTAACCCGACGA CACTGGCGCCGGCGCCTGCCGTTTCTGCCTGCGATGTTGCGGCGGGTGCT GGCGCGCCACCAGCGGCGTCTGCCCGTCCGGCGTCGCACAGCCGCGCGTC ACTGGTTGCCTCCCGTAGCGGCTTCTGCGCGCCTTCCCCGGCGCTGCGCTC TCAGCGCACCTCTACTGTCGCGCCGGCGCGCCGCGCCGCGTCGGCGCCGC GTGCGAGCGCAGTTGACGATATTCAACGTGCTCTGAGCACCGCTGGAAGC CCGGTATCCGGTAAACAGTACGATTACATCCTGGTGGGTGGCGGCACCGC GGCATGCGTTTTGGCTAACCGTTTAACCGCGGACGGTAGCAAACGTGTAC TGGTGCTGGAAGCGGGTGCGGACAACGTGAGCCGCGATGTTAAAGTCCC GGCTGCGATCACCCGTTTGTTCCGTTCACCGTTGGATTGGAACTTGTTCAG CGAATTGCAGGAACAGCTGGCTGCACGTCAGATCTATATGGCTCGCGGCC GCTTGCTGGGTGGGTCTAGCGCGACCAATGCTACTCTTTACCACCGTGGC GCGGCGGCGGATTATGATGCGTGGGGCGTGCCGGGCTGGGGCGCAGCTG ACGTGCTGCCATGGTTCGTTAAGGCCGAAACCAACGCGGAGTTTGCGGCG GGCAAATATCACGGCGCAGGTGGTAACATGCGCGTTGAGAATCCGCGCT ACTCCAACCCGCAGCTGCACGGTGCTTTCTTTGCAGCTGCGCAGCAGATG GGTCTGCCGCAGAATACCGACTTCAACAATTGGGATCAGGATCATGCAGG CTTTGGCACTTTTCAGGTTATGCAGGAAAAAGGCACCCGCGCTGATATGT ACCGCCAGTATCTTAAACCAGCTCTTGGTCGTCCGAACCTGCAGGTTCTG ACCGGTGCGTCTGTGACCAAAGTTCATATCGATAAAGCTGGCGGTAAACC GCGTGCTCTGGGCGTAGAGTTTTCTCTGGATGGTCCGGCTGGTGAACGTA TGGCAGCAGAGCTGGCGCCGGGCGGTGAAGTTCTCATGTGCGCTGGCGCC GTGCATAGCCCGCACATTCTGCAGCTGTCTGGCGTTGGTTCGGCGGCTAC TCTGGCAGACCACGGCATCGCAGCAGTGGCAGATCTGCCAGGTGTTGGTG CGAACATGCAGGACCAGCCGGCCTGCCTGACAGCGGCTCCCCTGAAAGA CAAATACGATGGCATTTCGCTGACCGATCATATCTATAATAGCAAAGGCC AGATTCGCAAACGCGCTATCGCGTCCTACCTGCTTCAGGGTAAAGGTGGT CTGACGTCAACTGGCTGCGACCGTGGCGCGTTTGTACGTACCGCAGGCCA GGCACTGCCGGACCTGCAGGTGCGTTTCGTGCCAGGCATGGCACTGGATG CAGATGGTGTGTCCACCTACGTCCGTTTCGCAAAATTTCAGTCTCAGGGC CTGAAATGGCCGTCTGGCATCACCGTACAGCTTATTGCGTGTCGCCCGCA CAGCAAAGGTTCTGTTGGCCTGAAAAACGCGGACCCGTTCACCCCGCCGA AACTGCGTCCGGGCTACCTGACCGACAAAGCGGGTGCGGATCTGGCGAC CCTGCGCTCTGGTGTTCATTGGGCCCGTGATCTGGCATCTAGCGGTCCGCT GAGCGAATTTCTTGAAGGCGAACTGTTTCCGGGTAGCCAAGTTGTTTCCG ATGATGATATTGATTCTTACATTCGTCGTACCATTCACTCCAGCAACGCGA TTGTGGGCACCTGTCGTATGGGCGCGGCGGGTGAAGCGGGTGTTGTTGTG GATAACCAGCTGCGCGTTCAGGGTGTTGATGGTCTGCGTGTTGTTGACGC GAGCGTAATGCCGCGTATCCCAGGTGGTCAGGTGGGTGCGCCGGTTGTGA TGCTGGCCGAACGTGCAGCAGCGATGCTGACCGGTCAGGCAGCGCTGGCT GGTGCTAGCGCTGCAGCTCCGCCGACCCCGGTCGCGGCT

