WO2016062171A1 - 基因及其用途、合成奇数中链脂肪醛的方法以及合成偶数中链脂肪烃的方法 - Google Patents

基因及其用途、合成奇数中链脂肪醛的方法以及合成偶数中链脂肪烃的方法 Download PDF

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WO2016062171A1
WO2016062171A1 PCT/CN2015/089232 CN2015089232W WO2016062171A1 WO 2016062171 A1 WO2016062171 A1 WO 2016062171A1 CN 2015089232 W CN2015089232 W CN 2015089232W WO 2016062171 A1 WO2016062171 A1 WO 2016062171A1
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gene
vector
synthesizing
medium chain
plasmid
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French (fr)
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元英进
曹英秀
肖文海
刘夺
丁明珠
谢泽雄
张金来
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天津大学
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)
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    • C12P5/00—Preparation of hydrocarbons or halogenated hydrocarbons
    • C12P5/02—Preparation of hydrocarbons or halogenated hydrocarbons acyclic
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    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
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    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/24—Preparation of oxygen-containing organic compounds containing a carbonyl group
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    • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
    • C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
    • C12Y101/01021—Aldehyde reductase (1.1.1.21), i.e. aldose-reductase
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    • C12Y401/00—Carbon-carbon lyases (4.1)
    • C12Y401/99—Other Carbon-Carbon Lyases (1.4.99)
    • C12Y401/99005—Octadecanal decarbonylase (4.1.99.5)

Definitions

  • the present invention relates to the field of biotechnology, and in particular to genes, proteins encoded therein and uses thereof, gene elements, methods for synthesizing odd-chain mid-chain fatty aldehydes, and methods for synthesizing even-chain intermediate aliphatic hydrocarbons.
  • Medium-chain fatty alcohols have very important applications in the industry due to their amphiphilic properties, and can be used in surfactants, pharmaceuticals, cosmetics, and energy fields, with market capitalization of $1-120 million.
  • the aliphatic hydrocarbon molecules with a carbon chain length of 6-16 are the main components of aviation kerosene, and have the advantages of high calorific value, low vapor pressure, low freezing point and low hygroscopicity.
  • 50% of commercial fatty alcohols are extracted from plant seeds or animal fats, and the remaining fatty alcohols and all hydrocarbons are refined from petroleum. Either way, it does not meet the sustainable and environmentally friendly production requirements of modern society.
  • genetically engineered strains can utilize renewable energy such as sugar, xylan or glycerol to specifically synthesize the desired products.
  • fatty alcohols and hydrocarbons are mainly derived from fatty acid synthesis pathways.
  • Three molecules of fatty acyl-ACP/CoA and free fatty acids can be used as synthetic precursors, respectively.
  • hydrocarbon alcohols the conversion of fatty acyl-ACP/CoA or fatty acids to fatty aldehydes is a critical step, followed by reduction of the fatty aldehydes to fatty alcohols or decarbonylation to hydrocarbons with less than one molecule of carbon.
  • Microbial synthesis of fatty alcohols/hydrocarbons with fatty acyl ACP/CoA as precursors has been reported since 2010.
  • the synthetic system for the synthesis of medium-chain hydrocarbons using free fatty acids as a substrate did not appear until 2013.
  • Howard et al. overexpress thioesterase from Cinnamomum camphora in E. coli for a specific length
  • the free fatty acid is released from the fatty acyl-ACP and simultaneously expresses the luxC, luxD, luxE gene-derived fatty acid reductase (FAR) of Photorhabdus luminescens and from Nostoc punctiforme PCC73102
  • FAR luxC, luxD, luxE gene-derived fatty acid reductase
  • the fatty aldehyde decarbonylase which converts free fatty acids into fatty aldehydes and decarboxylates them into hydrocarbon molecules with one carbon atom, constructs a hydrocarbon synthesis system capable of synthesizing relatively free fatty acids as a substrate.
  • CAR carboxylic acid reductase
  • E.coli BL21(DE3) strain can synthesize up to 350mg/L in a minimal medium with glucose as a carbon source when this pathway is combined intracellularly with a thioesterase that produces a specific chain length free fatty acid. Fatty alcohol.
  • the present invention provides a gene, a protein encoded thereby, use thereof, a gene element, a method of synthesizing an odd chain medium fatty aldehyde, and a method of synthesizing an even medium chain aliphatic hydrocarbon.
  • Alpha-dioxygenase converts the precursor fatty acid molecule into an important intermediate metabolite.
  • the process of fatty aldehyde is an oxidation reaction, which does not require cells to provide additional reducing power and energy, reducing the burden of cell production; filling the fatty alcohol
  • the chain length of the product is only an even number, and the chain length of the aliphatic hydrocarbon product is mostly an odd technical limit, which can make the bio-based bulk chemicals and biofuel molecules more compatible with petroleum-based products.
  • the present invention provides the following technical solutions:
  • the invention provides a gene having:
  • (III) a sequence which encodes the same protein as the nucleotide sequence of (I) or (II) but differs from the nucleotide sequence of (I) or (II) due to the degeneracy of the genetic code;
  • the invention also provides the use of the above genes for the synthesis of fatty aldehydes, odd medium chain fatty acids, odd medium chain fatty alcohols, and even medium chain aliphatic hydrocarbons.
