WO2021077543A1 - 一种亚油酸异构酶及其在共轭亚油酸生产中的应用 - Google Patents

一种亚油酸异构酶及其在共轭亚油酸生产中的应用 Download PDF

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WO2021077543A1
WO2021077543A1 PCT/CN2019/121822 CN2019121822W WO2021077543A1 WO 2021077543 A1 WO2021077543 A1 WO 2021077543A1 CN 2019121822 W CN2019121822 W CN 2019121822W WO 2021077543 A1 WO2021077543 A1 WO 2021077543A1
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linoleic acid
host cell
conjugated linoleic
seq
cla
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PCT/CN2019/121822
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French (fr)
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陈海琴
杨波
高鹤
赵建新
陈永泉
张灏
陈卫
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江南大学
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Priority claimed from CN201911011728.3A external-priority patent/CN112695024B/zh
Priority claimed from CN201911025170.4A external-priority patent/CN112708570B/zh
Priority claimed from CN201911020755.7A external-priority patent/CN112708587B/zh
Application filed by 江南大学 filed Critical 江南大学
Publication of WO2021077543A1 publication Critical patent/WO2021077543A1/zh
Priority to US17/497,963 priority Critical patent/US20220017886A1/en

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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y502/00—Cis-trans-isomerases (5.2)
    • C12Y502/01—Cis-trans-Isomerases (5.2.1)
    • C12Y502/01005—Linoleate isomerase (5.2.1.5)
    • C—CHEMISTRY; METALLURGY
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
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    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6409—Fatty acids
    • C12P7/6427—Polyunsaturated fatty acids [PUFA], i.e. having two or more double bonds in their backbone
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    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6409—Fatty acids
    • C12P7/6427—Polyunsaturated fatty acids [PUFA], i.e. having two or more double bonds in their backbone
    • C12P7/6431—Linoleic acids [18:2[n-6]]
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    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/185—Escherichia
    • C12R2001/19—Escherichia coli
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    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/225—Lactobacillus
    • C12R2001/25—Lactobacillus plantarum
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    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi

Definitions

  • the invention relates to a linoleic acid isomerase and its application in the production of conjugated linoleic acid, belonging to the technical field of protein engineering and microbial engineering.
  • Conjugated linoleic acid is a general term for a series of fatty acids that contain conjugated double bonds and have multiple positions and geometric isomers. Studies have shown that conjugated linoleic acid has anti-cancer, lipid-lowering, anti-atherosclerotic, physiological effects such as regulating energy metabolism, enhancing immunity and promoting growth and development. It is widely used in medicine, food, cosmetics and other fields.
  • cis9, trans11-CLA and trans10, cis12-CLA are the two most physiologically active isomers of conjugated linoleic acid. Therefore, there is great demand for cis9, trans11-CLA and trans10, cis12-CLA in the market.
  • Natural conjugated linoleic acid mainly exists in rumen animals, certain plants and marine organisms, and these natural conjugated linoleic acids mainly exist in the form of cis9, trans11-CLA, and their physiological activities are extremely high. However, they The content is very small, and it is difficult to meet the market demand for conjugated linoleic acid. Therefore, people have gradually developed a method of artificially synthesizing conjugated linoleic acid.
  • the main methods of artificially synthesizing conjugated linoleic acid are chemical synthesis and microbial synthesis.
  • chemical synthesis will cause the production of many toxic by-products, which are toxic to the environment and the human body.
  • conjugated linoleic acid isomers prepared by chemical synthesis, making it difficult to carry out effective Therefore, the chemical synthesis method cannot truly realize the large-scale industrial production of conjugated linoleic acid.
  • microbial synthesis has the advantages of less pollution and a single type of conjugated linoleic acid isomers. Therefore, microbial synthesis is a promising method for large-scale industrial production of conjugated linoleic acid. Methods.
  • conjugated linoleic acid production strains for industrial production, for example, Vibrio cellulolyticus butyric acid , Propionibacterium and Clostridium sporogenum, etc.;
  • conjugated linoleic acid by some microorganisms that can produce high conjugated linoleic acid is not high. If it is used as a conjugated linoleic acid production strain for industrial production, the production efficiency is too low, for example, Lactobacillus plantarum ZS2058 (see details Reference: Qi Hui, Yang Bo, etc. Study on the mechanism of biotransformation of conjugated linoleic acid by Lactobacillus plantarum ZS2058[D], Jiangnan University, 2017);
  • Lactobacillus plantarum ZS2058 see references for details: Qi Hui, Yang Bo, etc. Study on the mechanism of biotransformation of conjugated linoleic acid by Lactobacillus plantarum ZS2058 [D], Jiangnan University, 2017),
  • the present invention provides a linoleic acid isomerase (EC 5.2.1.5), characterized in that the linoleic acid isomerase is:
  • (b) A protein derived from (a) in which the amino acid sequence in (a) has been substituted, deleted or added one or several amino acids and has linoleate isomerase activity.
  • the linoleic acid isomerase composed of the amino acid sequence shown in SEQ ID No. 1 is derived from Bifidobacterium breve, and the linoleic acid isomerase is derived from Bifidobacterium breve (Bifidobacterium breve).
  • the linoleate isomerase composed of the amino acid sequence shown is derived from Bifidobacterium longum
  • the linoleate isomerase composed of the amino acid sequence shown in SEQ ID No. 3 is derived from pseudo-small chains Bifidobacterium pseudocatenulatum
  • the linoleic acid isomerase composed of the amino acid sequence shown in SEQ ID No. 4 is derived from Bifidobacterium dentium.
  • the present invention also provides a gene encoding the above-mentioned linoleate isomerase.
  • the nucleotide sequence of the gene is such as SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7, SEQ ID No. 8, SEQ ID No. 17 or SEQ ID No.18 is shown.
  • the present invention also provides a recombinant plasmid, which carries the above-mentioned gene.
  • the vector of the recombinant plasmid is pET-28a(+) plasmid, pINA 1312sp plasmid or pNZ44 plasmid.
  • the present invention also provides a host cell which carries the above-mentioned gene or the above-mentioned recombinant plasmid.
  • the host cell is Escherichia coli, Yarrowia lipolytica or Lactobacillus plantarum.
  • the host cell when the host cell is Escherichia coli, the host cell uses the pET-28a(+) plasmid as a vector to carry nucleotide sequences such as SEQ ID No. 5, SEQ ID No. 6, The gene encoding the above-mentioned linoleic acid isomerase shown in SEQ ID No. 7 or SEQ ID No. 8;
  • the host cell uses the pINA 1312sp plasmid as a vector to carry the gene encoding the above-mentioned linoleate isomerase with a nucleotide sequence as shown in SEQ ID No. 17;
  • the host cell When the host cell is Lactobacillus plantarum, the host cell uses the pNZ44 plasmid as a vector to carry the gene encoding the above-mentioned linoleate isomerase as shown in SEQ ID No. 18.
  • the present invention also provides the application of the above-mentioned linoleic acid isomerase or the above-mentioned gene or the above-mentioned recombinant plasmid or the above-mentioned host cell in the production of conjugated linoleic acid.
  • the present invention also provides a method for producing conjugated linoleic acid, which method uses the above-mentioned host cell;
  • the method is to inoculate the above-mentioned host cell into a culture medium, and culture it at a temperature of 35-40°C and a rotation speed of 150-250 rpm to an OD 600 of 0.4-0.6 to obtain a culture solution A; Add IPTG with a final concentration of 0.01-1.0 mM to culture solution A, and induce culture for 12-16 hours at a temperature of 15-20°C and a rotation speed of 150-250 rpm to obtain culture solution B; centrifuge culture solution B , Collect wet bacteria; use linoleic acid as a substrate, add wet bacteria to the reaction system containing linoleic acid, and react at a temperature of 35 to 40°C and a rotation speed of 150 to 250 rpm to obtain a rich A reaction solution of conjugated linoleic acid; extracting the reaction solution rich in conjugated linoleic acid to obtain conjugated linoleic acid;
  • the method is to inoculate the above host cell into a medium containing linoleic acid and/or glycerides at a temperature of 35-40°C and a rotation speed of 150-250 rpm. Culture the host cells rich in conjugated linoleic acid, and then extract the host cells rich in conjugated linoleic acid to obtain conjugated linoleic acid;
  • the method is to inoculate the above-mentioned host cell into a medium containing linoleic acid, and statically cultivate it at a temperature of 37°C to obtain a culture rich in conjugated linoleic acid Liquid; extract the culture solution rich in conjugated linoleic acid to obtain conjugated linoleic acid.
  • the method when the host cell is Escherichia coli, the method is to inoculate the above host cell into a culture medium, and cultivate it at a temperature of 37° C. and a rotation speed of 200 rpm until the OD 600 is 0.4. ⁇ 0.6 to obtain culture solution A; add IPTG with a final concentration of 0.01 to 1.0 mM to culture solution A, and induce culture for 12 to 16 hours at a temperature of 18°C and a rotation speed of 200 rpm to obtain culture solution B; B.
  • Centrifuge to collect the wet bacteria; using linoleic acid as the substrate, add the wet bacteria to the reaction system containing linoleic acid, and perform the reaction at a temperature of 37°C and a rotation speed of 200 rpm to obtain a rich conjugate
  • the reaction liquid of linoleic acid; the reaction liquid rich in conjugated linoleic acid is extracted to obtain conjugated linoleic acid.
  • the glyceride when the host cell is Yarrowia lipolytica, is safflower seed oil, linseed oil, cottonseed oil, and/or soybean oil.
  • the glyceride when the host cell is Yarrowia lipolytica, is safflower seed oil.
  • the reaction system when the host cell is Escherichia coli, the reaction system includes a buffer and linoleic acid.
  • the pH of the buffer is 6-7.
  • the pH of the buffer is 6.5.
  • the buffer when the host cell is Escherichia coli, the buffer is potassium phosphate buffer.
  • the concentration of the linoleic acid in the reaction system is 0.05-0.15 mg/mL.
  • the concentration of the linoleic acid in the reaction system is 0.1 mg/mL.
  • the concentration of the wet bacteria in the reaction system is 0.5-2 mg/mL.
  • the concentration of the wet bacteria in the reaction system is 1 mg/mL.
  • the conjugated linoleic acid when the host cell is Escherichia coli, the conjugated linoleic acid is cis9,trans11-CLA and/or trans9,trans11-CLA; when the host cell is Yarrowia lipolytica The conjugated linoleic acid is cis9, trans11-CLA, trans10, cis12-CLA and/or trans9, trans11-CLA; when the host cell is Lactobacillus plantarum, the conjugated linoleic acid is cis9, trans11- CLA.
  • the conjugated linoleic acid is cis9, trans11-CLA.
  • the medium when the host cell is Escherichia coli, the medium is LB medium; when the host cell is Yarrowia lipolytica, the medium is YPD medium; when the host cell In the case of Lactobacillus plantarum, the medium is MRS medium.
  • the present invention also provides the application of the above-mentioned host cell in the production of linoleate isomerase.
  • the amino acid sequence of the linoleate isomerase is shown in SEQ ID No. 1.
  • the present invention also provides a method for producing the above-mentioned linoleic acid isomerase, characterized in that the above-mentioned host cell is used;
  • the method is to add the above host cell to the culture medium and culture it at a temperature of 35-40°C and a rotation speed of 150-250 rpm to obtain a host rich in linoleic acid isomerase Then extract the host cell rich in linoleate isomerase to obtain linoleate isomerase;
  • the method is to inoculate the above-mentioned host cell into a culture medium and cultivate it at a temperature of 35-40°C and a rotation speed of 150-250 rpm to obtain isoforms rich in linoleic acid.
  • the method is to inoculate the above-mentioned host cell into the inoculation medium, and statically culture it at a temperature of 37°C to obtain a host cell rich in linoleic acid isomerase. Then the host cells rich in linoleate isomerase are extracted to obtain linoleate isomerase.
  • the medium when the host cell is Escherichia coli, the medium is LB medium; when the host cell is Yarrowia lipolytica, the medium is YPD medium; when the host cell In the case of Lactobacillus plantarum, the medium is MRS medium.
  • the amino acid sequence of the present invention derived from Bifidobacterium breve is shown in SEQ ID No. 1.
  • the linoleic acid isomerase can be used to produce conjugated linoleic acid and will contain the linoleic acid of the present invention.
  • Recombinant Escherichia coli with isomerase was added to the reaction system containing linoleic acid for 3 hours to increase the conversion rate of conjugated linoleic acid as high as 42.1%, and the content of cis9, trans11-CLA in conjugated linoleic acid As high as 89.1%, this result provides substantial theoretical support for further obtaining microorganisms with high safety and high yield through genetic engineering methods, and most of the conjugated linoleic acid monomers produced are cis9,trans11-CLA.
  • the amino acid sequence of the present invention derived from Bifidobacterium longum is shown in SEQ ID No. 2.
  • the linoleic acid isomerase can be used to produce conjugated linoleic acid, which will contain the linoleic acid of the present invention.
  • Recombinant Escherichia coli with isomerase was added to the reaction system containing linoleic acid for 3 hours to increase the conversion rate of conjugated linoleic acid as high as 12.1%, and the content of cis9, trans11-CLA in conjugated linoleic acid As high as 84.3%, this result provides substantial theoretical support for further obtaining microorganisms with high safety and high yield through genetic engineering methods, and the conjugated linoleic acid monomers produced are mostly cis9,trans11-CLA.
  • the amino acid sequence of the present invention derived from Bifidobacterium pseudocatenulatum (Bifidobacterium pseudocatenulatum) is shown in SEQ ID No. 3, and the linoleic acid isomerase can be used to produce conjugated linoleic acid, which will contain the amino acid sequence of the present invention.
  • Recombinant Escherichia coli with oleate isomerase was added to the reaction system containing linoleic acid to react for 3 hours, which can make the conversion rate of conjugated linoleic acid as high as 19.5%, and can make cis9,trans11-CLA in conjugated linoleic acid The content is as high as 88.9%. This result provides substantial theoretical support for further obtaining microorganisms with high safety and high yield through genetic engineering methods, and most of the conjugated linoleic acid monomers produced are cis9,trans11-CLA.