SEQ ID NO.2 (amino acid sequence of fatty acid decarboxylase McFAP):

MAEMAGGGEGDGMLMGGAGSANTTDACYSDPSNPDCAAFERSDDDWAAD IELLCSAMPFMPGCTLAEQCMNGTAAGEYCEMSSLAGNICLDMPGMKGCEA WNALCGAASAVEQCSSPGPVVALPTTALAKEGLESLCSTHCMDGCPDCEMG KLWNTCTDPLSVLAWMCYAMPDMPECLAAPQGSGMVVACGDAEVAATFP LVCAQPPTPAANFQHRLRTCRTAGVAASASGSPAVTMAGLSTVLAVLALLPS PVAMAMTPMPTPALAPGPAIDDIGGNCPLLGRGNMEAPCYSDPSAAACVSFE RSDAGWADDLSQLCSAMPYAVGCWLWHLCKTGAASGTYCALPSLTANVC VDAPLVNATSAPGCEAWAALCGAQGSVVAQCSAPGPLPDIINTLTTRDGINS LCGMHYMDGCNECTPHEGPAVHDFAACADPGPLPTLAHQCYAMPEMGECT QTGITAMCSGAEARATFPTVCVDPPNPTTLAPAPAVSACDVAAGAGAPPAAS ARPASHSRASLVASRSGFCAPSPALRSQRTSTVAPARRAASAPRASAVDDIQR ALSTAGSPVSGKQYDYILVGGGTAACVLANRLTADGSKRVLVLEAGADNVS RDVKVPAAITRLFRSPLDWNLFSELQEQLAARQIYMARGRLLGGSSATNATL YHRGAAADYDAWGVPGWGAADVLPWFVKAETNAEFAAGKYHGAGGNMR VENPRYSNPQLHGAFFAAAQQMGLPQNTDFNNWDQDHAGFGTFQVMQEK GTRADMYRQYLKPALGRPNLQVLTGASVTKVHIDKAGGKPRALGVEFSLDG PAGERMAAELAPGGEVLMCAGAVHSPHILQLSGVGSAATLADHGIAAVADL PGVGANMQDQPACLTAAPLKDKYDGISLTDHIYNSKGQIRKRAIASYLLQGK GGLTSTGCDRGAFVRTAGQALPDLQVRFVPGMALDADGVSTYVRFAKFQSQ GLKWPSGITVQLIACRPHSKGSVGLKNADPFTPPKLRPGYLTDKAGADLATL RSGVHWARDLASSGPLSEFLEGELFPGSQVVSDDDIDSYIRRTIHSSNAIVGTC RMGAAGEAGVVVDNQLRVQGVDGLRVVDASVMPRIPGGQVGAPVVMLAE RAAAMLTGQAALAGASAAAPPTPVAA

the mutant of fatty acid light decarboxylase McFAP (named as McFAP-S in the following) is obtained by shortening the N end of McFAP for deletion mutation, and the McFAP mutant pure enzyme (the nucleotide sequence is shown as SEQ ID NO.3, and the amino acid sequence is shown as SEQ ID NO. 4) is obtained by nickel column purification. The catalytic effect of the McFAP mutant is greatly improved, and the decarboxylation of medium-chain fatty acid with 6-12 carbon atoms can be catalyzed. Enriches the variety of FAP and supplements the FAP enzyme variety of the photocatalytic decarboxylation short-chain fatty acid.