  • the fatty aldehyde is an odd medium chain fatty aldehyde.
  • the medium chain fatty acid, the medium chain fatty alcohol, and the medium chain aliphatic hydrocarbon of the present invention each represent a fatty acid having from 8 to 14 carbon atoms, a fatty alcohol, and an aliphatic hydrocarbon molecule.
  • the fatty aldehyde is an odd medium chain fatty aldehyde.
  • the fatty aldehyde is 1-undecaldehyde, tridecanal or pentadecalaldehyde.
  • the odd medium chain fatty alcohol is 1-undecyl alcohol, 1-tridecyl alcohol or 1-pentadecanol.
  • the even number of medium chain aliphatic hydrocarbons are aliphatic hydrocarbons having a chain length of C12 and C14.
  • the present invention also provides a vector comprising the gene ( ⁇ -dox) represented by SEQ ID NO: 1.
  • the invention also provides a host cell comprising the above vector.
  • the host cell is E. coli.
  • the present invention also provides a genetic element for synthesizing a fatty aldehyde comprising the gene ( ⁇ -dox) represented by SEQ ID NO: 1.
  • the present invention also provides a genetic element for synthesizing an odd-numbered medium chain fatty aldehyde comprising the gene ( ⁇ -dox) and the thioesterase gene represented by SEQ ID NO: 1.
  • the gene element for synthesizing odd-chain mid-chain fatty aldehyde provided by the present invention is pACYC-(T7-Dox-tesA') plasmid (numbered YX135 in the present invention), pACYC-(T7-Dox-BTE) plasmid (present invention The plasmid numbered YX104), pACYC-(T7-Dox-BnFatA) (numbered YX105 in the present invention).
  • the present invention also provides a method for constructing a genetic element for synthesizing odd medium chain fatty aldehydes, comprising the following contents:
  • ⁇ -dox ⁇ -dioxygenase gene represented by SEQ ID NO: 1 into the pET21a plasmid to form a pET21a-Dox plasmid;
  • pACYC-Trc-tesA was used as a vector, and digested with SpeI and BamHI, and the fragment template was 21A-Dox.
  • the pET21a-Dox plasmid was digested with XbaI and BamHI, and the gel was recovered and ligated with the vector to construct pACYC.
  • -Trc-tesA-Dox (CYX134) plasmid.
  • the present invention also provides a genetic element for synthesizing a fatty alcohol comprising the gene ( ⁇ -dox) represented by SEQ ID NO: 1.
  • the present invention also provides a genetic element for synthesizing an odd-numbered medium chain fatty alcohol comprising the ⁇ -dioxygenase gene ( ⁇ -dox), thioesterase gene and aldehyde group reduction represented by SEQ ID NO: 1. Enzyme gene.
  • the aldehyde reductase gene is selected from the group consisting of a gene having the nucleotide sequence set forth in SEQ ID No. 2, adhP, yjgB, yqhD or adhE.
  • the gene element for synthesizing an odd-numbered medium chain fatty alcohol provided by the present invention is pACYC-Trc-tesA-Dox plasmid (numbered CYX134 in the present invention), pACYC-(T7-Dox)-(T7-tesA') plasmid (numbered YX220 in the present invention), pACYC-(T5-Dox)-( T7-tesA') plasmid (numbered YX232 in the present invention), pACYC-(Trc-Dox)-(T7-tesA') plasmid (numbered YX233 in the present invention), pACYC-(LacUV5-Dox)-(T7- tesA') plasmid (numbered YX234 in the present invention), pACYC-(BAD-Dox)-(T7-tesA') plasmid (numbered YX235 in the present invention), pACYC-(T7-
  • a method for constructing a genetic element for synthesizing an odd-numbered medium-chain fatty alcohol characterized by: different types or sources of aldehyde dehydrogenase genes (adhP, yjgB, yqhD, adhE, and SEQ) Spl1(2) shown by ID NO: 2 is ligated into the pET28a plasmid to form 28a-AdhP, 28a-YjgB, pET28a-YqhD, pET28a-AdhE, pET28a-Slr1192 plasmid;
  • CYX134 pACYC-Trc-tesA-Dox
  • digestion with SpeI and BamHI was carried out and purified.
  • 28a-AdhP, 28a-YjgB, pET28a-YqhD, pET28a-AdhE, pET28a-Slr1192 as a template, XbaI and BamHI were used for digestion, and the gel was recovered and ligated to the vector.
  • the present invention also provides a genetic element for synthesizing an even-numbered medium chain aliphatic hydrocarbon, which comprises the gene of claim 1, a thioesterase gene, and a carbonyl decarbonylase gene.
  • the carbonyl decarbonylase gene is selected from the group consisting of a gene having the nucleotide sequence set forth in SEQ ID No. 3, 4 or 5 or ad73102.