  • the linoleic acid isomerase with the amino acid sequence derived from Bifidobacterium dentium of the present invention as shown in SEQ ID No. 4 can be used to produce conjugated linoleic acid, which will contain the linoleic acid of the present invention.
  • Recombinant Escherichia coli with isomerase was added to the reaction system containing linoleic acid for 3 hours to increase the conversion rate of conjugated linoleic acid as high as 13.5% and the content of cis9, trans11-CLA in conjugated linoleic acid As high as 87.1%, this result provides substantial theoretical support for further obtaining microorganisms with high safety and high yield through genetic engineering methods, and the conjugated linoleic acid monomers produced are mostly cis9,trans11-CLA.
  • the recombinant Yarrowia lipolytic yeast strain of the present invention can be transformed to produce conjugated linoleic acid using glycerides such as safflower seed oil as a substrate, and can produce high conjugated linoleic acid, and the produced conjugated linoleic acid Oleic acid isomers are mostly cis9, trans11-CLA; adding the recombinant Yarrowia lipolytica strain of the present invention to a culture medium containing free linoleic acid for 36 hours can increase the yield of conjugated linoleic acid in the fermentation broth.
  • the output of cis9,trans11-CLA is as high as 1.5mg/L, accounting for about 25% of the total conjugated linoleic acid output;
  • the recombinant Yarrowia lipolytic yeast strain of the present invention is added to the safflower seed oil-containing Cultured in the medium for 36 hours, the output of conjugated linoleic acid in the fermentation broth can be as high as 751.7mg/L, among which the output of cis9 and trans11-CLA is as high as 224.0mg/L, accounting for about 29.8% of the total output of conjugated linoleic acid. .
  • Yarrowia lipolytica is a microorganism that is generally regarded as safe (GRAS), and Yarrowia lipolytica has been recognized by the European Union as a safe strain that can be used in food. Therefore, the present invention is recombinant The conjugated linoleic acid produced by the lipolytic yeast strain is relatively safer.
  • Yarrowia lipolytic yeast belongs to strict aerobic bacteria, which is easier to realize industrial cultivation than strict anaerobic bacteria, and the industrial production process using Yarrowia lipolytic yeast as the production strain has been very mature. Therefore, the reorganization of the present invention Yarrowia lipolytic yeast strains are more suitable for large-scale industrial production.
  • the recombinant Yarrowia lipolytica strain of the present invention can use safflower seed oil and other glycerides as a substrate to transform and produce conjugated linoleic acid, with high yield And the produced conjugated linoleic acid isomers are mostly cis9, trans11-CLA. Therefore, the recombinant Yarrowia lipolytic yeast strain of the present invention has lower cost and is more suitable for large-scale industrial production.
  • the Lactobacillus plantarum engineered bacteria of the present invention can produce high conjugated linoleic acid and the conjugated linoleic acid isomers produced are mostly cis9, trans11-CLA.
  • the engineered Lactobacillus plantarum bacteria of the present invention are added Cultivated in a medium containing linoleic acid for 72 hours, the conversion rate of conjugated linoleic acid can be as high as 89.9%, and the content of cis9, trans11-CLA in conjugated linoleic acid can be as high as 100%.
  • Lactobacillus plantarum is a kind of probiotics, and has been included in the "List of Bacteria that Can be Used in Food" issued by the Ministry of Health. Therefore, the conjugated linoleic acid pair produced by the engineered Lactobacillus plantarum of the present invention As far as the human body is concerned, the safety is higher.
  • Lactobacillus plantarum is a facultative aerobic bacteria, which is easier to cultivate than strict anaerobic bacteria and is suitable for large-scale industrial production.
  • Figure 1 IPTG concentration versus recombinant E.coli BL21(DE3)/pET28a-bbi, E.coli BL21(DE3)/pET28a-bli, E.coli BL21(DE3)/pET28a-bpi and E.coli BL21( The influence of DE3)/pET28a-bdi conversion rate of conjugated linoleic acid.
  • Figure 2 Types of conjugated linoleic acid isomers in conjugated linoleic acid produced by recombinant E. coli BL21(DE3)/pET28a-bbi and the proportion of each conjugated linoleic acid isomer.
  • Figure 3 Types of conjugated linoleic acid isomers in conjugated linoleic acid produced by recombinant E. coli BL21(DE3)/pET28a-bli and the proportion of each conjugated linoleic acid isomer.
  • Figure 4 Types of conjugated linoleic acid isomers in conjugated linoleic acid produced by recombinant E. coli BL21(DE3)/pET28a-bpi and the proportion of each conjugated linoleic acid isomer.
  • Figure 5 Types of conjugated linoleic acid isomers in conjugated linoleic acid produced by recombinant E. coli BL21(DE3)/pET28a-bdi and the proportion of each conjugated linoleic acid isomer.
  • Figure 6 CAI map of the uncodon-optimized bbi sequence and the codon-optimized bbi sequence.
  • Figure 7 Plasmid map of recombinant plasmid pINA 1312sp-obbi.
  • Figure 8 PCR verification results of the recombinant plasmid pINA 1312sp-obbi.
  • Figure 9 PCR verification result of the recombinant Yarrowia lipolytica strain Yarrowia lipolytica/pINA 1312sp-obbi.
  • Figure 10 Yield of recombinant Yarrowia lipolytica/pINA 1312sp-obbi using free fatty acids as a substrate to produce cis9, trans11-CLA, trans10, cis12-CLA and trans9, trans11-CLA.
  • Figure 11 The contents of cis9, trans11-CLA, trans10, cis12-CLA and trans9, trans11-CLA produced by recombinant Yarrowia lipolytica/pINA 1312sp-obbi using free fatty acids as a substrate account for the total amount of production The ratio of conjugated fatty acid content.
  • Figure 12 Yield of recombinant Yarrowia lipolytica/pINA 1312sp-obbi using safflower seed oil as a substrate to produce cis9, trans11-CLA, trans10, cis12-CLA and trans9, trans11-CLA.
  • Figure 13 The production of cis9, trans11-CLA, trans10, cis12-CLA and trans9, trans11-CLA produced by recombinant Yarrowia lipolytica/pINA 1312sp-obbi using safflower seed oil as a substrate accounts for the production The ratio of total conjugated fatty acid production.
  • Figure 14 GC-MS identification map of the recombinant Yarrowia lipolytica/pINA 1312sp-obbi produced conjugated linoleic acid under different substrates; among them, (a) represents the recombinant Yarrowia lipolytica strain Yarrowia lipolytica/pINA 1312sp-obbi uses free fatty acids as a substrate to produce conjugated linoleic acid.
  • (b) represents the recombinant Yarrowia lipolytica strain Yarrowia lipolytica/pINA 1312sp-obbi uses safflower seed oil as The GC-MS identification spectrum of conjugated linoleic acid obtained from the production of the substrate
  • (c) represents the composition chromatogram of the fatty acid in the sample (the sample here is a standard product of conjugated linoleic acid purchased from Sigma), numbers 1, 2 , 3 respectively represent cis9, trans11-CLA, trans10, cis12-CLA and trans9, trans11-CLA.
  • Figure 15 The effect of safflower seed oil concentration on the yield of cis9 and trans11-CLA produced by recombinant Yarrowia lipolytica/pINA 1312sp-obbi.
  • Figure 16 The effect of safflower seed oil concentration on the yield of cis9 and trans11-CLA produced by recombinant Yarrowia lipolytica/pINA 1312sp-obbi after 84 hours of fermentation.
  • Figure 17 Safflower seed oil concentration versus the ratio of the yield of cis9 and trans11-CLA produced by the recombinant Yarrowia lipolytica/pINA 1312sp-obbi strain Yarrowia lipolytica/pINA 1312sp-obbi to the total yield of conjugated fatty acids produced during 84 hours of fermentation.
  • Figure 18 The effect of safflower seed oil concentration on the yield and conversion rate of cis9, trans11-CLA produced by recombinant Yarrowia lipolytica/pINA 1312sp-obbi.
  • Figure 19 PCR verification results of recombinant plasmids pNZ44-bbi and pNZ44-bbi(U).
  • Figure 20 GC-MS identification chromatogram of conjugated linoleic acid produced by the engineered strain Lactobacillus plantarum ST-III/pNZ44-bbi(U).
  • Figure 21 GC-MS mass spectrum fragments of conjugated linoleic acid produced by Lactobacillus plantarum ST-III/pNZ44-bbi(U).
  • Escherichia coli DH5 ⁇ and E.coli BL21(DE3) involved in the following examples were purchased from General Biotechnology Co., Ltd.; the pET-28a(+) vector involved in the following examples was purchased from Invitrogen; The bacterial genomic DNA extraction kit and plasmid extraction kit involved in the above examples were purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd., and the models were DP302 and DP103 respectively; the free linoleic acid involved in the following examples was purchased from Sigma Company; the safflower seed oil involved in the following examples was purchased from COFCO (Changji) Cereals and Oils Industry Co., Ltd.; the Yarrowia lipolytica involved in the following examples was purchased from Bina Biotech, the product number is : BNCC193899; the pINA 1312 plasmid involved in the following examples was purchased from the BioVector plasmid vector strain cell gene collection center; the construction method of the pINA 1312
  • Microb Cell Fact. 12: 70 The construction method of the pNZ44 plasmid involved in the above examples is described in the document "McGrath, S. et al., 2001. Improvement and optimization of two engineered phage resistance mechanisms in Lactobacoccus lactic. Applied and Environmental Microbiology, 67(2): 608- 616.”
  • MRS solid medium peptone 10g/L, beef extract 10g/L, glucose 20g/L, sodium acetate 2g/L, yeast powder 5g/L, diammonium hydrogen citrate 2g/L, K 2 HPO 4 ⁇ 3H 2 O 2.6g/L, MgSO 4 ⁇ 7H 2 O 0.1g/L, MnSO 4 ⁇ H 2 O 0.05g/L, Tween 80 1mL/L, agar 15g/L, cysteine salt 0.5g/L .
  • MRS liquid medium peptone 10g/L, beef extract 10g/L, glucose 20g/L, sodium acetate 2g/L, yeast powder 5g/L, diammonium hydrogen citrate 2g/L, K 2 HPO 4 ⁇ 3H 2 O 2.6 g/L, MgSO 4 ⁇ 7H 2 O 0.1 g/L, MnSO 4 ⁇ H 2 O 0.05 g/L, Tween 80 1 mL/L, and cysteine salt 0.5 g/L.
  • LB liquid medium tryptone 10g/L, yeast extract 5g/L, sodium chloride 10g/L, add 100 ⁇ g/mL kanamycin before use.
  • LB solid medium tryptone 10g/L, yeast extract 5g/L, sodium chloride 10g/L, agar 15g/L, add 100 ⁇ g/mL kanamycin before use.
  • YNBD solid medium yeast containing nitrogen bases (without amino acids) 6.7g/L, glucose 20g/L, agar 15g/L, pH 5.5.
  • YNBD liquid medium Yeast contains 6.7 g/L nitrogen bases (without amino acids), glucose 20 g/L, and the pH is 5.5.
  • YPD medium peptone 20g/L, yeast extract 10g/L, glucose 20g/L, pH 6.5.
  • Method for detecting specific enzyme activity of linoleic acid isomerase Collect the bacteria, add the bacteria to KPB buffer (pH 6.5), and break the bacteria with glass beads to obtain a cell disruption solution; add the cell disruption solution to 8000g Centrifuge for 10 minutes to collect the supernatant to obtain the crude enzyme solution; adjust the protein content of the crude enzyme solution to 0.5 mg/mL, and divide the adjusted crude enzyme solution into 6 reaction glass bottles, each with 1 mL; Add linoleic acid with a final concentration of 0.1 mg/mL to the glass bottle, and react at 37°C for 60 minutes to obtain a reaction solution; after the reaction, quickly add isopropanol and n-hexane to the reaction solution to extract fatty acids and determine the content of fatty acids.
  • the specific enzyme activity of linoleic acid isomerase is defined as the amount of enzyme required to convert to 1 mg of conjugated linoleic acid in 1 min under the conditions of 37°C and pH 6.5, and the unit is U/mg.
  • the conversion rate of conjugated linoleic acid in the reaction solution the types of conjugated linoleic acid isomers in conjugated linoleic acid, and the proportion of each conjugated linoleic acid isomer in conjugated linoleic acid.
  • Detection method according to 1mL reaction solution +1mL isopropanol + 2mL n-hexane, add isopropanol and n-hexane to the reaction solution to obtain a mixed solution; vortex the mixture for 30s; stand still for layering; transfer the upper n-hexane layer to a clean In the spiral glass bottle, blow nitrogen to dry; then add 400 ⁇ L of methanol and vortex for 30 seconds; add 40 ⁇ L of diazomethane to each glass bottle for methyl esterification. At this time, the solution is yellow-green and react for 15 minutes.
  • the conjugated linoleic acid conversion rate (the mass of conjugated linoleic acid/the mass of linoleic acid in the control group) ⁇ 100%.
  • Intracellular conjugated linoleic acid production production of each conjugated linoleic acid isomer, conjugated linoleic acid conversion rate, conversion rate of each conjugated linoleic acid isomer, conjugated linoleic acid in conjugated linoleic acid
  • the type of isomer and the detection method of the proportion of each conjugated linoleic acid isomer use the hydrochloric acid-methanol method to methylate the fatty acids in the recombinant Yarrowia lipolytic yeast cell: weigh 20-25mg freeze-dried bacterial powder, Place in a 5mL glass bottle, add 100 ⁇ L C17:0 fatty acid internal standard (2.000g/L), 1mL 10% hydrochloric acid-methanol, 60°C water bath for 3h (vibrate every 30min for 1min); add 1mL n-hexane after cooling to room temperature Shake and mix with 1mL saturated NaCl.
  • conjugated linoleic acid (conjugated linoleic acid peak area/internal standard peak area) ⁇ 0.1mL ⁇ 2.0mg/mL;
  • Yield of each conjugated linoleic acid isomer (peak area of each conjugated linoleic acid isomer/internal standard peak area) ⁇ 0.1mL ⁇ 2.0mg/mL;
  • Conjugated linoleic acid conversion rate (the mass of conjugated linoleic acid/the mass of linoleic acid in the control group) ⁇ 100%;
  • Conversion rate of each conjugated linoleic acid isomer (mass of each conjugated linoleic acid isomer/mass of linoleic acid in the control group) ⁇ 100%.