SEQ ID NO.3 (encoding gene for a mutant of fatty acid decarboxylase McFAP):

CGTGCGAGCGCAGTTGACGATATTCAACGTGCTCTGAGCACCGCTGGAAG CCCGGTATCCGGTAAACAGTACGATTACATCCTGGTGGGTGGCGGCACCG CGGCATGCGTTTTGGCTAACCGTTTAACCGCGGACGGTAGCAAACGTGTA CTGGTGCTGGAAGCGGGTGCGGACAACGTGAGCCGCGATGTTAAAGTCC CGGCTGCGATCACCCGTTTGTTCCGTTCACCGTTGGATTGGAACTTGTTCA GCGAATTGCAGGAACAGCTGGCTGCACGTCAGATCTATATGGCTCGCGGC CGCTTGCTGGGTGGGTCTAGCGCGACCAATGCTACTCTTTACCACCGTGG CGCGGCGGCGGATTATGATGCGTGGGGCGTGCCGGGCTGGGGCGCAGCT GACGTGCTGCCATGGTTCGTTAAGGCCGAAACCAACGCGGAGTTTGCGGC GGGCAAATATCACGGCGCAGGTGGTAACATGCGCGTTGAGAATCCGCGC TACTCCAACCCGCAGCTGCACGGTGCTTTCTTTGCAGCTGCGCAGCAGAT GGGTCTGCCGCAGAATACCGACTTCAACAATTGGGATCAGGATCATGCAG GCTTTGGCACTTTTCAGGTTATGCAGGAAAAAGGCACCCGCGCTGATATG TACCGCCAGTATCTTAAACCAGCTCTTGGTCGTCCGAACCTGCAGGTTCTG ACCGGTGCGTCTGTGACCAAAGTTCATATCGATAAAGCTGGCGGTAAACC GCGTGCTCTGGGCGTAGAGTTTTCTCTGGATGGTCCGGCTGGTGAACGTA TGGCAGCAGAGCTGGCGCCGGGCGGTGAAGTTCTCATGTGCGCTGGCGCC GTGCATAGCCCGCACATTCTGCAGCTGTCTGGCGTTGGTTCGGCGGCTAC TCTGGCAGACCACGGCATCGCAGCAGTGGCAGATCTGCCAGGTGTTGGTG CGAACATGCAGGACCAGCCGGCCTGCCTGACAGCGGCTCCCCTGAAAGA CAAATACGATGGCATTTCGCTGACCGATCATATCTATAATAGCAAAGGCC AGATTCGCAAACGCGCTATCGCGTCCTACCTGCTTCAGGGTAAAGGTGGT CTGACGTCAACTGGCTGCGACCGTGGCGCGTTTGTACGTACCGCAGGCCA GGCACTGCCGGACCTGCAGGTGCGTTTCGTGCCAGGCATGGCACTGGATG CAGATGGTGTGTCCACCTACGTCCGTTTCGCAAAATTTCAGTCTCAGGGC CTGAAATGGCCGTCTGGCATCACCGTACAGCTTATTGCGTGTCGCCCGCA CAGCAAAGGTTCTGTTGGCCTGAAAAACGCGGACCCGTTCACCCCGCCGA AACTGCGTCCGGGCTACCTGACCGACAAAGCGGGTGCGGATCTGGCGAC CCTGCGCTCTGGTGTTCATTGGGCCCGTGATCTGGCATCTAGCGGTCCGCT GAGCGAATTTCTTGAAGGCGAACTGTTTCCGGGTAGCCAAGTTGTTTCCG ATGATGATATTGATTCTTACATTCGTCGTACCATTCACTCCAGCAACGCGA TTGTGGGCACCTGTCGTATGGGCGCGGCGGGTGAAGCGGGTGTTGTTGTG GATAACCAGCTGCGCGTTCAGGGTGTTGATGGTCTGCGTGTTGTTGACGC GAGCGTAATGCCGCGTATCCCAGGTGGTCAGGTGGGTGCGCCGGTTGTGA TGCTGGCCGAACGTGCAGCAGCGATGCTGACCGGTCAGGCAGCGCTGGCT GGTGCTAGCGCTGCAGCTCCGCCGACCCCGGTCGCGGCT

SEQ ID NO.4 (amino acid sequence of a mutant of fatty acid light decarboxylase McFAP):