  • the gene element for synthesizing even-chain mid-chain aliphatic hydrocarbons is pACYC-(Trc-tesA'-Dox-CER1) plasmid (numbered as CYX148 in the present invention), pACYC-(Trc-tesA'-Dox-AD9313 Plasmid (numbered CYX149 in the present invention), pACYC-(Trc-tesA'-Dox-AD7942) plasmid (numbered CYX150 in the present invention), pACYC-(Trc-tesA'-Dox-AD73102) plasmid (in the present invention) The number is CYX151).
  • the present invention also provides a method for constructing a genetic element for synthesizing even-chain intermediate aliphatic hydrocarbons.
  • the aldehyde-based decarbonylase gene of different types or sources (cer1, SEQ ID NO: 4 shown in SEQ ID NO: 3)
  • the shown ad9313, ad7942, ad73102 shown in SEQ ID NO: 5 were ligated into the pET28a plasmid to form pET28a-CER1, pET28a-AD9313, pET28a-AD7942 and pET28a-AD73102 plasmids;
  • CYX134 is used as a carrier.
  • pET28a-CER1 pET28a-AD9313, pET28a-AD7942 and pET28a-AD73102 as templates, the enzyme was digested, the gel was recovered, and the vector was ligated.
  • the present invention provides a method of synthesizing an odd number of medium chain fatty aldehydes, comprising the steps of:
  • Step 1 ligating the above gene into a vector to construct an expression vector
  • Step 2 The expression vector is transformed into a host cell, expressed, and the expression product is collected.
  • the ⁇ -dioxygenase gene was ligated from the pET21a-Dox plasmid with the RBS dox gene into the pACYC-Trc-tesA plasmid to form the CYX134 plasmid.
  • the invention also provides a method for synthesizing an odd number of medium chain fatty alcohols, comprising the steps of:
  • Step 1 constructing a first vector containing a thioesterase gene and the thioesterase gene promoter
  • Step 2 ligating the above gene to the first vector by enzyme digestion to construct a second vector
  • Step 3 ligating the aldehyde-reductase gene to the second vector by restriction enzyme digestion to construct an expression vector
  • Step 4 The expression vector is transformed into a host cell, expressed, and the expression product is collected.
  • aldehyde-reductase genes of different origins were digested with XbaI and BamHI from the plasmids of 28a-AdhP, 28a-YjgB, pET28a-YqhD, pET28a-AdhE, pET28a-Slr1192 and ligated into SpeI and BamHI, respectively.
  • CYX143, CYX144, CYX145, CYX146, CYX147 plasmids are formed in the CYX134 plasmid;
  • Each plasmid was transformed into E. coli BL21 (DE3) strain, fermented, and the product was collected.
  • the invention also provides a method for synthesizing even-chain intermediate aliphatic hydrocarbons, comprising the steps of:
  • Step 1 constructing a first vector containing a thioesterase gene and the thioesterase gene promoter
  • Step 2 The gene of claim 1 is cleaved to the first vector to construct a second vector;
  • Step 3 ligating the carbonyl decarbonylase gene to the second vector by restriction enzyme digestion to construct an expression vector
  • Step 4 The expression vector is transformed into a host cell, expressed, and the expression product is collected.
  • the invention provides a method for synthesizing an odd number of intermediate fatty alcohols by fed-batch fermentation, wherein the CYX144 plasmid and the FadR plasmid are heat-shocked into a host cell and fed in batches;
  • the CYX144 plasmid is pACYC-(Trc-tesA'-Dox-yjgB);
  • the FadR plasmid pTrcHis2A-fadR The FadR plasmid pTrcHis2A-fadR.
  • the present invention provides a method for synthesizing an odd number of intermediate fatty alcohols by fed-batch fermentation, wherein the CYX144 and FadR plasmids are heat-shocked into E. coli BL21 (DE3) strain and 30° C. on LB solid plates. After overnight culture, the recombinant single colony was inoculated in 2 mL of LB medium and cultured at 30 ° C until the OD was 2.5-4, and transferred to 20 mL of M9 medium at a ratio of 1:100, and cultured at 30 ° C until the OD was 2.5-4. And transferred to 800 mL of M9 medium again at a ratio of 1:100.
  • the culture solution was centrifuged to 50 mL, and inoculated with a 2.5 L fermentor for fed-batch fermentation.
  • induction was carried out with 10 ⁇ M of IPTG. Samples were taken every 4 hours, and 15 mL each was used for analysis of cell density, glycerol, acetic acid, and fatty alcohol concentration.
  • the content of each antibiotic in the solid and liquid medium was chloramphenicol 34 ⁇ g/mL and ampicillin 100 ⁇ g/mL.
  • the cell density was measured at a wavelength of 600 using a TU-1810 UV-Vis spectrophotometer (Beijing General Instrument Co., Ltd.).
  • the invention provides a gene having:
  • (III) a sequence which encodes the same protein as the nucleotide sequence of (I) or (II) but differs from the nucleotide sequence of (I) or (II) due to the degeneracy of the genetic code;
  • the present invention Compared with the existing microbial synthesis pathways of fatty alcohols and aliphatic hydrocarbons, the present invention has the following two advantages: (1) the process by which ⁇ -dioxygenase converts the fatty acid molecules of the precursor into an important intermediate metabolite, the fatty aldehyde. For the oxidation reaction, the cells do not need to provide additional reducing power and energy, which reduces the burden of cell production; (2) the technical limitation that the fatty acid product chain length is only an even number and the aliphatic hydrocarbon product chain length is mostly odd, which can make Bio-based bulk chemicals and biofuels are more closely matched to petroleum-based products.