  • Example 1 Screening of genes encoding linoleate isomerase
  • the transcriptomics data of Bifidobacterium breve CGMCC No.11828 (documented in the patent application with publication number CN105925514A) under linoleic acid stress was collected through the PacBio sequencing platform. The sampling time points were 3h and 8h, respectively. 15h. According to the analysis of biological information, there are 8 genes in Bifidobacterium breve CGMCCNo.11828 with increased gene transcription levels at three time points. These 8 genes are respectively annotated as codes according to the change in the transformation level.
  • Unknown protein 1 "melibiose carrier protein”, “ribokinase”, linoleic acid hydratase, "unknown protein 2”, "transcription regulatory protein”, “ribose binding ABC channel protein 1” and “ribose binding ABC channel”
  • the transcription level of the gene encoding "Unknown protein 1" at 8h was 68 times higher than that at 3h, and the transcription level at 15h and 8h was up-regulated by 3.5 times and 8.2 times compared with 3h. It forms a gene cluster with other genes. Therefore, it is speculated that this gene is more likely to participate in CLA transformation (the amino acid sequence of "unknown protein 1" is shown in SEQ ID No. 1, and the nucleotide of the gene encoding "unknown protein 1" The sequence is shown in SEQ ID No. 5).
  • genes that may be involved in CLA transformation were obtained from Bifidobacterium longum, Bifidobacterium pseudocatenulatum, and Bifidobacterium dentium.
  • Genes obtained from Bifidobacterium longum, Bifidobacterium pseudocatenulatum, and Bifidobacterium dentium that may be involved in CLA transformation are respectively annotated as encoding "unknown protein 3", "unknown protein 4", The gene of "unknown protein 5", wherein the amino acid sequence of "unknown protein 3" is shown in SEQ ID No. 2, and the nucleotide sequence of the gene encoding "unknown protein 3" is shown in SEQ ID No.
  • Bifidobacterium breve (Bifidobacterium breve) CGMCC No. 11828 from the bacterial preservation tube, streak it on the MRS solid medium, culture it in a constant temperature anaerobic workstation at 37°C for 48 hours to obtain a single colony; pick a single colony for inoculation In the MRS liquid medium, continue to stand for 24 hours in a constant temperature anaerobic workstation at 37°C, and activate continuously for 3 generations to obtain an activated bacterial solution; the activated bacterial solution is 1% (v/v) inoculum Inoculate into MRS liquid medium, culture for 24h in a constant temperature anaerobic workstation at 37°C to obtain a bacterial suspension; centrifuge the obtained bacterial suspension at 25°C and 12000g for 10 minutes to obtain a wet bacterial body; use bacterial genomic DNA
  • the extraction kit extracts genomic DNA from the wet bacteria, and amplifies bbi by PCR; after the PCR reaction, the
  • the amplified product band is verified by 1% agarose gel electrophoresis Size to obtain bbi (this bbi gene is the gene encoding "unknown protein 1"); among them, the primers used to amplify bbi are shown in Table 1;
  • PCR reaction system includes: KOD 1 ⁇ L, ddH2O 29 ⁇ L, upstream and downstream primers 1 ⁇ L, genomic DNA 1 ⁇ L, dNTP 5 ⁇ L, 10 ⁇ reaction buffer 5 ⁇ L and Mg 2+ 3 ⁇ L;
  • the PCR reaction conditions were: 95°C, 5min; (95°C, 30s; 55°C, 30s; 68°C, 1min) 30 cycles; 68°C, 5min; 12°C, 5min.
  • Example 3 Expression of linoleate isomerase in Escherichia coli
  • the pET-28a(+) vector was introduced into E.coli DH5 ⁇ to obtain E.coli DH5 ⁇ /pET28a; E.coli DH5 ⁇ /pET28a was streaked on LB solid medium (containing 10 ⁇ g/mL card Namycin), cultured in a constant temperature incubator at 37°C for 18h to obtain a single colony; pick a single colony and inoculate it in LB liquid medium (containing 10 ⁇ g/mL kanamycin), shake at 37°C, 200rpm Cultivate in the bed for 14 hours and activate continuously for 3 generations to obtain an activated bacterial solution; inoculate the activated bacterial solution into LB liquid medium (containing 10 ⁇ g/mL kanamycin) at an inoculum amount of 1% (v/v) ), cultured in a shaker at 37°C and 200 rpm for 14 hours to obtain a bacterial suspension; centrifuged the obtained bacterial suspension at 25°C and 12000 g for 10 minutes to obtain wet
  • the obtained ligation product was ligated overnight at 16°C for 15 hours, and then transformed into E. coli DH5 ⁇ competent cells; the transformed E. coli DH5 ⁇ competent cells were coated with LB solid medium (containing 10 ⁇ g/mL kanamycin ), inverted culture at 37°C for 24h; picked the positive transformants, extracted the plasmids, the sequencing verification results showed that the ligation was successful, and the recombinant plasmids pET28a-bbi, pET28a-bli, pET28a-bpi and pET28a-bdi were obtained.
  • the obtained recombinant plasmids pET28a-bbi, pET28a-bli, pET28a-bpi and pET28a-bdi were respectively introduced into E. coli BL21(DE3) to obtain recombinant E.coli BL21(DE3)/pET28a-bbi, E .coli BL21(DE3)/pET28a-bli, E.coli BL21(DE3)/pET28a-bpi and E.coli BL21(DE3)/pET28a-bdi.
  • E.coli BL21(DE3)/pET28a-bbi, E.coli BL21(DE3)/pET28a-bli, E.coli BL21(DE3)/pET28a-bpi and E.coli BL21(DE3)/ pET28a-bdi were streaked on LB solid medium and cultured in a constant temperature incubator at 37°C for 18 hours to obtain single colonies; single colonies were picked and inoculated into LB liquid medium and cultured in a shaker at 37°C and 200 rpm 14h, continuously activated for 3 generations to obtain the activated bacteria solution; inoculate the activated bacteria solution into the LB liquid medium at the inoculum amount of 1% (v/v), at a temperature of 37°C and a rotation speed of 200 rpm Cultivate for 12 hours under the conditions to obtain fermentation broth; centrifuge the fermentation broth at 4°C and 12000g for 10 minutes to obtain wet bacteria; crush the wet bacteria and centrifuge at
  • the enzyme activity of linoleate isomerase in the cell disruption supernatant obtained from the fermentation of recombinant E. coli BL21(DE3)/pET28a-bbi was 6.7 U/mg, and the recombinant E.
  • the enzyme activity of linoleate isomerase in the cell disruption supernatant obtained by the fermentation of bli was 1.7 U/mg
  • the enzyme activity in the cell disruption supernatant obtained by the recombinant E.coli BL21(DE3)/pET28a-bpi fermentation was 1.8 U/mg
  • the enzyme activity of linoleate isomerase in the cell disruption supernatant obtained by fermentation of recombinant E.coli BL21(DE3)/pET28a-bdi was 1.4 U/mg. mg.
  • E.coli BL21(DE3)/pET28a-bbi E.coli BL21(DE3)/pET28a-bli
  • E.coli BL21(DE3)/pET28a-bpi E.coli BL21(DE3)/pET28a-bdi
  • E.coli BL21(DE3)/pET28a -bdi can successfully express linoleate isomerase.
  • E.coli BL21(DE3)/pET28a-bbi, E.coli BL21(DE3)/pET28a-bli, E.coli BL21(DE3)/pET28a-bpi and E.coli BL21( DE3)/pET28a-bdi activated bacteria solution was inoculated into LB liquid medium at an inoculum amount of 1% (v/v), and cultivated at a temperature of 37°C and a rotation speed of 200 rpm until the OD 600 was 0.4 After ⁇ 0.6, add IPTG with final concentrations of 0mM, 0.05mM, 0.1mM, 0.3mM, 0.5mM, 0.8mM, 1.0mM to the culture medium, and continue to induce and culture for 15h under the conditions of 18°C and 200rpm to obtain the culture medium ; Centrifuge the culture solution at 25°C and 12000g for 10 minutes to obtain wet bacteria; resuspend the wet bacteria at the concentration of 0.5m
  • the conversion rate of conjugated linoleic acid in the reaction solution obtained by the recombinant E.coli BL21(DE3)/pET28a-bbi reaction can reach 42.1%, of which, 89.1% are cis9, trans11-CLA, 1% are trans10, cis12-CLA, and 9.9% are trans9, trans11-CLA;
  • the conversion rate of conjugated linoleic acid in the reaction solution obtained by the recombinant E.coli BL21(DE3)/pET28a-bli reaction is 12.1%, of which 84.3% is cis9, trans11-CLA , 1.2% is trans10, cis12-CLA, 4.5% is trans9, trans11-CLA;
  • the conversion rate of conjugated linoleic acid in the reaction solution obtained by the recombinant E.coli BL21(DE3)/pET28a-bpi reaction is 19.5%, of which 88.9% is cis9, trans11-CLA , 0.98% is trans10, cis12-CLA, 10.1% is trans9, trans11-CLA;
  • the conversion rate of conjugated linoleic acid in the reaction solution obtained by the recombinant E.coli BL21(DE3)/pET28a-bdi reaction is 13.5%, of which 87.1% is cis9, trans11-CLA , 1.3% are trans10, cis12-CLA, 11.6% are trans9, trans11-CLA.
  • Example 5 Expression of linoleic acid isomerase in Yarrowia lipolytica strain
  • the bbi gene was optimized using Genscript OptimumGeneTM software. After optimization, the codon adaptation index increased from 0.80 to 0.96, and the optimized gene was named obbi gene.
  • the obbi gene was fully synthesized by Nanjing GenScript Biotechnology Co., Ltd. and cloned into the vector pUC57 to obtain the recombinant plasmid pUC57-obbi; the nucleotide sequence of the bbi gene is shown in SEQ ID No. 5, and the obbi gene The nucleotide sequence of is shown in SEQ ID No. 17, and the CAI map of bbi gene and obbi gene is shown in Fig. 6.
  • the obtained recombinant plasmid pINA 1312sp-obbi was introduced into Yarrowia lipolytica; the transformed Yarrowia lipolytica was streaked on YNBD solid medium and cultured in a constant temperature incubator at 28°C for 2 to 3 days ; Pick the positive transformants and inoculate them in YNBD liquid medium, culture for 2 days at 28°C, 200rpm/min, collect the bacteria, extract the genome, use the verification primer P1/P2(P1 nucleotide sequence as shown in SEQ ID No.6 Shows: The nucleotide sequence of ATACAAGAGCGTTTGCCAGC/P2 is shown in SEQ ID No. 7: CCTTGGTCCAGGGGTTGA).
  • the recombinant Yarrowia lipolytica/pINA 1312sp-obbi was streaked on YNBD solid medium, and cultured in a constant temperature incubator at 28°C for 2 to 3 days; picked and inoculated into YNBD liquid medium at 28°C, 200rpm Cultivate for 2 days per minute; inoculate the seed solution into 5mL YPD medium at an inoculum of 1% (v/v), incubate at 28°C and 200rpm/min for 36h to obtain fermentation broth; incubate the fermentation broth at 25°C, 12000g Centrifuge for 10 min under the conditions to obtain wet bacteria; crush the wet bacteria and centrifuge for 10 min at 25°C and 12000g to obtain the cell disruption supernatant; detect the linoleic acid isomers in the obtained cell disruption supernatant
  • the enzyme is more active than the enzyme, the test results are as follows:
  • the specific enzyme activity of linoleate isomerase in the cell crush supernatant obtained from the fermentation of recombinant Yarrowia lipolytica/pINA 1312sp-obbi strain Yarrowia lipolytica/pINA 1312sp-obbi was 2.31 U/mg. It can be seen that the recombinant Yarrowia lipolytica strain Yarrowia lipolytica/pINA 1312sp-obbi can successfully express linoleate isomerase.
  • the recombinant Yarrowia lipolytica/pINA 1312sp containing an empty plasmid as a control the recombinant Yarrowia lipolytica/pINA 1312sp-obbi was streaked on the YNBD solid medium and kept at a constant temperature incubator at 28°C Culture for 2 ⁇ 3d in medium; pick a single colony and inoculate it in YNBD liquid medium, culture at 28°C, 200rpm/min for 2d; inoculate the seed liquid with 1% (v/v) inoculum into 5mL YPD medium, After culturing at 28°C and 200 rpm/min for 36 hours, add free linoleic acid with a final concentration of 0.5 g/L to the medium, and continue culturing at 28°C and 200 rpm/min for 36 hours to obtain fermentation broth; Centrifuge at 12000g for 10 minutes to obtain wet bacteria; wash the wet bacteria twice with 0.85% Na
  • Figures 10-11 and 14 show that the recombinant Yarrowia lipolytica/pINA 1312sp-obbi uses free fatty acids as a substrate to produce cis9, trans11-CLA, trans10, cis12-CLA and trans9, trans11-CLA respectively. 1.5mg/L, 0.5mg/L, 3.8mg/L, among them, the content of cis9, trans11-CLA accounted for 25% of the total conjugated fatty acid content, trans10, cis12-CLA content accounted for 9% of the total conjugated fatty acid content, The content of trans9 and trans11-CLA accounted for 66% of the total conjugated fatty acid content.
  • the recombinant Yarrowia lipolytica/pINA 1312sp containing an empty plasmid as a control the recombinant Yarrowia lipolytica/pINA 1312sp-obbi was streaked on the YNBD solid medium and kept at a constant temperature incubator at 28°C Culture for 2 ⁇ 3d in medium; pick a single colony and inoculate it in YNBD liquid medium, culture at 28°C, 200rpm/min for 2d; inoculate the seed liquid with 1% (v/v) inoculum into 5mL YPD medium, After culturing at 28°C and 200rpm/min for 36 hours, add safflower seed oil with a final concentration of 20g/L to the medium, and continue culturing at 28°C and 200rpm/min for 36 hours to obtain fermentation broth; Centrifuge for 10 min under the conditions of 5% to obtain wet bacteria; wash the wet bacteria twice with 0.85% NaCl and
  • Figure 12-14 shows that the recombinant Yarrowia lipolytica/pINA 1312sp-obbi uses safflower seed oil as a substrate to produce cis9, trans11-CLA, trans10, cis12-CLA and trans9, trans11-CLA respectively.