RASAVDDIQRALSTAGSPVSGKQYDYILVGGGTAACVLANRLTADGSKRVL VLEAGADNVSRDVKVPAAITRLFRSPLDWNLFSELQEQLAARQIYMARGRLL GGSSATNATLYHRGAAADYDAWGVPGWGAADVLPWFVKAETNAEFAAGK YHGAGGNMRVENPRYSNPQLHGAFFAAAQQMGLPQNTDFNNWDQDHAGF GTFQVMQEKGTRADMYRQYLKPALGRPNLQVLTGASVTKVHIDKAGGKPR ALGVEFSLDGPAGERMAAELAPGGEVLMCAGAVHSPHILQLSGVGSAATLA DHGIAAVADLPGVGANMQDQPACLTAAPLKDKYDGISLTDHIYNSKGQIRK RAIASYLLQGKGGLTSTGCDRGAFVRTAGQALPDLQVRFVPGMALDADGVS TYVRFAKFQSQGLKWPSGITVQLIACRPHSKGSVGLKNADPFTPPKLRPGYLT DKAGADLATLRSGVHWARDLASSGPLSEFLEGELFPGSQVVSDDDIDSYIRR TIHSSNAIVGTCRMGAAGEAGVVVDNQLRVQGVDGLRVVDASVMPRIPGGQ VGAPVVMLAERAAAMLTGQAALAGASAAAPPTPVAA

in the following examples, the contents of fatty acids and alkanes before and after decarboxylation were measured by Agilent Technologies, Palo Alto, Calif., USA, gas chromatography 7890BThe analytical column is KB-FFAP (30m × 0.25mm, 0.25 μm), and the specific chromatographic analysis method is as follows: sample introduction volume: 1 mu L of the solution; sample injector temperature: 250 ℃; the split ratio is as follows: 30: 1; detector temperature: 280 ℃; the temperature rising procedure is as follows: the initial temperature was 110 deg.C, held for 3.4min, followed by 25 deg.C min ^-1 At a rate of 190 ℃ for 2.1min, and then again at 25 ℃ for a further min ^-1 At a rate of 230 deg.C for 2min and finally at 30 deg.C for 30min ^-1 The rate of (2) was increased to 250 ℃ and maintained for 12 min. The method comprises the steps of adopting various fatty acid and alkane standard substances to carry out qualitative determination of chromatographic peak time, adopting the standard substances to prepare standard solutions with different concentrations, 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 Biotechnology engineering (Shanghai) Inc.; the empty plasmid pET28a is stored in the laboratory of the applicant; coli BL21(DE3) competent cells, purchased from Biotech Limited; plasmid extraction kit, purchased from bio-engineering (Shanghai) Co., Ltd; all other chemicals were purchased from Sigma-Aldrich, TCI or Aladdin and were used in the highest purity without further purification.

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

Example 1 preparation of McFAP @ E.coli and validation of photocatalytic decarboxylation of catalytic fatty acids

Coli BL21(DE3) strain containing plasmid pET28a-McFAP was cultured in super Broth (Terrific Broth, TB) containing 50. mu.g/mL kanamycin at 37 ℃ when OD was measured ₆₀₀ When 0.7-0.8 was reached, 0.5mM isopropyl beta-D-1-thiogalactoside (IPTG) was added and the cells were incubated at 17 ℃ for 20 h. Centrifuging at 4000rpm at 4 deg.C for 30min to collect bacteria; washing with Tris-HCl buffer (50mM, pH 8, containing 100mM NaCl) and re-centrifuging (10000rpm, 20min, 4 ℃); the cell particles were mixed as follows 1: 2(w/v) was suspended in the same buffer, 1mM benzylxanthoyl chloride (PMSF) and 5% glycerol (w/v) were added, frozen in liquid nitrogen, and stored at-80 ℃ until use.

To verify the catalytic decarboxylation of McFAP, E.coli cells (denoted empty WC) containing the empty plasmid pET28a vector were prepared in the same manner.

500. mu.L of McFAP @ E.coli at a wet weight concentration of 0.5g/mL, 300. mu.L of 170mM fatty acid DMSO solution, 200. mu.L of LTris-HCl buffer (100mM, pH 8.0) was added to a 5mL transparent reaction flask, and the total reaction volume was 1 mL; then placing the mixture into a self-made photocatalytic reaction device (a blue light irradiation device is added on a common catalytic reaction device), and reacting for 12 hours under the irradiation of blue light (10W, 220V) at 500rpm and 30 ℃; after the reaction is finished, taking the reaction mixture to a 2mL EP tube, and adding 1mL of ethyl acetate solution of 25mM n-octanol internal standard for extraction, namely extracting the mixture in a volume ratio of 1: 1, after the extracted mixture was centrifuged at 11000rpm for 4min, the upper organic phase was taken for GC analysis in a 2mL chromatography flask. The same reaction system is placed in the dark, other conditions are unchanged, and decarboxylation of fatty acid is catalyzed.