  • the invention provides a microbial synthesis route and a construction method of an odd-numbered medium-chain fatty alcohol and an even-numbered medium-chain hydrocarbon by means of synthetic biology, and the invention also provides an engineering large intestine for preparing an odd-numbered medium-chain fatty alcohol and an even-numbered medium-chain hydrocarbon by the above path. Bacillus.
  • Figure 1 is a gas chromatogram of the product of Example 4 containing E. coli BL21 (DE3) containing pACYC-Trc-tesA ( Figure 1A) or CYX134 plasmid ( Figure 1B) at 30 °C for 30 h; wherein 1:C 11 aldehyde; 2: C 11 alcohol; 3: C 13 aldehyde; 4: C 13 alcohol; 5: C 15: 1 aldehyde; 6: C 15 alcohol; 7: C 16 alcohol (internal standard, IS); (Fig. 1C) C 11 aldehyde mass spectrum; (Fig. 1D) C 11 alcohol mass spectrum; (Fig. 1E) C 13 aldehyde mass spectrum; (Fig.
  • Figure 3 shows the results of the production of aliphatic hydrocarbons after 40 hours of fermentation induced by E. coli BL21 (DE3) containing CYX148, CYX149, CYX150, and CYX151 plasmids in Example 6;
  • Fig. 3A Comparison of fatty hydrocarbon yields of different engineering strains;
  • Fig. 3B Gas chromatogram of product after induction of fermentation by E. coli BL21 (DE3) containing CYX148 plasmid at 30 °C for 30 h;
  • Fig. 3C E. coli BL21 (DE3) containing CYX151 plasmid after induction of fermentation at 40 °C for 40 h
  • Fig. 3D C 12 hydrocarbon mass spectrum;
  • Fig. 3E C 14 hydrocarbon mass spectrum;
  • Figure 4 shows the results of metabolic flow optimization between ⁇ -dioxygenase and endogenous thioesterase in Example 7;
  • FIG. 5 shows that Example 8 demonstrates that alpha-dioxygenase has extensive substrate selectivity and regulatable ability in the cell.
  • 5A shows the results of fatty acid production ratios of different chain lengths after 40 hours of induction of fermentation by E. coli BL21 (DE3) containing YX101, YX102, and YX103 plasmids in Example 8;
  • FIG. 5B shows that YX135 and YX104 are contained in Example 8.
  • FIG. 5D shows E. coli BL21 (DE3) containing YX135, YX104, YX105 plasmid in Example 8 at 30 ° C to induce fatty aldehydes and fatty alcohols after 40 h of fermentation
  • Figure 6 shows the results of Example 9 for adjusting the expression intensity of the upstream fatty acid synthesis pathway to increase the synthesis ability of the odd-chain fatty alcohol
  • Figure 7 shows the results of the fed-batch fermentation in Example 10.
  • 7A is a graph showing changes in biomass (OD600), glycerin remaining amount, acetic acid content, and fatty alcohol content with time in the fed-batch fermentation process in Example 10;
  • FIG. 7B shows the batch fed fermentation in Example 10 for 9 hours. After 17 hours, 27.5 hours, the ratio of different chain length fatty alcohol content in the fermentation broth.
  • the invention discloses a gene, a protein encoded thereby and a use thereof, a gene original, a method for synthesizing an odd-numbered medium chain fatty aldehyde, a method for synthesizing an odd-numbered medium-chain fatty alcohol, and a method for synthesizing an even-numbered medium-chain aliphatic hydrocarbon, and those skilled in the art can learn from the contents of the paper. Appropriately improve the implementation of process parameters. It is specifically noted that all such similar substitutions and modifications will be apparent to those skilled in the art. They are all considered to be included in the present invention.
  • the method and the application of the present invention have been described by the preferred embodiments, and it is obvious that the method and application described herein may be modified or appropriately modified and combined without departing from the scope of the present invention. The technique of the present invention is applied.
  • the gene, the encoded protein and use thereof, the gene original, the synthetic odd-chain fatty aldehyde, the method for synthesizing the odd-numbered medium-chain fatty alcohol, and the raw materials and reagents used in the method for synthesizing the even-chain intermediate aliphatic hydrocarbon are all commercially available. .
  • Thioesterase (TesA'): E. coli endogenous thioesterase gene I (tesA') was purchased from addgene (Plasmid 24636). This gene was assigned to pKS1 with a replicon of p15A and a promoter of placUV5. And removed the 75 bp nucleotide after the start codon ATG. The amino acid sequence encoded by the removed nucleotide is a signal peptide that is used to localize the enzyme to the intercellular substance. This signal peptide sequence was removed, but the thioesterase was enriched in the cells and E. coli was produced with a large amount of free fatty acids.