  • trans10, cis12-CLA content accounts for 10% of the total conjugated fatty acid content
  • the content of trans9 and trans11-CLA accounted for 60% of the total conjugated fatty acid content.
  • the recombinant Yarrowia lipolytica/pINA 1312sp-obbi was streaked on YNBD solid medium and cultured in a constant temperature incubator at 28°C for 2 to 3 days; a single colony was picked and inoculated into YNBD liquid medium at 28°C , 200rpm/min culture for 2d; the seed solution was inoculated into 50mL YPD medium with 1% (v/v) inoculum, and after 36h incubation at 28°C and 200rpm/min, the final concentration of 20g/min was added to the medium.
  • the recombinant Yarrowia lipolytica/pINA 1312sp-obbi uses safflower seed oil as a substrate to produce cis9, trans11-CLA, trans10, cis12-CLA and trans9, and the yields of trans11-CLA are 224.0mg/L and 73.7, respectively mg/L, 454.0mg/L, of which the content of cis9, trans11-CLA accounted for 29.8% of the total conjugated fatty acid content, the content of trans10, cis12-CLA accounted for 9.8% of the total conjugated fatty acid content, and the content of trans9, trans11-CLA It accounts for 60.4% of the total conjugated fatty acid content.
  • the recombinant Yarrowia lipolytica/pINA 1312sp-obbi was streaked on YNBD solid medium and cultured in a constant temperature incubator at 28°C for 2 to 3 days; a single colony was picked and inoculated into YNBD liquid medium at 28°C , 200rpm/min culture for 2d; the seed solution was inoculated into 50mL YPD medium with 1% (v/v) inoculum, and after 36h incubation at 28°C and 200rpm/min, the final concentration of 10g was added to the medium.
  • the concentration of safflower seed oil is related to the production of cis9 by the recombinant Yarrowia lipolytica/pINA 1312sp-obbi
  • the effect of trans11-CLA output is shown in Figure 15.
  • the effect of safflower seed oil concentration on the yield of cis9 and trans11-CLA produced by the recombinant Yarrowia lipolytica/pINA 1312sp-obbi fermentation for 84 hours is shown in Figure 15. 16.
  • the ratio of safflower seed oil concentration to the yield of cis9 and trans11-CLA produced when the recombinant Yarrowia lipolytica/pINA 1312sp-obbi is fermented for 84 hours to the total yield of conjugated fatty acids produced is shown in Figure 17.
  • the effect of safflower seed oil concentration on the production of cis9, trans11-CLA and the conversion rate of recombinant Yarrowia lipolytica/pINA 1312sp-obbi is shown in Figure 18.
  • Example 7 Expression of linoleate isomerase in Lactobacillus plantarum
  • the pNZ44 plasmid was introduced into E.coli DH5 ⁇ to obtain E.coli DH5 ⁇ /pNZ44; E.coli DH5 ⁇ /pNZ44 was streaked on LB solid medium (containing 10 ⁇ g/mL kanamycin) Incubate in a constant temperature incubator at 37°C for 18 hours to obtain a single colony; pick a single colony and inoculate it in LB liquid medium (containing 10 ⁇ g/mL kanamycin), and cultivate it in a shaker at 37°C and 200 rpm for 14 hours.
  • plasmid small extraction kit to extract the recombinant plasmid pU57-bbi(U) from the recombinant E.coli DH5 ⁇ /pU57-bbi(U); use 50 ⁇ L of ddH 2 O for the obtained recombinant plasmid pU57-bbi(U) Dissolve and store at -20°C.
  • the obtained ligation product was ligated overnight at 16°C for 15 hours, and then transformed into E.coli DH5 ⁇ competent cells; the transformed E.coli DH5 ⁇ competent cells were coated with LB solid medium (containing 10 ⁇ g/ mL of chloramphenicol), inverted culture at 37°C for 24h; picked the positive transformants, extracted the plasmids, and the sequencing verification results showed that the connection was successful, and the recombinant plasmid pNZ44-bbi and recombinant plasmid pNZ44-bbi(U) were obtained.
  • the verification results are shown in Figure 19. .
  • the obtained recombinant plasmid pNZ44-bbi and recombinant plasmid pNZ44-bbi(U) were respectively introduced into Lactobacillus plantarum ST-III to obtain the engineered Lactobacillus plantarum ST-III/pNZ44-bbi and Lactobacillus plantarum ST-III/pNZ44-bbi. Lactobacillus plantarum ST-III/pNZ44-bbi(U).
  • the obtained engineering bacteria Lactobacillus plantarum ST-III/pNZ44-bbi and engineering bacteria Lactobacillus plantarum ST-III/pNZ44-bbi(U) were respectively streaked on MRS solid medium and cultured at 37°C at a constant temperature Cultivate in the box for 18 hours to obtain a single colony; pick single colonies and inoculate them into MRS liquid medium, culture them in a shaker at 37°C and 200 rpm for 14 hours, and activate continuously for 3 generations to obtain an activated bacterial solution;
  • the bacterial solution was inoculated into LB liquid medium at an inoculum amount of 1% (v/v), and incubated at 37°C for 12 hours to obtain a fermentation broth; the fermentation broth was placed at 4°C, 12000g Centrifuge for 10 minutes to obtain wet bacteria; crush the wet bacteria and centrifuge at 4°C and 12000g for 10 minutes to obtain cell disruption supernatant; detect the linoleic acid isome
  • the linoleic acid isomerase enzyme activity in the cell disruption supernatant obtained by fermentation of the engineered Lactobacillus plantarum ST-III/pNZ44-bbi is 2.5 U/mg
  • the enzyme activity of linoleate isomerase in the cell crush supernatant obtained by the fermentation of bbi(U) was 10.5 U/mg.
  • Lactobacillus plantarum ST-III/pNZ44-bbi and the engineered Lactobacillus plantarum ST-III/pNZ44-bbi(U) can successfully express linoleic acid isomerase, but the engineered Lactobacillus plantarum ST-III/pNZ44-bbi(U) can successfully express linoleic acid isomerase. Lactobacillus plantarum ST-III/pNZ44-bbi(U) has stronger expression ability.
  • the obtained Lactobacillus plantarum ST-III/pNZ44-bbi and the Lactobacillus plantarum ST-III/pNZ44-bbi (U) activated bacteria liquid are 1% (v/v)
  • the amount of inoculation was respectively inoculated into MRS liquid medium containing 0.5mg/mL free linoleic acid, and allowed to stand for 72 hours at 37°C to obtain fermentation broth; to detect the conversion rate of conjugated linoleic acid in the fermentation broth,
  • the types of conjugated linoleic acid isomers and the proportion of each conjugated linoleic acid isomer in the detected conjugated linoleic acid are shown in Figure 20-21.
  • the fermentation broth obtained by the fermentation of the engineered Lactobacillus plantarum ST-III/pNZ44-bbi does not contain conjugated linoleic acid; the engineered Lactobacillus plantarum ST-III/pNZ44-bbi(U)
  • the conversion rate of conjugated linoleic acid in the fermentation broth obtained by fermentation can reach 89.9%.