Coli was changed to empty WC with other conditions unchanged, catalyzing the decarboxylation of fatty acids.

The results of the experiment are shown in FIG. 1. As can be seen from fig. 1, empty WC has no decarboxylation effect on fatty acids, McFAP @ e.coli has no decarboxylation effect on fatty acids under the condition of no blue light, and McFAP @ e.coli has decarboxylation effect on fatty acids under the condition of blue light, thereby verifying that McFAP @ e.coli catalyzes the decarboxylation reaction of fatty acid substrates under the condition of blue light.

Example 2 construction and purification of McFAP-S mutant

The total length of the McFAP gene is 3438bp, the total length is 1146 amino acids, the total amino acids from the 551 th amino acid from the N end to the C end are 596 amino acids which are used as the constructed mutant McFAP-S, the amino acid sequence is shown as SEQ ID NO.4, and the nucleotide sequence is shown as SEQ ID NO. 3.

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

TABLE 1 primers for construction of McFAP-S

TABLE 2PCR reaction System and procedure

And (3) recovering the amplified target gene and the amplified vector according to an operating manual for recovering the column type PCR product gel of the foundry SanPrep, determining the DNA concentrations of the target gene and the vector, performing seamless cloning according to the requirements of a seamless cloning kit (a reaction system and a program are shown in table 2), performing transformation culture on the obtained plasmid after the seamless cloning, and sending the obtained plasmid to a sample for sequencing, wherein the sequencing result is the constructed deletion mutant McFAP-S.

The instrument and the chromatographic column used for McFAP-S purification and the purification chromatographic column used for loading and collecting samples are as follows: his Prep ^TM FF16/10；HiPrep ^TM 26/10 changing salt column. The column was equilibrated with loading buffer (50mM Tris-HCl 300mM NaCl 10mM imidazole 5% (v/v) glycerol pH 9), after equilibration the crude enzyme solution was pumped in and after loading was complete the column was equilibrated with loading buffer. Elution was then performed with elution buffer (50mM Tris-HCl 300mM NaCl 500mM imidazole 5% (v/v) glycerol pH 9), and peak samples were collected and examined by SDS-PAGE electrophoresis. And (3) carrying out salt exchange on the sample containing the peak corresponding to the target protein by using a salt exchange column. Adding the protein into a salt exchange column well balanced by a salt exchange buffer solution (50mM Tris-HCl 150mM NaCl 5% (v/v) glycerol pH 9), continuously eluting by the salt exchange buffer solution, collecting eluted protein, concentrating, subpackaging, pre-freezing by liquid nitrogen, and storing at-80 ℃ for later use. The whole flow velocity is 5mLmin ^-1 。

The SDS-PAGE protein map of McFAP-S after nickel column purification and 0.5M imidazole elution is shown in figure 2, and the McFAP-S obtains better expression and purification effects from figure 2.

Example 3 characterization of the enzymatic Properties of McFAP-S

FAP enzyme activity definition: the amount of enzyme required to catalyze the reaction of 1. mu. mol of n-octanoic acid to produce 1. mu. mol of n-heptane within 1min under irradiation with blue light (10W, 220V) at 30 ℃ and 500rpm was defined as 1U.

1. Optimization of reaction time

In this example, 12 reaction times were selected to optimize the process of decarboxylation of n-octanoic acid catalyzed by McFAP-S (5min, 10min, 15min, 20min, 25min, 30min, 35min, 40min, 45min, 50min, 55min and 60min), wherein 150. mu.L of 140mM DMSO solution of n-octanoic acid (20 mM concentration of n-octanoic acid in the reaction system, 15% addition of DMSO), 40. mu.M enzyme addition, 1mL of Tris-HCl buffer (100mM, pH 9.0) was added to a 5mL transparent reaction flask. Then placing the mixture into a self-made photocatalytic reaction device, and reacting for a certain time under the irradiation of blue light (10W, 220V) at the speed of 500rpm and the temperature of 30 ℃; after the reaction is finished, taking the reaction mixture to a 2mL EP tube, and adding 1mL of ethyl acetate solution of 25mM n-octanol internal standard for extraction, namely extracting the mixture in a volume ratio of 1: 1, after the extracted mixture was centrifuged at 11000rpm for 4min, the upper organic phase was taken for GC analysis in a 2mL chromatography flask.