  • Alpha-dioxygenase (Dox): according to the reported protein sequence of ⁇ -dioxygenase (NCBI Reference Sequence: NP_001066718.1) in rice (Oryza sativa), according to the large intestine The bacillus is codon-optimized, and the optimized DNA molecule encoding ⁇ -dioxygenase has the nucleotide sequence shown in SEQ ID NO: 1, and the gene is synthesized in Jinweizhi Company.
  • Plasmid pTrcHis2A was purchased from Invitrogen.
  • Plasmid pACYCDuet-1 was purchased from Novagen.
  • Plasmid pET21a was purchased from Novagen.
  • the vector is pTrcHis2A, digested with NcoI and BamHI, and the length is 4400, purified.
  • the template was pKS1, amplified with primers NcoI-tesA-fwd and BamHI-SpeI-tesA-rev, length 575, recovered by gelatinization, digested with NcoI and BamHI, purified, and ligated to the vector.
  • Colony PCR primers pTrcHis2A-F and primer pTrcHis2A-R have a correct length of 894
  • pACYCDuet-1 was amplified with primers AflII-pACYC-fwd and PstI-pACYC-rev, the length was 3810, and the gel was recovered and digested with PstI and AflII to purify.
  • the fragment template is pHisTrc-tesA
  • PCR is carried out with PstI-Gibson-pHisTrc-fwd and AflII-Gibson-rrnBT1-rev, the length is 1190, the gel is recovered, and the carrier is Gibson-ligated.
  • the vector is pET21a, digested with NdeI and BamHI, and the length is 5350.
  • the fragment template is a synthetic dox gene, amplified with primers NdeI-Dox-fwd and BamHI-SpeI-Dox-rev, length 1885, recovered by gelatinization, digested with NdeI and BamHI, purified, and ligated to the vector.
  • the vector is pET28a, digested with NdeI and BamHI;
  • the fragment template is a synthetic dox gene, amplified with primers NdeI-Dox-fwd and BamHI-SpeI-Dox-rev, length 1885, recovered by gelatinization, digested with NdeI and BamHI, purified, and ligated to the carrier;
  • the vector is pET21a, digested with NdeI and BamHI;
  • the fragment template is pKS1, amplified with primers NdeI-tesA-fwd and BamHI-SpeI-tesA-rev, recovered by gelatinization, digested with NdeI and BamHI, purified, and ligated to the carrier;
  • AdhE acetaldehyde coenzyme A reductase/iron ion-dependent alcohol dehydrogenase from the E. coli BL21 (DE3) genome (NCBI-GeneID:8180074), with a single point mutation replacing the NcoI restriction site in the sequence.
  • AdhP ethanol active dehydrogenase/acetaldehyde active reductase from the E. coli BL21 (DE3) genome (NCBI-GeneID: 8181169).
  • YqdD NADPH-dependent acetaldehyde reductase from the E. coli BL21 (DE3) genome (NCBI-GeneID: 8180496), with a single point mutation replacing the NdeI restriction site in the sequence.
  • YjgB alcohol dehydrogenase (atypical zinc-type alcohol dehydrogenase-like protein, zinc and NADPH-dependent) from the E. coli BL21 (DE3) genome (NCBI-GeneID: 8182107).
  • Slr1192 Alcohol dehydrogenase containing zinc, derived from Synechocystis sp. PCC6803 strain (Synechocystis sp. PCC 6803), the protein sequence is NCBI Reference Sequence: NP_443028.1, codon optimized according to E. coli, optimized encoding Slr1192 DNA molecule having the nucleotide sequence shown in SEQ ID NO: 2, which is synthesized in the laboratory because.
  • Plasmid pET28a was purchased from Novagen.
  • the vector is pTrcHis2A, digested with NcoI and BamHI, and the length is 4400, purified.
  • the template was pKS1, amplified with primers NcoI-tesA-fwd and BamHI-SpeI-tesA-rev, length 575, recovered by gelatinization, digested with NcoI and BamHI, purified, and ligated to the vector.
  • Colony PCR primers pTrcHis2A-F and primer pTrcHis2A-R have a correct length of 894
  • pACYCDuet-1 was amplified with primers AflII-pACYC-fwd and PstI-pACYC-rev, the length was 3810, and the gel was recovered and digested with PstI and AflII to purify.
  • the fragment template is pHisTrc-tesA
  • PCR is carried out with PstI-Gibson-pHisTrc-fwd and AflII-Gibson-rrnBT1-rev, the length is 1190, the gel is recovered, and the carrier is Gibson-ligated.
  • the vector is pET21a, digested with NdeI and BamHI, and the length is 5350.
  • the fragment template is a synthetic dox gene, amplified with primers NdeI-Dox-fwd and BamHI-SpeI-Dox-rev, length 1885, recovered by gelatinization, digested with NdeI and BamHI, purified, and ligated to the carrier;
  • the vector is pET28a, digested with NdeI and BamHI, and the length is 5400.
  • the fragment template is E. coli BL21 (DE3) genome, amplified with primers NdeI-AdhP-fwd and BamHI-SpeI-AdhP-rev, length 1036
  • the vector is pET28a-AdhP, digested with NdeI and SpeI, and the fragment is cut to recover a fragment of 5350 in length.