  • 100% of the conjugated linoleic acid obtained by fermentation of Lactobacillus plantarum ST-III/pNZ44-bbi(U) is cis9,trans11-CLA.

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Abstract

一种亚油酸异构酶及其在共轭亚油酸生产中的应用，属于蛋白质工程和微生物工程技术领域。来源于双岐杆菌的亚油酸异构酶可用于生产共轭亚油酸，将含有亚油酸异构酶的重组大肠杆菌加入含亚油酸的反应体系中反应3h，即可使共轭亚油酸的转化率高达12.1～42.1％，且可使共轭亚油酸中cis9,trans11-CLA的含量高达84.3～89.1％，为通过基因工程手段进一步获得安全性高、产量高且所产得的共轭亚油酸单体多为cis9,trans11-CLA的微生物提供了理论支持。

Description

一种亚油酸异构酶及其在共轭亚油酸生产中的应用 技术领域
本发明涉及一种亚油酸异构酶及其在共轭亚油酸生产中的应用，属于蛋白质工程和微生物工程技术领域。
背景技术
共轭亚油酸(Conjugated linoleic acid,CLA)是一系列含有共轭双键、具有多种位置和几何异构体的脂肪酸的总称。研究表明，共轭亚油酸具有抗癌、降脂、抗动脉粥状硬化、调节能量代谢、增强机体免疫力与促进生长发育等生理作用，被广泛应用于医药、食品、化妆品等领域，而cis9,trans11-CLA和trans10,cis12-CLA是共轭亚油酸异构体中最具有生理活性的两种异构体。因此，市场上对cis9,trans11-CLA和trans10,cis12-CLA的需求极大。
天然的共轭亚油酸主要存在于瘤胃动物、某些植物和海洋生物中，并且，这些天然的共轭亚油酸主要以cis9,trans11-CLA的形式存在，生理活性极高，但是，它们含量极少，难以满足市场对于共轭亚油酸的需求。因此，人们逐渐开发了人工合成共轭亚油酸的方法。
目前，人工合成共轭亚油酸的方法主要有化学合成法以及微生物合成法。其中，化学合成法会导致很多有毒性的副产物的产生，对环境以及人体均具有毒害作用，并且，通过化学合成法制备得到的共轭亚油酸异构体种类多，很难进行有效的分离，因此，化学合成法无法真正实现共轭亚油酸的大规模工业化生产。与化学合成法相比，微生物合成法具有污染少、所得共轭亚油酸异构体种类单一的优点，因此，微生物合成法是一种极具潜力的可实现共轭亚油酸大规模工业化生产的方法。
不过，现有的微生物合成法仍存在以下缺陷：
第一，大部分可高产共轭亚油酸的微生物均为致病菌，存在极大的安全性问题，不能直接作为共轭亚油酸生产菌株进行工业化生产，例如，溶纤维丁酸弧菌、丙酸杆菌以及产芽孢梭状芽孢杆菌等；
第二，部分可高产共轭亚油酸的微生物生产共轭亚油酸产量不高，若作为共轭亚油酸生产菌株进行工业化生产，生产效率过低，例如，植物乳杆菌ZS2058(具体可见参考文献：齐慧,杨波等.植物乳杆菌ZS2058生物转化共轭亚油酸机理的研究[D],江南大学,2017)；
第三，大部分可高产共轭亚油酸的微生物为严格厌氧菌，在工业或实验室中不易培养且产量较低，难以广泛应用到食品和药品中，例如，溶纤维丁酸弧菌以及双歧杆菌等；
第四，部分生产CLA的菌种多严格依赖亚油酸作为底物，过量游离的亚油酸和副产物的生成会抑制CLA菌的生长，从而影响其转化率，导致CLA的生产效率过低，例如，植物乳杆菌ZS2058(具体可见参考文献：齐慧,杨波等.植物乳杆菌ZS2058生物转化共轭亚油酸机理的研究[D],江南大学,2017)，
上述缺陷均使得现有的微生物合成法无法真正实现共轭亚油酸的大规模工业化生产，因此，急需找到一种具有非严格厌氧、安全性高、不严格依赖亚油酸和/或产量高的优势的共轭亚油酸生产菌株以克服上述缺陷。
发明内容
为解决上述问题，本发明提供了一种亚油酸异构酶(Linoleic acid isomerase,EC 5.2.1.5)，其特征在于，所述亚油酸异构酶为：
(a)由SEQ ID No.1、SEQ ID No.2、SEQ ID No.3或SEQ ID No.4所示的氨基酸序列组成的蛋白质；或者，
(b)在(a)中的氨基酸序列经过取代、缺失或添加一个或几个氨基酸且具有亚油酸异构酶活性的由(a)衍生的蛋白质。
在本发明的一种实施方式中，所述由SEQ ID No.1所示的氨基酸序列组成的亚油酸异构酶来源于短双岐杆菌(Bifidobacterium breve)，所述由SEQ ID No.2所示的氨基酸序列组成的亚油酸异构酶来源于长双歧杆菌(Bifidobacterium longum)，所述由SEQ ID No.3所示的 氨基酸序列组成的亚油酸异构酶来源于假小链双歧杆菌(Bifidobacterium pseudocatenulatum)，所述由SEQ ID No.4所示的氨基酸序列组成的亚油酸异构酶来源于齿双歧杆菌(Bifidobacterium dentium)。
本发明还提供了一种基因，所述基因编码上述亚油酸异构酶。
在本发明的一种实施方式中，所述基因的核苷酸序列如SEQ ID No.5、SEQ ID No.6、SEQ ID No.7、SEQ ID No.8、SEQ ID No.17或SEQ ID No.18所示。
本发明还提供了一种重组质粒，所述重组质粒携带上述基因。
在本发明的一种实施方式中，所述重组质粒的载体为pET-28a(+)质粒、pINA 1312sp质粒或pNZ44质粒。
本发明还提供了一种宿主细胞，所述宿主细胞携带上述基因或上述重组质粒。
在本发明的一种实施方式中，所述宿主细胞为大肠杆菌、耶氏解脂酵母或植物乳杆菌。
在本发明的一种实施方式中，当宿主细胞为大肠杆菌时，所述宿主细胞以pET-28a(+)质粒为载体携带核苷酸序列如SEQ ID No.5、SEQ ID No.6、SEQ ID No.7或SEQ ID No.8所示的编码上述亚油酸异构酶的基因；
当宿主细胞为耶氏解脂酵母时，所述宿主细胞以pINA 1312sp质粒为载体携带核苷酸序列如SEQ ID No.17所示的编码上述亚油酸异构酶的基因；
当宿主细胞为植物乳杆菌时，所述宿主细胞以pNZ44质粒为载体携带核苷酸序列如SEQ ID No.18所示的编码上述亚油酸异构酶的基因。
本发明还提供了上述亚油酸异构酶或上述基因或上述重组质粒或上述宿主细胞在生产共轭亚油酸方面的应用。
本发明还提供了一种生产共轭亚油酸的方法，所述方法为使用上述宿主细胞；
当宿主细胞为大肠杆菌时，所述方法为将上述宿主细胞接种至培养基中，于温度为35～40℃、转速为150～250rpm的条件下培养至OD 600为0.4～0.6，得到培养液A；在培养液A中加入终浓度为0.01～1.0mM的IPTG，于温度为15～20℃、转速为150～250rpm的条件下诱导培养12～16h，得到培养液B；将培养液B离心，收集湿菌体；以亚油酸为底物，在含亚油酸的反应体系中加入湿菌体，于温度为35～40℃、转速为150～250rpm的条件下进行反应，得到富含共轭亚油酸的反应液；将富含共轭亚油酸的反应液进行提取，得到共轭亚油酸；
当宿主细胞为耶氏解脂酵母时，所述方法为将上述宿主细胞接种至含有亚油酸和/或甘油酯的培养基中，于温度为35～40℃、转速为150～250rpm的条件下进行培养，得到富含共轭亚油酸的宿主细胞，然后将富含共轭亚油酸的宿主细胞进行提取，得到共轭亚油酸；
当宿主细胞为植物乳杆菌时，所述方法为将上述宿主细胞接种至含有亚油酸的培养基中，于温度为37℃的条件下静置培养，得到富含共轭亚油酸的培养液；将富含共轭亚油酸的培养液进行提取，得到共轭亚油酸。
在本发明的一种实施方式中，当宿主细胞为大肠杆菌时，所述方法为将上述宿主细胞接种至培养基中，于温度为37℃、转速为200rpm的条件下培养至OD 600为0.4～0.6，得到培养液A；在培养液A中加入终浓度为0.01～1.0mM的IPTG，于温度为18℃、转速为200rpm的条件下诱导培养12～16h，得到培养液B；将培养液B离心，收集湿菌体；以亚油酸为底物，在含亚油酸的反应体系中加入湿菌体，于温度为37℃、转速为200rpm的条件下进行反应，得到富含共轭亚油酸的反应液；将富含共轭亚油酸的反应液进行提取，得到共轭亚油酸。
在本发明的一种实施方式中，当宿主细胞为耶氏解脂酵母时，所述甘油酯为红花籽油、亚麻仁油、棉籽油、和/或大豆油。
在本发明的一种实施方式中，当宿主细胞为耶氏解脂酵母时，所述甘油酯为红花籽油。
在本发明的一种实施方式中，当宿主细胞为大肠杆菌时，所述反应体系包含缓冲液以及亚油酸。
在本发明的一种实施方式中，当宿主细胞为大肠杆菌时，所述缓冲液的pH为6～7。
在本发明的一种实施方式中，当宿主细胞为大肠杆菌时，所述缓冲液的pH为6.5。
在本发明的一种实施方式中，当宿主细胞为大肠杆菌时，所述缓冲液为磷酸钾缓冲液。
在本发明的一种实施方式中，当宿主细胞为大肠杆菌时，所述亚油酸在反应体系中的浓度为0.05～0.15mg/mL。
在本发明的一种实施方式中，当宿主细胞为大肠杆菌时，所述亚油酸在反应体系中的浓度为0.1mg/mL。
在本发明的一种实施方式中，当宿主细胞为大肠杆菌时，所述湿菌体在反应体系中的浓度为0.5～2mg/mL。
在本发明的一种实施方式中，当宿主细胞为大肠杆菌时，所述湿菌体在反应体系中的浓度为1mg/mL。
在本发明的一种实施方式中，当宿主细胞为大肠杆菌时，所述共轭亚油酸为cis9,trans11-CLA和/或trans9,trans11-CLA；当宿主细胞为耶氏解脂酵母时，所述共轭亚油酸为cis9,trans11-CLA、trans10,cis12-CLA和/或trans9,trans11-CLA；当宿主细胞为植物乳杆菌时，所述共轭亚油酸为cis9,trans11-CLA。
在本发明的一种实施方式中，当宿主细胞为大肠杆菌、耶氏解脂酵母或植物乳杆菌时，所述共轭亚油酸为cis9,trans11-CLA。
在本发明的一种实施方式中，当宿主细胞为大肠杆菌时，所述培养基为LB培养基；当宿主细胞为耶氏解脂酵母时，所述培养基为YPD培养基；当宿主细胞为植物乳杆菌时，所述培养基为MRS培养基。
本发明还提供了上述宿主细胞在生产亚油酸异构酶方面的应用，所述亚油酸异构酶的氨基酸序列如SEQ ID No.1所示。
本发明还提供了一种生产上述亚油酸异构酶的方法，其特征在于，使用上述宿主细胞；
当宿主细胞为大肠杆菌时，所述方法为将上述宿主细胞加入培养基中，于温度为35～40℃、转速为150～250rpm的条件下培养，得到富含亚油酸异构酶的宿主细胞，然后将富含亚油酸异构酶的宿主细胞进行提取，得到亚油酸异构酶；
当宿主细胞为耶氏解脂酵母时，所述方法为将上述宿主细胞接种至培养基中，于温度为35～40℃、转速为150～250rpm的条件下培养，得到富含亚油酸异构酶的宿主细胞，然后将富含亚油酸异构酶的宿主细胞进行提取，得到亚油酸异构酶；
当宿主细胞为植物乳杆菌时，所述方法为将上述宿主细胞接种至接种至培养基中，于温度为37℃的条件下静置培养，得到富含亚油酸异构酶的宿主细胞，然后将富含亚油酸异构酶的宿主细胞进行提取，得到亚油酸异构酶。
在本发明的一种实施方式中，当宿主细胞为大肠杆菌时，所述培养基为LB培养基；当宿主细胞为耶氏解脂酵母时，所述培养基为YPD培养基；当宿主细胞为植物乳杆菌时，所述培养基为MRS培养基。
有益效果：
(1)本发明的来源于短双岐杆菌(Bifidobacterium breve)的氨基酸序列如SEQ ID No.1所示的亚油酸异构酶可用于生产共轭亚油酸，将含有本发明亚油酸异构酶的重组大肠杆菌加入含亚油酸的反应体系中反应3h，即可使共轭亚油酸的转化率高达42.1％，且可使共轭亚油酸中cis9,trans11-CLA的含量高达89.1％，该结果为通过基因工程手段进一步获得安全性高、产量高且所产得的共轭亚油酸单体多为cis9,trans11-CLA的微生物提供了充实的理论支持。
(2)本发明的来源于长双歧杆菌(Bifidobacterium longum)的氨基酸序列如SEQ ID No.2所示的亚油酸异构酶可用于生产共轭亚油酸，将含有本发明亚油酸异构酶的重组大肠杆菌加入含亚油酸的反应体系中反应3h，即可使共轭亚油酸的转化率高达12.1％，且可使共轭亚油酸中cis9,trans11-CLA的含量高达84.3％，该结果为通过基因工程手段进一步获得安全性高、产量高且所产得的共轭亚油酸单体多为cis9,trans11-CLA的微生物提供了充实的理论支持。
(3)本发明的来源于假小链双歧杆菌(Bifidobacterium pseudocatenulatum)的氨基酸序列如SEQ ID No.3所示的亚油酸异构酶可用于生产共轭亚油酸，将含有本发明亚油酸异构酶 的重组大肠杆菌加入含亚油酸的反应体系中反应3h，即可使共轭亚油酸的转化率高达19.5％，且可使共轭亚油酸中cis9,trans11-CLA的含量高达88.9％，该结果为通过基因工程手段进一步获得安全性高、产量高且所产得的共轭亚油酸单体多为cis9,trans11-CLA的微生物提供了充实的理论支持。
(4)本发明的来源于齿双歧杆菌(Bifidobacterium dentium)的氨基酸序列如SEQ ID No.4所示的亚油酸异构酶可用于生产共轭亚油酸，将含有本发明亚油酸异构酶的重组大肠杆菌加入含亚油酸的反应体系中反应3h，即可使共轭亚油酸的转化率高达13.5％，且可使共轭亚油酸中cis9,trans11-CLA的含量高达87.1％，该结果为通过基因工程手段进一步获得安全性高、产量高且所产得的共轭亚油酸单体多为cis9,trans11-CLA的微生物提供了充实的理论支持。
(5)本发明的重组耶氏解脂酵母菌株可以以红花籽油等甘油酯为底物转化生产共轭亚油酸，可高产共轭亚油酸，且其所产得的共轭亚油酸异构体多为cis9,trans11-CLA；将本发明的重组耶氏解脂酵母菌株加入含游离亚油酸的培养基中培养36h，即可使发酵液中共轭亚油酸的产量高达5.8mg/L，其中，cis9,trans11-CLA的产量高达1.5mg/L，约占总共轭亚油酸产量的25％；将本发明的重组耶氏解脂酵母菌株加入含红花籽油的培养基中培养36h，即可使发酵液中共轭亚油酸的产量高达751.7mg/L，其中，cis9,trans11-CLA的产量高达224.0mg/L，约占总共轭亚油酸产量的29.8％。
(6)耶氏解脂酵母属于广泛认为是安全的(generally regarded as safe,GRAS)微生物，并且，耶氏解脂酵母已被欧盟认定为可用于食品中的安全菌株，因此，本发明重组耶氏解脂酵母菌株所产得的共轭亚油酸相对而言更为安全。
(7)耶氏解脂酵母属于严格好氧菌，相较严格厌氧菌更易实现工业化培养，并且，以耶氏解脂酵母作为生产菌株的工业化生产工艺已十分成熟，因此，本发明的重组耶氏解脂酵母菌株更适用于大规模工业化生产。
(8)红花籽油等甘油酯的来源广泛、价格低廉，本发明的重组耶氏解脂酵母菌株可以以红花籽油等甘油酯为底物转化生产共轭亚油酸，产量较高，且其所产得的共轭亚油酸异构体多为cis9,trans11-CLA，因此，本发明的重组耶氏解脂酵母菌株成本更低，更适用于大规模工业化生产。
(9)本发明的植物乳杆菌工程菌可高产共轭亚油酸且其所产得的共轭亚油酸异构体多为cis9,trans11-CLA，将本发明的植物乳杆菌工程菌加入含亚油酸的培养基中培养72h，即可使共轭亚油酸的转化率高达89.9％，且可使共轭亚油酸中cis9,trans11-CLA的含量高达100％。