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

2. Optimization of the amount of reaction enzyme

In this example, the addition of 4 enzymes was selected to optimize the process of McFAP-S catalyzed palmitic acid decarboxylation (final concentrations of 6. mu.M, 12. mu.M, 24. mu.M and 36. mu.M for the reaction enzymes), and the conditions and treatment were the same as above.

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

3. Optimization of reaction substrate concentration

In this example, 5 substrate concentrations were selected to optimize the process of McFAP-S catalyzed palmitic acid decarboxylation (reaction substrate concentrations 10mM, 20mM, 30mM, 40mM, 50mM, respectively), and other conditions and treatment were the same as above.

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

4. Optimization of reaction temperature

In this example, 5 reaction temperatures were selected to optimize the process of McFAP-S catalyzed palmitic acid decarboxylation (reaction temperatures 20 deg.C, 30 deg.C, 40 deg.C, 45 deg.C, 50 deg.C, respectively), and other conditions and treatment methods were unchanged, as above.

The result is shown in figure 6, the optimum temperature of the MCFAP-S for catalyzing the decarboxylation of the caprylic acid is 40 ℃, the relative enzyme activity is more than 80% at the temperature of 30-45 ℃, and the relative enzyme activity is rapidly reduced when the temperature is higher than 45 ℃.

5. Optimization of reaction pH

In this example, 5 phs were selected to optimize the process of catalytic palmitic acid decarboxylation by McFAP-S (the pH of the reaction system was 6, 7, 8, 9, and 10, respectively), and the other conditions and treatment methods were unchanged, and the procedure was the same as above.

As shown in FIG. 7, the optimum temperature of the MCFAP-S for catalyzing the decarboxylation of the n-caprylic acid is 8-9, but the conversion rate is more than 80% within the range of pH 6-10, i.e., the MCFAP-S has good catalytic effect for catalyzing the decarboxylation of the n-caprylic acid within the range of pH 6-10.

6. Investigation of storage stability

In this example, the storage stability of enzyme activity of McFAP-S catalyzing palmitic acid decarboxylation under dark condition at 4 ℃ is selected to be examined, the residual enzyme activity of McFAP-S catalyzing palmitic acid decarboxylation is selected to be examined for 12 storage times (the storage time is 10min, 30min, 1h, 3h, 6h, 12h, 1d, 2d, 3d, 5d, 7d, 10d), and other conditions and treatment modes are not changed, and the steps are the same as above.

As shown in FIG. 8, the residual activity of McFAP-S for catalyzing decarboxylation of n-octanoic acid is still more than 70% when the McFAP-S is stored at 4 ℃ for 10 days in the dark.

7. Optimizing pH tolerance

In this example, 5 phs were selected for incubation of the McFAP-S enzyme solution (pH of the incubation system was 6, 7, 8, 9, and 10, respectively), the incubation condition was 4 ℃ in a dark condition, and other conditions and treatment methods were not changed, and the procedure was as above.

The result is shown in figure 9, under the dark condition of 4 ℃, the residual activity of the MCFAP-S for catalyzing decarboxylation of the caprylic acid is still more than 50% after incubation for 5d within the range of pH 6-10. Meanwhile, the pH value is within the range of 6-10, the pH value has no obvious influence on the enzyme activity of McFAP-S, and the residual activity after incubation at the pH value of 6-8 is slightly better than that at the pH value of 9-10.

8. Investigation of light inactivation factor

In this example, the storage stability of the enzyme activity of the McFAP-S catalyzing palmitic acid decarboxylation under normal temperature storage under different illumination conditions is selected to be examined, 5 different illumination environments are selected to incubate the McFAP-S pure enzyme solution (blue light irradiation, sunlight irradiation, dark storage, red light irradiation, and red light irradiation co-incubation with 5% DMSO 10mM n-octanoic acid contained in the system), 5 storage times are selected to examine the residual enzyme activity of the McFAP-S catalyzing palmitic acid decarboxylation (the storage time is 10min, 30min, 1h, 2h, and 3h), other conditions and treatment modes are not changed, and the steps are the same as above.