  • the fragment template is the E.coli BL21 (DE3) genome, and the different gene fragments (*** represents the gene name) are amplified by the primers NdeI-***-fwd and SpeI-***-rev.
  • the primer sequences are shown in the table. 3, the fragment name, the length after PCR is shown in Table 2, where YqhD needs to replace the NdeI restriction site, and AdhE needs to replace the NcoI restriction site. Therefore, it is necessary to PCR-amplify the two parts with the mutation point as the center. After the recovery, the overlap is carried out, and finally the fragments of the NdeI and SpeI cleavage sites are recovered, and digested with NdeI and SpeI, purified, and ligated to the carrier;
  • Example 3 Construction of a genetic element for synthesizing even-chain mid-chain aliphatic hydrocarbons
  • CER1 fatty aldehyde decarbonylase, derived from Arabidopsis thaliana, the protein sequence is UniProtKB/Swiss-Prot: F4HVY0.1, codon optimized according to E. coli, and the optimized DNA molecule encoding CER1 has SEQ ID NO: The nucleotide sequence shown in 3, which synthesizes the gene in genscript.
  • AD9313 fatty aldehyde decarbonylase, derived from Prochlorococcus marinus MIT9313, protein sequence NCBI Reference Sequence: NP_895059.1, codon optimized according to E. coli, optimized DNA molecule encoding AD9313 with SEQ ID
  • the nucleotide sequence shown by NO: 4 synthesizes a gene in the laboratory.
  • AD7942 Fatty aldehyde decarbonylase, derived from Synechococcus elongatus PCC7942, protein sequence accession number: YP_400610, codon optimized according to E. coli, optimized DNA molecule encoding AD7942 has SEQ ID NO: 5
  • the nucleotide sequence shown is a synthetic gene in GENEART.
  • AD73102 Fatty aldehyde decarbonylase from Nocardia (Nostoc punctiforme PCC73102), protein sequence accession number: YP_001865325, codon optimized according to E. coli, optimized DNA molecule encoding AD73102, from Pennsylvania State University (The Pennsylvania) State University, USA) presented by the Squire J.Booker team.
  • the vector is pET28a-AdhP, digested with NdeI and SpeI, and the fragment is cut to recover a fragment of 5350 in length.
  • Example 4 Feasibility verification of ⁇ -addition dioxygenase for the synthesis of odd medium chain fatty alcohols and even medium chain aliphatic hydrocarbons
  • the vector is pACYC-Trc-tesA, digested with SpeI and BamHI, purified
  • the fragment template was 21A-Dox, digested with XbaI and BamHI, and the length was 1911, and the gel was recovered and linked to the vector.
  • Plasmid CYX134 was heat shock transformed into E. coli BL21 (DE3) strain and screened on LB solid plates. The cells were cultured in an incubator at 30 ° C, and the content of each antibiotic in the solid and liquid medium was chloramphenicol 34 ⁇ g / mL.
  • the E. coli BL21 (DE3) strain transformed with the plasmid pACYC-Dox-tesA' was subjected to fermentation.
  • the recombinant single colonies were inoculated in LB medium and cultured at 30 ° C overnight, and inoculated in 5 mL of M9 medium at a ratio of 1:100, and fermentation was carried out at 30 ° C in a shaker at 220 rpm.
  • OD600 1.0-1.2
  • 1 mM IPTG was added, and after induction of expression for 40 h, a fatty alcohol sample was taken for detection.
  • the GC/MS system involved in this experiment is the Waters GCT Premier MICROMASS system, which includes:
  • Time-of-flight mass spectrometer (TOF-MS, Waters Corp., USA)
  • J&W DB-5 capillary quartz column (30m length, I.D.0.25mm, Film 0.25 ⁇ m, Agilent Technologies, USA)
  • the GC conditions were as follows: a DB-5 gas chromatograph column was used with a injection volume of 1 ⁇ L, and a post-column splitting technique was used with a split ratio of 2:1.
  • the inlet temperature was 260 ° C and the GC interface temperature was 280 ° C.
  • High purity helium is carrier gas, constant pressure of 91Kpa.
  • the temperature ramping procedure for chromatographic separation was as follows: initial temperature 70 ° C, held for 2 min, ramped to 290 ° C at 8 ° C ⁇ min -1 and held at 290 ° C for 6 min.
  • mass spectrometry ionization mode was electro-impact ionization (EI+) in positive ion mode, the ionization voltage was 70 eV, and the source temperature was maintained at 250 °C.
  • the mass spectrum has a scan range of 50-800 m/z and a scan speed of 2 scan ⁇ s-1.
  • the pACYC-Trc-tesA and CYX134 plasmids were transferred into E. coli BL21 (DE3), and fermentation was induced at 30 ° C for 40 h.
  • the fermentation products were subjected to GC-MS detection, and the results are shown in FIG. 2 .
  • the strain transferred to the pACYC-Trc-tesA module only detected the production of fatty acids, which was obtained by hydrolysis of fatty acyl-ACP by thioesterase (TesA').