(10)植物乳杆菌是益生菌的一种，目前已被纳入卫生部下发的《可用于食品的菌种名单》，因此，本发明植物乳杆菌工程菌所产得的共轭亚油酸对人体而言，安全性更高。
(11)植物乳杆菌属于兼性好氧菌，相较严格厌氧菌更易培养，适用于大规模工业化生产。
附图说明
图1：IPTG浓度对重组大肠杆菌E.coli BL21(DE3)/pET28a-bbi、E.coli BL21(DE3)/pET28a-bli、E.coli BL21(DE3)/pET28a-bpi以及E.coli BL21(DE3)/pET28a-bdi的共轭亚油酸转化率的影响。
图2：重组大肠杆菌E.coli BL21(DE3)/pET28a-bbi所产共轭亚油酸中共轭亚油酸异构体类型及各共轭亚油酸异构体占比。
图3：重组大肠杆菌E.coli BL21(DE3)/pET28a-bli所产共轭亚油酸中共轭亚油酸异构体类型及各共轭亚油酸异构体占比。
图4：重组大肠杆菌E.coli BL21(DE3)/pET28a-bpi所产共轭亚油酸中共轭亚油酸异构体类型及各共轭亚油酸异构体占比。
图5：重组大肠杆菌E.coli BL21(DE3)/pET28a-bdi所产共轭亚油酸中共轭亚油酸异构体类型及各共轭亚油酸异构体占比。
图6：未经密码子优化的bbi序列以及经密码子优化的bbi序列的CAI图谱。
图7：重组质粒pINA 1312sp-obbi的质粒图谱。
图8：重组质粒pINA 1312sp-obbi的PCR验证结果。
图9：重组耶氏解脂酵母菌株Yarrowia lipolytica/pINA 1312sp-obbi的PCR验证结果。
图10：重组耶氏解脂酵母菌株Yarrowia lipolytica/pINA 1312sp-obbi以游离脂肪酸为底物生产cis9,trans11-CLA、trans10,cis12-CLA以及trans9,trans11-CLA的产量。
图11：重组耶氏解脂酵母菌株Yarrowia lipolytica/pINA 1312sp-obbi以游离脂肪酸为底物生产得到的cis9,trans11-CLA、trans10,cis12-CLA以及trans9,trans11-CLA的含量占生产得到的总共轭脂肪酸含量的比例。
图12：重组耶氏解脂酵母菌株Yarrowia lipolytica/pINA 1312sp-obbi以红花籽油为底物生产cis9,trans11-CLA、trans10,cis12-CLA以及trans9,trans11-CLA的产量。
图13：重组耶氏解脂酵母菌株Yarrowia lipolytica/pINA 1312sp-obbi以红花籽油为底物生产得到的cis9,trans11-CLA、trans10,cis12-CLA以及trans9,trans11-CLA的产量占生产得到的总共轭脂肪酸产量的比例。
图14：重组耶氏解脂酵母菌株Yarrowia lipolytica/pINA 1312sp-obbi在不同底物下生产得到共轭亚油酸的GC-MS鉴定图谱；其中，(a)表示重组耶氏解脂酵母菌株Yarrowia lipolytica/pINA 1312sp-obbi以游离脂肪酸为底物生产得到共轭亚油酸的GC-MS鉴定图谱，(b)表示重组耶氏解脂酵母菌株Yarrowia lipolytica/pINA 1312sp-obbi以红花籽油为底物生产得到共轭亚油酸的GC-MS鉴定图谱，(c)表示样品中脂肪酸的组成色谱图(此处样品为购自Sigma公司的共轭亚油酸标准品)，数字1、2、3分别表示cis9,trans11-CLA、trans10,cis12-CLA以及trans9,trans11-CLA。
图15：红花籽油浓度对重组耶氏解脂酵母菌株Yarrowia lipolytica/pINA 1312sp-obbi生产cis9,trans11-CLA的产量的影响。
图16：红花籽油浓度对重组耶氏解脂酵母菌株Yarrowia lipolytica/pINA 1312sp-obbi发酵84h时生产得到的cis9,trans11-CLA的产量的影响。
图17：红花籽油浓度对重组耶氏解脂酵母菌株Yarrowia lipolytica/pINA 1312sp-obbi发酵84h时生产得到的cis9,trans11-CLA的产量的占生产得到的总共轭脂肪酸产量的比例。
图18：红花籽油浓度对重组耶氏解脂酵母菌株Yarrowia lipolytica/pINA 1312sp-obbi生产cis9,trans11-CLA的产量以及转化率的影响。
图19：重组质粒pNZ44-bbi以及pNZ44-bbi(U)的PCR验证结果。
图20：植物乳杆菌工程菌Lactobacillus plantarum ST-III/pNZ44-bbi(U)所产共轭亚油酸的GC-MS鉴定色谱图。
图21：植物乳杆菌工程菌Lactobacillus plantarum ST-III/pNZ44-bbi(U)所产共轭亚油酸的GC-MS质谱碎片图。
具体实施方式
下述实施例中涉及的大肠杆菌(Escherichia coli)DH5α、E.coli BL21(DE3)购自通用生物技术有限公司；下述实施例中涉及的pET-28a(+)载体购自Invitrogen公司；下述实施例中涉及的细菌基因组DNA提取试剂盒以及质粒小提试剂盒购自天根生化科技(北京)有限公司，型号分别为DP302、DP103；下述实施例中涉及的游离亚油酸购自Sigma公司；下述实施例中涉及的红花籽油购自中粮(昌吉)粮油工业有限公司；下述实施例中涉及的耶氏解脂酵母(Yarrowia lipolytica)购自北纳生物，产品编号为：BNCC193899；下述实施例中涉及的pINA 1312质粒购自BioVector质粒载体菌种细胞基因保藏中心；下述实施例中涉及的pINA 1312sp质粒的构建方法记载于文献“Zhang B，Chen H，Li M，Gu Z，SongY，Ratledge C，Chen YQ，Zhang H，Chen W(2013)Genetic engineering of Yarrowia lipolytica for enhanced production of trans-10，cis-12conjugated linoleic acid.Microb Cell Fact.12：70”中；下述实施例中涉及的pNZ44质粒的构建方法记载于文献“McGrath,S.et al.,2001.Improvement and optimization of two engineered phage resistance mechanisms in Lactobacoccus lactic.Applied and  Environmental Microbiology,67(2):608-616.”中；
下述实施例中涉及的培养基如下：
MRS固体培养基：蛋白胨10g/L、牛肉膏10g/L、葡萄糖20g/L、乙酸钠2g/L、酵母粉5g/L、柠檬酸氢二铵2g/L、K 2HPO 4·3H 2O 2.6g/L、MgSO 4·7H 2O 0.1g/L、MnSO 4·H 2O 0.05g/L、吐温80 1mL/L、琼脂15g/L、半胱氨酸氨酸盐0.5g/L。
MRS液体培养基：蛋白胨10g/L、牛肉膏10g/L、葡萄糖20g/L、乙酸钠2g/L、酵母粉5g/L、柠檬酸氢二铵2g/L、K 2HPO 4·3H 2O 2.6g/L、MgSO 4·7H 2O 0.1g/L、MnSO 4·H 2O 0.05g/L、吐温80 1mL/L、半胱氨酸氨酸盐0.5g/L。
LB液体培养基：胰蛋白胨10g/L、酵母提取物5g/L、氯化钠10g/L，使用前添加100μg/mL的卡那霉素。
LB固体培养基：胰蛋白胨10g/L、酵母提取物5g/L、氯化钠10g/L、琼脂15g/L，使用前添加100μg/mL的卡那霉素。
YNBD固体培养基：酵母含氮碱基(不含氨基酸)6.7g/L、葡萄糖20g/L、琼脂15g/L，pH为5.5。
YNBD液体培养基：酵母含氮碱基(不含氨基酸)6.7g/L、葡萄糖20g/L，pH为5.5。
YPD培养基：蛋白胨20g/L、酵母提取物10g/L、葡萄糖20g/L，pH为6.5。
下述实施例中涉及的检测方法如下：
亚油酸异构酶比酶活的检测方法：收集菌体，将菌体加入KPB缓冲液(pH 6.5)中，并将菌体用玻璃珠破碎，得到细胞破碎液；将细胞破碎液于8000g离心10min收集上清，得到粗酶液；调整粗酶液中蛋白含量为0.5mg/mL，并将调整后的粗酶液分装至6个反应玻璃瓶中，每个玻璃瓶1mL；分别在玻璃瓶中加入终浓度为0.1mg/mL亚油酸，于37℃反应60min，得到反应液；反应结束后，迅速在反应液中加入异丙醇以及正己烷，对脂肪酸进行萃取，测定脂肪酸的含量变化(脂肪酸含量变化的检测方法参考下述共轭亚油酸转化率、共轭亚油酸中共轭亚油酸异构体类型以及共轭亚油酸中各共轭亚油酸异构体占比的检测方法)，从而计算比酶活；比酶活(U/mg)＝W/(T×M)，其中，W为反应生成的共轭亚油酸的质量(μg)，T为反应时间(min)，M为待测样品质量(mg)
其中，亚油酸异构酶比酶活的定义为：于37℃、pH 6.5的条件下，1min内转化生成1mg共轭亚油酸所需的酶量，单位为U/mg。
反应液中共轭亚油酸转化率、共轭亚油酸中共轭亚油酸异构体类型以及共轭亚油酸中各共轭亚油酸异构体占比的检测方法：按照1mL反应液+1mL异丙醇+2mL正己烷的比例，在反应液中加入异丙醇和正己烷，得到混合液；将混合液涡旋振荡30s；静置分层；移取上层的正己烷层至干净的螺旋玻璃瓶内，氮吹至干；接着加入400μL的甲醇，涡旋30s；每个玻璃瓶中加入40μL的重氮甲烷，进行甲酯化，此时溶液为黄绿色，反应15min，如果颜色不褪色，代表甲酯化比较充分；充分甲酯化后的液体经过氮吹至干，分别加入200μL的正己烷进行回溶，离心后，将上清液转移至色谱进样瓶中，暂存至GC-MS检测；
其中，共轭亚油酸转化率＝(共轭亚油酸的质量/对照组中亚油酸的质量)×100％。
细胞内共轭亚油酸产量、各共轭亚油酸异构体产量、共轭亚油酸转化率、各共轭亚油酸异构体转化率、共轭亚油酸中共轭亚油酸异构体类型以及各共轭亚油酸异构体占比的检测方法：采用盐酸-甲醇法甲酯化重组耶氏解脂酵母菌体中的脂肪酸：称取20～25mg冻干菌粉，置于5mL玻璃瓶中，加入100μL C17:0脂肪酸内标(2.000g/L)、1mL 10％的盐酸-甲醇，60℃水浴3h(每隔30min振荡1min)；冷却至室温后加入1mL正己烷和1mL饱和NaCl振荡混匀，3000×g离心3min，吸取上层溶液；再向原体系中加入1mL正己烷振荡混匀，3000g离心3min，吸取并合并上层溶液，氮气吹干后，加入1mL正己烷混匀，转入气相瓶，进行气相色谱分析；其中，脂肪酸分析方法参考文献“杨波，陈海琴，宋元达，等.动物双歧杆菌肌球交叉反应抗原MCRA酶学功能的研究[J].中国生物工程杂志，2012，32(12):30-36.”；
其中，共轭亚油酸产量＝(共轭亚油酸峰面积/内标峰面积)×0.1mL×2.0mg/mL；
各共轭亚油酸异构体产量＝(各共轭亚油酸异构体的峰面积/内标峰面积)×0.1mL×2.0mg/mL；
共轭亚油酸转化率＝(共轭亚油酸的质量/对照组中亚油酸的质量)×100％；
各共轭亚油酸异构体转化率＝(各共轭亚油酸异构体的质量/对照组中亚油酸的质量)×100％。
实施例1：编码亚油酸异构酶的基因的筛选
通过PacBio测序平台采集短双歧杆菌(Bifidobacterium breve)CGMCCNo.11828(记载于公开号为CN105925514A的专利申请文本中)在亚油酸胁迫下的转录组学数据，采样时间点分别为3h，8h，15h。经过生信分析发现，短双歧杆菌(Bifidobacterium breve)CGMCCNo.11828中三个时间点处基因转录水平均增大的基因共8个，根据转化水平变化大小将这8个基因分别注释为编码“未知蛋白1”、“蜜二糖载体蛋白”、“核糖激酶”、亚油酸水合酶、“未知蛋白2”、“转录调控蛋白”、“核糖结合ABC通道蛋白1”以及“核糖结合ABC通道蛋白2”的基因，其中，编码“未知蛋白1”的基因在8h时的转录水平较3h上升68倍，15h和8h时的转录水平较3h上调了3.5倍和8.2.倍，并且，其未与其他基因形成基因簇，因此，推测该基因参与CLA转化的可能性比较大(“未知蛋白1”的氨基酸序列如SEQ ID No.1所示、编码“未知蛋白1”的基因的核苷酸序列如SEQ ID No.5所示)。
通过同样的方法分别从长双歧杆菌(Bifidobacterium longum)、假小链双歧杆菌(Bifidobacterium pseudocatenulatum)以及齿双歧杆菌(Bifidobacterium dentium)中获得其他可能参与CLA转化的基因(将分别从长双歧杆菌(Bifidobacterium longum)、假小链双歧杆菌(Bifidobacterium pseudocatenulatum)以及齿双歧杆菌(Bifidobacterium dentium)中获得的可能参与CLA转化的基因分别注释为编码“未知蛋白3”、“未知蛋白4”、“未知蛋白5”的基因，其中，“未知蛋白3”的氨基酸序列如SEQ ID No.2所示、编码“未知蛋白3”的基因的核苷酸序列如SEQ ID No.6所示，“未知蛋白4”的氨基酸序列如SEQ ID No.3所示、编码“未知蛋白4”的基因的核苷酸序列如SEQ ID No.7所示，“未知蛋白5”的氨基酸序列如SEQ ID No.4所示、编码“未知蛋白5”的基因的核苷酸序列如SEQ ID No.8所示)。
实施例2：编码亚油酸异构酶的基因的克隆
从保菌管中挑取短双岐杆菌(Bifidobacterium breve)CGMCC No.11828的菌液划线于MRS固体培养基上，于37℃恒温厌氧工作站中培养48h，获得单菌落；挑取单菌落接种于MRS液体培养基中，于37℃恒温厌氧工作站中继续静置培养24h，连续活化3代，获得活化好的菌液；将活化好的菌液按1％(v/v)的接种量接种至MRS液体培养基中，于37℃恒温厌氧工作站中培养24h，获得菌悬液；将获得的菌悬液于25℃、12000g的条件下离心10min，获得湿菌体；使用细菌基因组DNA提取试剂盒提取湿菌体中的基因组DNA，并通过PCR反应扩增bbi；PCR反应结束，得到扩增产物，对扩增产物进行纯化后通过1％琼脂糖凝胶电泳验证扩增产物条带大小，获得bbi(此bbi基因即为编码“未知蛋白1”的基因)；其中，扩增bbi所用引物见表1；
PCR反应体系包含：KOD 1μL、ddH2O 29μL、上下游引物各1μL、基因组DNA 1μL、dNTP 5μL、10×reaction buffer 5μL以及Mg 2+3μL；
PCR反应条件为：95℃，5min；(95℃，30s；55℃，30s；68℃，1min)循环30次；68℃，5min；12℃，5min。
通过与获得bbi同样的方法分别从长双歧杆菌(Bifidobacterium longum)、假小链双歧杆菌(Bifidobacterium pseudocatenulatum)以及齿双歧杆菌(Bifidobacterium dentium)中获得bli(此bli基因即为编码“未知蛋白3”的基因)、bpi(此bpi基因即为编码“未知蛋白4”的基因)以及bdi(此bdi基因即为编码“未知蛋白5”的基因)；其中，扩增bli、bpi以及bdi所用引物见表1。