The result is shown in figure 10, under the condition of normal temperature, the influence of blue light irradiation on the activity of pure enzyme of McFAP-S is the largest, and the residual enzyme activity is less than 10% after 10 min; after 3 hours of sunlight irradiation, the residual enzyme activity of McFAP-S is less than 50 percent; under the conditions of darkness and red light irradiation, the residual enzyme activity of McFAP-S is still over 90 percent after 3 hours; and the McFAP-S under the condition of co-incubation of 10mM n-caprylic acid substrate and 5% DMSO is added, the residual enzyme activity is still more than 90% after 3 hours of sunlight irradiation, namely, the photo-inactivation of the McFAP-S is obviously inhibited by adding the substrate for co-incubation. For the above reasons, the following examples were conducted in the form of whole-cell catalysis to perform the research experiments on the substrate expansion of saturated fatty acids with different chain lengths.

Example 4McFAP @ E.coli and McFAP-S @ E.coli catalyzed substrate extension studies of fatty acids

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

The result is shown in fig. 11, both McFAP @ e.coli and McFAP-S @ e.coli can catalyze the fatty acid decarboxylation effect of C6: 0-C18: 0, and under the same whole cell addition amount, the efficiency of McFAP-S @ e.coli in catalyzing the fatty acid decarboxylation of C7: 0-C12: 0 is significantly higher than that of McFAP @ e.coli in catalyzing the fatty acid decarboxylation of C7: 0-C12: 0.

Example 5McFAP @ E.coli vs. CvFAP @ E.coli catalytic palmitic acid decarboxylation efficiency comparison

In this example, 6 reaction times were selected to compare the decarboxylation efficiencies (1, 2, 3, 4, 5, and 6h) of McFAP @ e.coli and CvFAP @ e.coli catalyzed palmitic acid, wherein the addition amounts of McFAP @ e.coli and CvFAP @ e.coli were 0.25g/mL, and other relevant reaction conditions were performed according to the procedure of example 1.

The result is shown in figure 12, after the reaction for catalyzing the decarboxylation of the palmitic acid by McFAP @ E.coli for 6 hours, the conversion rate of the catalytic decarboxylation of the palmitic acid can reach more than 90%. Under the same reaction condition, 30mM of product is generated after the CvFAP @ E. coli reaction is carried out for 6 hours, and the conversion rate is only about 60%. Therefore, under the condition of the same whole cell addition amount, the decarboxylation efficiency of the palmitic acid catalyzed by McFAP @ E.

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Sequence listing

<110> university of south China's Rich Industrial science, Guangdong excellent enzyme Biomanufacturing research institute, Inc

<120> fatty acid light decarboxylase McFAP mutant 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 (10)

1. A mutant of fatty acid light decarboxylase McFAP is characterized in that the amino acid sequence of the mutant of the fatty acid light decarboxylase McFAP is shown as SEQ ID NO. 4.

2. The gene encoding the fatty acid decarboxylase McFAP mutant as claimed in claim 1, wherein the nucleotide sequence of the encoding gene is shown in SEQ ID No. 3.

3. The mutant of fatty acid decarboxylase McFAP as claimed in claim 1 or the use of the coding gene as claimed in claim 2 for catalyzing the decarboxylation of fatty acid.

4. The use according to claim 3, wherein the fatty acid has 6 to 12 carbon atoms.

5. The use according to claim 4, wherein the fatty acid has 7 to 8 carbon atoms.

6. Use according to any one of claims 3 to 5, wherein the fatty acid is a saturated straight chain fatty acid.

7. A recombinant expression vector into which the coding gene of claim 2 is inserted.

8. A recombinant engineered strain transformed with the recombinant expression vector of claim 7.

9. Use of the recombinant expression vector of claim 7, or the recombinant engineered strain of claim 8, for catalyzing the decarboxylation of a fatty acid.

10. A method for catalyzing decarboxylation of fatty acid, which comprises performing a catalytic reaction using the whole cell of the fatty acid decarboxylase McFAP mutant according to claim 1.

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