  • strains transferred to the CYX134 module detected three fatty alcohols of 1-undecyl alcohol, 1-tridecyl alcohol and 1-pentadecanol at retention times (RT) of 12.37 min, 15.75 min, and 18.80 min, respectively.
  • RT retention times
  • These three fatty alcohols are products in which the fatty aldehydes corresponding to the carbon chains are spontaneously reduced in the cells.
  • the mass spectrum of each product is shown in Figures 1C-H.
  • the matching factor and the reverse matching factor of each substance were both 850 or more, which confirmed the accuracy of the characterization of the odd medium chain fatty aldehyde/alcohol molecule of the present invention.
  • plasmid Different aldehyde-based reductases (AdhP, YjgB, YqhD, AdhE, Slr1192) were ligated into the CYX134 plasmid. Specifically, CYX134 was used as a vector, and SpeI and BamHI were used for enzymatic digestion and purification. Using 28a-AdhP, 28a-YjgB, pET28a-YqhD, pET28a-AdhE, pET28a-Slr1192 as a template, XbaI and BamHI were used for digestion, and the gel was recovered and ligated to the vector. The specific fragment length is shown in Table 6.
  • Each plasmid was heat shock transformed into E. coli BL21 (DE3) strain and screened on LB solid plates. The cells were cultured in an incubator at 30 ° C, and the content of each antibiotic in the solid and liquid medium was chloramphenicol 34 ⁇ g / mL.
  • Each plasmid was heat shock transformed into E. coli BL21 (DE3) strain and screened on LB solid plates. The cells were cultured in an incubator at 30 ° C, and the content of each antibiotic in the solid and liquid medium was chloramphenicol 34 ⁇ g / mL.
  • Each plasmid was transferred to E. coli BL21 (DE3), and fermentation was induced at 30 ° C for 40 h, and the fermentation product was subjected to GC-MS detection, and the results are shown in FIG. It can be seen that the decarbonylase CER1 derived from Arabidopsis does not allow the engineered Escherichia coli to synthesize even-chain aliphatic hydrocarbons. After expressing three decarbonylases derived from cyanobacteria, the engineered E. coli has a chain length of The aliphatic hydrocarbons of C12 and C14 demonstrate a method of synthesizing medium chain aliphatic hydrocarbons using Escherichia coli in this patent.
  • FIG. 3B shows the position of the product producing the aliphatic hydrocarbons in the gas chromatogram after transfer of the CYX151 plasmid. Of these, 8 is a twelve hydrocarbon and 9 is a tetradecene hydrocarbon. The mass spectrum of each product is shown in Figures 3D and E. The matching factor and reverse matching factor of each substance were both 850 or more, which confirmed the accuracy of qualitative analysis of the even-chain intermediate aliphatic hydrocarbon product of the present invention.
  • the present invention provides a microbial synthesis route and a construction method of an odd-numbered medium-chain fatty alcohol and an even-numbered medium-chain hydrocarbon by means of synthetic biology, and the present invention also provides an intermediate medium-chain fatty alcohol and an even number in the above path.
  • Example 7 Optimizing metabolic flux between alpha-dioxygenase and thioesterase
  • Plasmid pBAD33 was purchased from ATCC.
  • the vector is pACYCDute-1, amplified with the primers AflII-pACYC-fwd and the primer PstI-pACYC-rev in Table 8.
  • the fragment templates were pQE-80L, pKS1, pTrcHis2A and pBAD33, and the remaining primers in Table 8 were used for amplification, and ligated with the vector to construct YX210, YX211, YX212 and YX213 plasmids, which simultaneously had two promoters.
  • One promoter is T7 and the other promoters are T5, pLacUV5, Trc and BAD. Specific information of these plasmids is shown in Table 9.
  • the vector is pACYCDute-1, YX210, YX211, YX212 and YX213, which are digested with NdeI and KpnI, and purified;
  • the fragment template was pKS1 plasmid, which was amplified with primers NdeI-tesA-fwd and KpnI-tesA-rev.
  • the primer sequences are shown in Table 8. After PCR, the cells were digested, purified, ligated to the vector, and the correct transformants were picked.
  • the vector is the 5 plasmids produced in step 2, and the polyclonal cleavage site in the first promoter is cleaved by NcoI and BamHI, and purified.
  • the fragment template is a synthetic dox gene amplified with primers NcoI-Dox-fwd and BamHI-SpeI-Dox-rev.
  • the primer sequences are shown in Table 8.
  • the length after PCR is 1874, which is digested, purified, and ligated to the vector. Take the correct transformant.
  • the first promoters (T7, T5, pLacUV5, Trc and BAD) in YX220, YX232, YX233, YX234, YX235 control the dox gene without the 6*His tag
  • the second promoter (T7) controls the tesA' gene.
  • the vector is the 5 plasmids produced in step 2, and the polyclonal cleavage site in the first promoter is cleaved by NcoI and BamHI, and purified.
  • the fragment template was 21a-dox plasmid, digested with NcoI and BamHI, the gel was recovered, and ligated to the vector to pick the correct transformant.
  • the first promoters (T7, T5, pLacUV5, Trc and BAD) in YX221, YX222, YX223, YX224, YX225 control the dox gene containing the 6*His tag, and the second promoter (T7) controls the tesA' gene.