表1 引物序列
Figure PCTCN2019121822-appb-000001
Figure PCTCN2019121822-appb-000002
实施例3：亚油酸异构酶在大肠杆菌中的表达
将pET-28a(+)载体导入大肠杆菌E.coli DH5α中，获得大肠杆菌E.coli DH5α/pET28a；将大肠杆菌E.coli DH5α/pET28a划线于LB固体培养基(含有10μg/mL的卡那霉素)上，于37℃恒温培养箱中培养18h，获得单菌落；挑取单菌落接种于LB液体培养基(含有10μg/mL的卡那霉素)中，于37℃、200rpm的摇床中培养14h，连续活化3代，获得活化好的菌液；将活化好的菌液按1％(v/v)的接种量接种至LB液体培养基(含有10μg/mL的卡那霉素)中，于37℃、200rpm的摇床中培养14h，获得菌悬液；将获得的菌悬液于25℃、12000g的条件下离心10min，获得湿菌体；使用质粒小提试剂盒提取湿菌体中的pET-28a(+)载体；将获得的pET-28a(+)载体用50μL的ddH 2O回溶，于-20℃下储存。
使用限制性内切酶HindⅢ和Nde I对获得的pET-28a(+)载体以及实施例2获得的bbi、bli、bpi、bdi基因进行酶切，然后利用T 4连接酶将经酶切、纯化后的DNA进行连接，获得连接产物，具体连接体系如表2。
将获得的连接产物于16℃下过夜连接15h后，转化至大肠杆菌DH5α感受态细胞中；将转化后的大肠杆菌DH5α感受态细胞涂布LB固体培养基(含有10μg/mL的卡那霉素)，37℃倒置培养24h；挑取阳性转化子，提取质粒，测序验证结果表明连接成功，获得重组质粒pET28a-bbi、pET28a-bli、pET28a-bpi以及pET28a-bdi。
将获得的重组质粒pET28a-bbi、pET28a-bli、pET28a-bpi以及pET28a-bdi分别导入大肠杆菌E.coli BL21(DE3)中，获得重组大肠杆菌E.coli BL21(DE3)/pET28a-bbi、E.coli BL21(DE3)/pET28a-bli、E.coli BL21(DE3)/pET28a-bpi以及E.coli BL21(DE3)/pET28a-bdi。
将获得的重组大肠杆菌E.coli BL21(DE3)/pET28a-bbi、E.coli BL21(DE3)/pET28a-bli、E.coli BL21(DE3)/pET28a-bpi以及E.coli BL21(DE3)/pET28a-bdi分别划线于LB固体培养基上，于37℃恒温培养箱中培养18h，获得单菌落；挑取单菌落分别接种于LB液体培养基中，于37℃、200rpm的摇床中培养14h，连续活化3代，获得活化好的菌液；将活化好的菌液按1％(v/v)的接种量分别接种至LB液体培养基中，于温度为37℃、转速为200rpm的条件下培养12h，获得发酵液；将发酵液于4℃、12000g的条件下离心10min，获得湿菌体；将湿菌体进行破碎后于4℃、12000g的条件下离心10min，获得细胞破碎上清液；检测获得的细胞破碎上清液中的亚油酸异构酶酶活，检测结果如下：
重组大肠杆菌E.coli BL21(DE3)/pET28a-bbi发酵获得的细胞破碎上清液中的亚油酸异构酶酶活为6.7U/mg、重组大肠杆菌E.coli BL21(DE3)/pET28a-bli发酵获得的细胞破碎上清液中的亚油酸异构酶酶活为1.7U/mg、重组大肠杆菌E.coli BL21(DE3)/pET28a-bpi发酵获得的细胞破碎上清液中的亚油酸异构酶酶活为1.8U/mg、重组大肠杆菌E.coli BL21(DE3)/pET28a-bdi发酵获得的细胞破碎上清液中的亚油酸异构酶酶活为1.4U/mg。可见，重组大肠 杆菌E.coli BL21(DE3)/pET28a-bbi、E.coli BL21(DE3)/pET28a-bli、E.coli BL21(DE3)/pET28a-bpi以及E.coli BL21(DE3)/pET28a-bdi均可成功表达亚油酸异构酶。
表2 连接体系
Figure PCTCN2019121822-appb-000003
实施例4：重组大肠杆菌的应用
将实施例3获得的重组大肠杆菌E.coli BL21(DE3)/pET28a-bbi、E.coli BL21(DE3)/pET28a-bli、E.coli BL21(DE3)/pET28a-bpi以及E.coli BL21(DE3)/pET28a-bdi的活化好的菌液按1％(v/v)的接种量分别接种至LB液体培养基中，于温度为37℃、转速为200rpm的条件下培养至OD 600为0.4～0.6后，在培养基中分别加入终浓度为0mM、0.05mM、0.1mM、0.3mM、0.5mM、0.8mM、1.0mM的IPTG继续于18℃、200rpm的条件下诱导培养15h，得到培养液；将培养液分别于25℃、12000g的条件下离心10min，获得湿菌体；将湿菌体按0.5mg湿菌体/mL的浓度分别回悬至KPB缓冲溶液(pH＝6.5)中，然后在KPB缓冲溶液中分别加入终浓度为0.01mg/mL、0.05mg/mL、0.1mg/mL、0.5mg/mL的亚油酸，于37℃、200rpm的条件下反应3h；反应结束后，检测反应液中共轭亚油酸的转化率，并且，检测所得共轭亚油酸中共轭亚油酸异构体的类型及各共轭亚油酸异构体占比，检测结果见图1-5。
如图1可知，当IPTG终浓度为0.1mM时，重组大肠杆菌E.coli BL21(DE3)/pET28a-bbi、E.coli BL21(DE3)/pET28a-bli、E.coli BL21(DE3)/pET28a-bpi以及E.coli BL21(DE3)/pET28a-bdi反应获得的反应液中共轭亚油酸的转化率最高。
如图2-5可知，当IPTG终浓度为0.1mM时，重组大肠杆菌E.coli BL21(DE3)/pET28a-bbi反应获得的反应液中共轭亚油酸的转化率可达42.1％，其中，89.1％为cis9,trans11-CLA、1％为trans10,cis12-CLA、9.9％为trans9,trans11-CLA；
当IPTG终浓度为0.1mM时，重组大肠杆菌E.coli BL21(DE3)/pET28a-bli反应获得的反应液中共轭亚油酸的转化率为12.1％，其中，84.3％为cis9,trans11-CLA、1.2％为trans10,cis12-CLA、4.5％为trans9,trans11-CLA；
当IPTG终浓度为0.1mM时，重组大肠杆菌E.coli BL21(DE3)/pET28a-bpi反应获得的反 应液中共轭亚油酸的转化率为19.5％，其中，88.9％为cis9,trans11-CLA、0.98％为trans10,cis12-CLA、10.1％为trans9,trans11-CLA；
当IPTG终浓度为0.1mM时，重组大肠杆菌E.coli BL21(DE3)/pET28a-bdi反应获得的反应液中共轭亚油酸的转化率为13.5％，其中，87.1％为cis9,trans11-CLA、1.3％为trans10,cis12-CLA、11.6％为trans9,trans11-CLA。
实施例5：亚油酸异构酶在耶氏解脂酵母菌株中的表达
根据耶氏解脂酵母的密码子偏好性，利用Genscript OptimumGene TM软件对bbi基因进行优化，优化后密码子适应指数从0.80提高至0.96，优化后的基因命名为obbi基因。
obbi基因由南京金斯瑞生物科技有限公司进行全基因合成，并克隆至载体pUC57上，得到重组质粒pUC57-obbi；其中，bbi基因的核苷酸序列如SEQ ID No.5所示，obbi基因的核苷酸序列如SEQ ID No.17所示、bbi基因和obbi基因的CAI图谱如图6所示。
使用限制性内切酶BamH I和Kpn I对pINA 1312sp质粒以及重组质粒pUC57-obbi进行酶切，然后利用T 4连接酶将经酶切、纯化后的DNA进行连接，获得连接产物；将获得的连接产物于16℃下过夜连接15h后，转化至大肠杆菌E.coli DH5α感受态细胞中；将转化后的大肠杆菌E.coli DH5α感受态细胞涂布LB固体培养基(含有10μg/mL的卡那霉素)，37℃倒置培养12～16h；挑取阳性转化子，提取质粒，测序验证结果表明连接成功，获得重组质粒pINA1312sp-obbi，重组质粒pINA 1312sp-obbi的质粒图谱见图7，验证结果见图8。
将获得的重组质粒pINA 1312sp-obbi导入耶氏解脂酵母(Yarrowia lipolytica)中；将转化后的耶氏解脂酵母划线于YNBD固体培养基上，于28℃恒温培养箱中培养2～3d；挑取阳性转化子接种于YNBD液体培养基中，28℃、200rpm/min培养2d，收集菌体，提取基因组，用验证引物P1/P2(P1的核苷酸序列如SEQ ID No.6所示：ATACAAGAGCGTTTGCCAGC/P2的核苷酸序列如SEQ ID No.7所示：CCTTGGTCCAGGGGTTGA)对转化子的基因组进行PCR验证，验证正确，获得重组耶氏解脂酵母菌株Yarrowia lipolytica/pINA 1312sp-obbi(共获得20个验证正确的转化子)，验证结果见图9。
将重组耶氏解脂酵母菌株Yarrowia lipolytica/pINA 1312sp-obbi划线于YNBD固体培养基上，于28℃恒温培养箱中培养2～3d；挑取接种于YNBD液体培养基中，28℃、200rpm/min培养2d；将种子液以1％(v/v)的接种量接种至5mL的YPD培养基中，28℃、200rpm/min培养36h，获得发酵液；将发酵液于25℃、12000g的条件下离心10min，获得湿菌体；将湿菌体进行破碎后于25℃、12000g的条件下离心10min，获得细胞破碎上清液；检测获得的细胞破碎上清液中的亚油酸异构酶比酶活，检测结果如下：
重组耶氏解脂酵母菌株Yarrowia lipolytica/pINA 1312sp-obbi发酵获得的细胞破碎上清液中的亚油酸异构酶比酶活为2.31U/mg。可见，重组耶氏解脂酵母菌株Yarrowia lipolytica/pINA 1312sp-obbi可成功表达亚油酸异构酶。
实施例6：重组耶氏解脂酵母菌株的应用
1、以亚油酸为底物
以含有空质粒的重组耶氏解脂酵母菌株Yarrowia lipolytica/pINA 1312sp作为对照，将重组耶氏解脂酵母菌株Yarrowia lipolytica/pINA 1312sp-obbi划线于YNBD固体培养基上，于28℃恒温培养箱中培养2～3d；挑取单菌落接种于YNBD液体培养基中，28℃、200rpm/min培养2d；将种子液以1％(v/v)的接种量接种至5mL的YPD培养基中，28℃、200rpm/min培养36h后，在培养基中添加终浓度为0.5g/L的游离亚油酸，继续于28℃、200rpm/min培养36h，获得发酵液；将发酵液于25℃、12000g的条件下离心10min，获得湿菌体；将湿菌体用0.85％NaCl洗涤两遍后检测细胞中cis9,trans11-CLA、trans10,cis12-CLA以及trans9,trans11-CLA的产量以及cis9,trans11-CLA、trans10,cis12-CLA以及trans9,trans11-CLA的含量占生产得到的总共轭脂肪酸含量的比例，检测结果见图10-11、14。
由图10-11、14可知，重组耶氏解脂酵母菌株Yarrowia lipolytica/pINA 1312sp-obbi以游离脂肪酸为底物生产cis9,trans11-CLA、trans10,cis12-CLA以及trans9,trans11-CLA的产量 分别为1.5mg/L、0.5mg/L、3.8mg/L，其中，cis9,trans11-CLA的含量占总共轭脂肪酸含量的25％、trans10,cis12-CLA的含量占总共轭脂肪酸含量的9％、trans9,trans11-CLA的含量占总共轭脂肪酸含量的66％。
2、以红花籽油为底物
以含有空质粒的重组耶氏解脂酵母菌株Yarrowia lipolytica/pINA 1312sp作为对照，将重组耶氏解脂酵母菌株Yarrowia lipolytica/pINA 1312sp-obbi划线于YNBD固体培养基上，于28℃恒温培养箱中培养2～3d；挑取单菌落接种于YNBD液体培养基中，28℃、200rpm/min培养2d；将种子液以1％(v/v)的接种量接种至5mL的YPD培养基中，28℃、200rpm/min培养36h后，在培养基中添加终浓度为20g/L的红花籽油，继续于28℃、200rpm/min培养36h，获得发酵液；将发酵液于25℃、12000g的条件下离心10min，获得湿菌体；将湿菌体用0.85％NaCl洗涤两遍后检测细胞中cis9,trans11-CLA、trans10,cis12-CLA以及trans9,trans11-CLA的产量以及cis9,trans11-CLA、trans10,cis12-CLA以及trans9,trans11-CLA的含量占生产得到的总共轭脂肪酸含量的比例，检测结果见图12-14。
由图12-14可知，重组耶氏解脂酵母菌株Yarrowia lipolytica/pINA 1312sp-obbi以红花籽油为底物生产cis9,trans11-CLA、trans10,cis12-CLA以及trans9,trans11-CLA的产量分别为146.8mg/L、50.9mg/L、310.7mg/L，其中，cis9,trans11-CLA的含量占总共轭脂肪酸含量的30％、trans10,cis12-CLA的含量占总共轭脂肪酸含量的10％、trans9,trans11-CLA的含量占总共轭脂肪酸含量的60％。
3、扩大培养
将重组耶氏解脂酵母菌株Yarrowia lipolytica/pINA 1312sp-obbi划线于YNBD固体培养基上，于28℃恒温培养箱中培养2～3d；挑取单菌落接种于YNBD液体培养基中，28℃、200rpm/min培养2d；将种子液以1％(v/v)的接种量接种至50mL的YPD培养基中，28℃、200rpm/min培养36h后，在培养基中添加终浓度为20g/L的红花籽油，继续于28℃、200rpm/min培养36h，获得发酵液；将发酵液于25℃、12000g的条件下离心10min，获得湿菌体；将湿菌体用0.85％NaCl洗涤两遍后检测细胞中cis9,trans11-CLA、trans10,cis12-CLA以及trans9,trans11-CLA的产量以及cis9,trans11-CLA、trans10,cis12-CLA以及trans9,trans11-CLA的含量占生产得到的总共轭脂肪酸含量的比例，检测结果如下：
重组耶氏解脂酵母菌株Yarrowia lipolytica/pINA 1312sp-obbi以红花籽油为底物生产cis9,trans11-CLA、trans10,cis12-CLA以及trans9,trans11-CLA的产量分别为224.0mg/L、73.7mg/L、454.0mg/L，其中，cis9,trans11-CLA的含量占总共轭脂肪酸含量的29.8％、trans10,cis12-CLA的含量占总共轭脂肪酸含量的9.8％、trans9,trans11-CLA的含量占总共轭脂肪酸含量的60.4％。
4、红花籽油浓度对重组耶氏解脂酵母菌株Yarrowia lipolytica/pINA 1312sp-obbi生产cis9,trans11-CLA的产量以及转化率的影响
将重组耶氏解脂酵母菌株Yarrowia lipolytica/pINA 1312sp-obbi划线于YNBD固体培养基上，于28℃恒温培养箱中培养2～3d；挑取单菌落接种于YNBD液体培养基中，28℃、200rpm/min培养2d；将种子液以1％(v/v)的接种量接种至50mL的YPD培养基中，28℃、200rpm/min培养36h后，在培养基中分别添加终浓度为10g/L、20g/L、30g/L、50g/L、70g/L的红花籽油，继续于28℃、200rpm/min培养，每隔12h取50mL发酵液；将发酵液于25℃、12000g的条件下离心10min，获得湿菌体；将湿菌体用0.85％NaCl洗涤两遍后检测细胞中cis9,trans11-CLA、trans10,cis12-CLA以及trans9,trans11-CLA的产量以及cis9,trans11-CLA、trans10,cis12-CLA以及trans9,trans11-CLA的含量占生产得到的总共轭脂肪酸含量的比例，其中，红花籽油浓度对重组耶氏解脂酵母菌株Yarrowia lipolytica/pINA 1312sp-obbi生产cis9,trans11-CLA的产量的影响结果见图15，红花籽油浓度对重组耶氏解脂酵母菌株Yarrowia lipolytica/pINA 1312sp-obbi发酵84h时生产得到的cis9,trans11-CLA的产量的影响见图16，红花籽油浓度对重组耶氏解脂酵母菌株Yarrowia lipolytica/pINA 1312sp-obbi发酵84h时生产 得到的cis9,trans11-CLA的产量的占生产得到的总共轭脂肪酸产量的比例见图17，红花籽油浓度对重组耶氏解脂酵母菌株Yarrowia lipolytica/pINA 1312sp-obbi生产cis9,trans11-CLA的产量以及转化率的影响见图18。
由图15-18可知，发酵时间为84h，红花籽油的添加浓度为50g/L时，cis9,trans11-CLA的含量最高，达到了350mg/L；此时cis9,trans11-CLA的占总CLA的比例达到了27.5％。
实施例7：亚油酸异构酶在植物乳杆菌中的表达
在不影响表达蛋白的前体下，减少实施例2获得的bbi中GC的含量，并且，使得对应的密码子更适合于乳杆菌的生物利用，密码子的优化以及基因序列的合成由通用生物系统(安徽)有限公司完成，序列两端的酶切位点分别为Kpn I以及Xba I，序列连接在pU57质粒中，质粒保存在大肠杆菌E.coli DH5α中，得到重组大肠杆菌E.coli DH5α/pU57-bbi(U)；其中，未经优化的bbi序列的核苷酸序列如SEQ ID No.5所示，经优化的bbi序列的核苷酸序列如SEQ ID No.18所示。
将pNZ44质粒导入大肠杆菌E.coli DH5α中，获得大肠杆菌E.coli DH5α/pNZ44；将大肠杆菌E.coli DH5α/pNZ44划线于LB固体培养基(含有10μg/mL的卡那霉素)上，于37℃恒温培养箱中培养18h，获得单菌落；挑取单菌落接种于LB液体培养基(含有10μg/mL的卡那霉素)中，于37℃、200rpm的摇床中培养14h，连续活化3代，获得活化好的菌液；将活化好的菌液按1％(v/v)的接种量接种至LB液体培养基(含有10μg/mL的卡那霉素)中，于37℃、200rpm的摇床中培养14h，获得菌悬液；将获得的菌悬液于25℃、12000g的条件下离心10min，获得湿菌体；使用质粒小提试剂盒提取湿菌体中的pNZ44质粒；将获得的pNZ44质粒用50μL的ddH 2O回溶，于-20℃下储存。
使用质粒小提试剂盒提取重组大肠杆菌E.coli DH5α/pU57-bbi(U)中的重组质粒pU57-bbi(U)；将获得的重组质粒pU57-bbi(U)用50μL的ddH 2O回溶，于-20℃下储存。
使用限制性内切酶Kpn I以及Xba I对获得的pNZ44质粒以及重组质粒pU57-bbi(U)进行酶切，然后利用T 4连接酶将经酶切、纯化后的DNA进行连接，获得连接产物，具体连接体系如表3。
将获得的连接产物于16℃下过夜连接15h后，转化至大肠杆菌E.coli DH5α感受态细胞中；将转化后的大肠杆菌E.coli DH5α感受态细胞涂布LB固体培养基(含有10μg/mL的氯霉素)，37℃倒置培养24h；挑取阳性转化子，提取质粒，测序验证结果表明连接成功，获得重组质粒pNZ44-bbi以及重组质粒pNZ44-bbi(U)，验证结果见图19。
将获得的重组质粒pNZ44-bbi以及重组质粒pNZ44-bbi(U)分别导入植物乳杆菌Lactobacillus plantarum ST-III中，获得植物乳杆菌工程菌Lactobacillus plantarum ST-III/pNZ44-bbi以及植物乳杆菌工程菌Lactobacillus plantarum ST-III/pNZ44-bbi(U)。
将获得的植物乳杆菌工程菌Lactobacillus plantarum ST-III/pNZ44-bbi以及植物乳杆菌工程菌Lactobacillus plantarum ST-III/pNZ44-bbi(U)分别划线于MRS固体培养基上，于37℃恒温培养箱中培养18h，获得单菌落；挑取单菌落分别接种于MRS液体培养基中，于37℃、200rpm的摇床中培养14h，连续活化3代，获得活化好的菌液；将活化好的菌液按1％(v/v)的接种量分别接种至LB液体培养基中，于温度为37℃的条件下静置培养12h，获得发酵液；将发酵液于4℃、12000g的条件下离心10min，获得湿菌体；将湿菌体进行破碎后于4℃、12000g的条件下离心10min，获得细胞破碎上清液；检测获得的细胞破碎上清液中的亚油酸异构酶酶活，检测结果如下：
植物乳杆菌工程菌Lactobacillus plantarum ST-III/pNZ44-bbi发酵获得的细胞破碎上清液中的亚油酸异构酶酶活为2.5U/mg、植物乳杆菌工程菌Lactobacillus plantarum ST-III/pNZ44-bbi(U)发酵获得的细胞破碎上清液中的亚油酸异构酶酶活为10.5U/mg。可见，植物乳杆菌工程菌Lactobacillus plantarum ST-III/pNZ44-bbi以及植物乳杆菌工程菌Lactobacillus plantarum ST-III/pNZ44-bbi(U)可成功表达亚油酸异构酶，但植物乳杆菌工程菌Lactobacillus plantarum ST-III/pNZ44-bbi(U)的表达能力更强。
表3 连接体系
Figure PCTCN2019121822-appb-000004
实施例8：植物乳杆菌工程菌的应用
将获得的植物乳杆菌工程菌Lactobacillus plantarum ST-III/pNZ44-bbi以及植物乳杆菌工程菌Lactobacillus plantarum ST-III/pNZ44-bbi(U)的活化好的菌液按1％(v/v)的接种量分别接种至含有0.5mg/mL游离亚油酸的MRS液体培养基中，于温度为37℃的条件下静置培养72h，得到发酵液；检测发酵液中共轭亚油酸的转化率，并且，检测所得共轭亚油酸中共轭亚油酸异构体的类型及各共轭亚油酸异构体占比，检测结果见图20-21。
由检测结果可知，植物乳杆菌工程菌Lactobacillus plantarum ST-III/pNZ44-bbi发酵得到的发酵液中并无共轭亚油酸；植物乳杆菌工程菌Lactobacillus plantarum ST-III/pNZ44-bbi(U)发酵得到的发酵液中共轭亚油酸的转化率可达89.9％。
如图20-21可知，植物乳杆菌工程菌Lactobacillus plantarum ST-III/pNZ44-bbi(U)发酵得到的共轭亚油酸100％为cis9,trans11-CLA。
虽然本发明已以较佳实施例公开如上，但其并非用以限定本发明，任何熟悉此技术的人，在不脱离本发明的精神和范围内，都可做各种的改动与修饰，因此本发明的保护范围应该以权利要求书所界定的为准。

Claims (20)

  1. 一种亚油酸异构酶，其特征在于，所述亚油酸异构酶为：
    (a)由SEQ ID No.1、SEQ ID No.2、SEQ ID No.3或SEQ ID No.4所示的氨基酸序列组成的蛋白质；或者，
    (b)在(a)中的氨基酸序列经过取代、缺失或添加一个或几个氨基酸且具有亚油酸异构酶活性的由(a)衍生的蛋白质。
  2. 一种基因，其特征在于，所述基因编码权利要求1所述的亚油酸异构酶。
  3. 如权利要求2所述的一种基因，其特征在于，所述基因的核苷酸序列如SEQ ID No.5、SEQ ID No.6、SEQ ID No.7、SEQ ID No.8、SEQ ID No.17或SEQ ID No.18所示。
  4. 一种重组质粒，特征在于，所述重组质粒携带权利要求2或3所述的基因。
  5. 如权利要求4所述的一种重组质粒，特征在于，所述重组质粒的载体为pET-28a(+)质粒、pINA 1312sp质粒或pNZ44质粒。
  6. 一种宿主细胞，其特征在于，所述宿主细胞携带权利要求2或3所述的基因或权利要求4所述的重组质粒。
  7. 如权利要求6所述的一种宿主细胞，其特征在于，所述宿主细胞为大肠杆菌、耶氏解脂酵母或植物乳杆菌。
  8. 如权利要求7所述的一种宿主细胞，其特征在于，当宿主细胞为大肠杆菌时，所述宿主细胞以pET-28a(+)质粒为载体携带核苷酸序列如SEQ ID No.5、SEQ ID No.6、SEQ ID No.7或SEQ ID No.8所示的编码权利要求1所述亚油酸异构酶的基因；
    当宿主细胞为耶氏解脂酵母时，所述宿主细胞以pINA 1312sp质粒为载体携带核苷酸序列如SEQ ID No.17所示的编码权利要求1所述亚油酸异构酶的基因；
    当宿主细胞为植物乳杆菌时，所述宿主细胞以pNZ44质粒为载体携带核苷酸序列如SEQ ID No.18所示的编码权利要求1所述亚油酸异构酶的基因。
  9. 权利要求1所述亚油酸异构酶或权利要求2或3所述基因或权利要求4或5所述重组质粒或权利要求6-8任一所述宿主细胞在生产共轭亚油酸方面的应用。
  10. 一种生产共轭亚油酸的方法，其特征在于，使用权利要求7或8所述的宿主细胞；
    当宿主细胞为大肠杆菌时，所述方法为将权利要求7或8所述的宿主细胞接种至培养基中，于温度为35～40℃、转速为150～250rpm的条件下培养至OD 600为0.4～0.6，得到培养液A；在培养液A中加入终浓度为0.01～1.0mM的IPTG，于温度为15～20℃、转速为150～250rpm的条件下诱导培养12～16h，得到培养液B；将培养液B离心，收集湿菌体；以亚油酸为底物，在含亚油酸的反应体系中加入湿菌体，于温度为35～40℃、转速为150～250rpm的条件下进行反应，得到富含共轭亚油酸的反应液；将富含共轭亚油酸的反应液进行提取，得到共轭亚油酸；
    当宿主细胞为耶氏解脂酵母时，所述方法为将权利要求7或8所述的宿主细胞接种至含有亚油酸和/或甘油酯的培养基中，于温度为35～40℃、转速为150～250rpm的条件下进行培养，得到富含共轭亚油酸的宿主细胞，然后将富含共轭亚油酸的宿主细胞进行提取，得到共轭亚油酸；
    当宿主细胞为植物乳杆菌时，所述方法为将权利要求7或8所述的宿主细胞接种至含有亚油酸的培养基中，于温度为37℃的条件下静置培养，得到富含共轭亚油酸的培养液；将富含共轭亚油酸的培养液进行提取，得到共轭亚油酸。
  11. 如权利要求10所述的一种生产共轭亚油酸的方法，其特征在于，当宿主细胞为大肠杆菌时，所述方法为将权利要求7或8所述的宿主细胞接种至培养基中，于温度为37℃、转速为200rpm的条件下培养至OD 600为0.4～0.6，得到培养液A；在培养液A中加入终浓度为0.01～1.0mM的IPTG，于温度为18℃、转速为200rpm的条件下诱导培养12～16h，得到培养液B；将培养液B离心，收集湿菌体；以亚油酸为底物，在含亚油酸的反应体系中加入湿菌体，于温度为37℃、转速为200rpm的条件下进行反应，得到富含共轭亚油酸的反应液；将富含共轭亚油酸的反应液进行提取，得到共轭亚油酸。
  12. 如权利要求10所述的一种生产共轭亚油酸的方法，其特征在于，当宿主细胞为耶氏解脂酵母时，所述甘油酯为红花籽油、亚麻仁油、棉籽油、和/或大豆油。
  13. 如权利要求10或11所述的一种生产共轭亚油酸的方法，其特征在于，当宿主细胞为大肠杆菌时，所述反应体系包含缓冲液以及亚油酸。
  14. 如权利要求10或11或13所述的一种生产共轭亚油酸的方法，其特征在于，当宿主细胞为大肠杆菌时，所述缓冲液的pH为6～7。
  15. 如权利要求10或11或13或14所述的一种生产共轭亚油酸的方法，其特征在于，当宿主细胞为大肠杆菌时，所述亚油酸在反应体系中的浓度为0.05～0.15mg/mL。
  16. 如权利要求10或11或13或14或15所述的一种生产共轭亚油酸的方法，其特征在于，当宿主细胞为大肠杆菌时，所述湿菌体在反应体系中的浓度为0.5～2mg/mL。
  17. 如权利要求10-16任一所述的一种生产共轭亚油酸的方法，其特征在于，当宿主细胞为大肠杆菌时，所述共轭亚油酸为cis9,trans11-CLA和/或trans9,trans11-CLA；当宿主细胞为耶氏解脂酵母时，所述共轭亚油酸为cis9,trans11-CLA、trans10,cis12-CLA和/或trans9,trans11-CLA；当宿主细胞为植物乳杆菌时，所述共轭亚油酸为cis9,trans11-CLA。
  18. 权利要求6-8任一所述宿主细胞在生产亚油酸异构酶方面的应用，其特征在于，所述亚油酸异构酶的氨基酸序列如SEQ ID No.1所示。
  19. 一种生产权利要求1所述亚油酸异构酶的方法，其特征在于，使用权利要求7或8所述的宿主细胞；
    当宿主细胞为大肠杆菌时，所述方法为将权利要求7或8所述的宿主细胞加入培养基中，于温度为35～40℃、转速为150～250rpm的条件下培养，得到富含亚油酸异构酶的宿主细胞，然后将富含亚油酸异构酶的宿主细胞进行提取，得到亚油酸异构酶；
    当宿主细胞为耶氏解脂酵母时，所述方法为将权利要求7或8所述的宿主细胞接种至培养基中，于温度为35～40℃、转速为150～250rpm的条件下培养，得到富含亚油酸异构酶的宿主细胞，然后将富含亚油酸异构酶的宿主细胞进行提取，得到亚油酸异构酶；
    当宿主细胞为植物乳杆菌时，所述方法为将权利要求7或8所述的宿主细胞接种至接种至培养基中，于温度为37℃的条件下静置培养，得到富含亚油酸异构酶的宿主细胞，然后将富含亚油酸异构酶的宿主细胞进行提取，得到亚油酸异构酶。
  20. 如权利要求19所述的一种生产权利要求1所述亚油酸异构酶的方法，其特征在于，当宿主细胞为大肠杆菌时，所述培养基为LB培养基；当宿主细胞为耶氏解脂酵母时，所述培养基为YPD培养基；当宿主细胞为植物乳杆菌时，所述培养基为MRS培养基。
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