  • the vectors are pACYCDute-1, YX210, YX211, YX212 and YX213, digested with NcoI and BamHI, and purified.
  • the fragment template is a synthetic dox gene, which is amplified with primers NcoI-Dox-fwd and BamHI-SpeI-Dox-rev.
  • the primer sequences are shown in Table 8. After PCR, the enzyme is digested, purified, and ligated to the vector to pick the correct transformation. child
  • the vector is the 5 plasmids produced in step 5, digested with SpeI and BamHI, and purified.
  • the fragment template 21a-tesA was digested with XbaI and BamHI, the gel was recovered, and ligated to the vector to pick the correct transformant.
  • the first promoters (T7, T5, pLacUV5, Trc and BAD) in YX135, YX136, YX137, YX138 and YX140 control both dox and tesA' genes
  • the vector is YX210, YX211, YX212 and YX213, which are digested with NcoI and BamHI and purified.
  • the fragment template is pKS1, amplified with primers NcoI-tesA-fwd and BamHI-SpeI-tesA-rev.
  • the primer sequences are shown in Table 8.
  • the enzyme is digested, purified, and ligated to the vector to pick the correct transformant.
  • tesA'-dox plasmids (YX131, YX132, YX133, YX134 and YX130) containing the T7, T5, pLacUV5, Trc and BAD promoters.
  • the vector is the 5 plasmids produced in step 7, which are digested with SpeI and BamHI and purified.
  • the fragment template 21a-Dox was digested with XbaI and BamHI, the gel was recovered, and ligated to the vector to pick the correct transformant.
  • the first promoters (T7, T5, pLacUV5, Trc and BAD) of YX131, YX132, YX133, YX134 and YX130 control the two genes tesA' and dox
  • Each plasmid was heat shock transformed into E. coli BL21 (DE3) strain and screened on LB solid plates. The cells were cultured in an incubator at 30 ° C, and the content of each antibiotic in the solid and liquid medium was chloramphenicol 34 ⁇ g / mL.
  • Each plasmid was transferred to E. coli BL21 (DE3), and fermentation was induced at 30 ° C for 40 h, and the fermentation product was subjected to GC-MS detection, and the results are shown in FIG. 4 .
  • the metabolic flow between TesA' and ⁇ -Dox is optimized in two parts.
  • TesA' and ⁇ -Dox were expressed in two open reading frames (ORFs). TesA' is controlled by the T7 promoter and ⁇ -Dox is expressed using five different promoters. When ⁇ -Dox is under the control of the strongest expression of T7 or the weakest expression of BAD promoter, the yield of fatty alcohol is relatively low. When the expression intensity of ⁇ -Dox is moderate (controlled by T5, LacUV5 and Trc promoters), the yield of fatty alcohol is relatively high. This indicates that the expression intensity between TesA' and ⁇ -Dox is too different, and when the expression level of ⁇ -Dox is slightly smaller than that of TesA', the metabolic flux is relatively balanced.
  • TesA' and ⁇ -Dox are expressed in an open reading frame to optimize the two bases. Due to the overall intensity of expression.
  • two expression structures of dox-tesA' and tesA'-dox were constructed, respectively.
  • the genes close to the promoter will have a relatively stronger expression intensity.
  • the yield of fatty alcohol is higher when tesA' is closer to the promoter.
  • the expression intensity of both tesA'-dox should not be too high or too low, and the yield of fatty alcohol was the highest (35.2 mg/L) under the control of the Trc promoter.
  • the CYX134 plasmid of Example 4 is a plasmid (CYX equivalent to YX) optimized by metabolic flow between tesA' and ⁇ -Dox in this example.
  • Example 8 Confirmation of ⁇ -dioxygenase with broad substrate selectivity and regulatable ability in cells
  • the vector is pACYCDute-1, digested with EcoRI and SacI, or digested with SalI and HindIII.
  • Fragment template is three different thioesterase genes: tesA' gene in pKS1 (Escherichia coli, E. coli source), or synthetic bte (Umbellularia californica, California Laurel source) and BnFatA (Brassica napus, European canola source) Gene, using the corresponding primers to amplify different gene fragments, the primer sequence is shown in Table 10, the fragment name, the length after PCR is shown in Table 11, digested, purified, and ligated to the carrier;
  • the vector is YX101, YX102 or YX103, which is digested with NcoI and BamHI and purified.
  • the fragment template is a synthetic dox gene amplified with primers NcoI-Dox-fwd and BamHI-SpeI-Dox-rev.
  • the length after PCR is 1874, which is digested, purified, and ligated to the vector to pick the correct transformant.
  • Table 10 is a list of primers required to demonstrate the broad substrate selectivity and regulatable ability of alpha-dioxygenase in cells.
  • the sequence under the straight underline is the enzyme cleavage site, and the bold or italic sequence is the start codon or stop codon.
  • the sequence is RBS. .
  • YX101, YX102, YX103 plasmids were heat shock transformed into E. coli BL21 (DE3) strains, respectively, and screened on LB solid plates. The cells were cultured in an incubator at 30 ° C, and the content of each antibiotic in the solid and liquid medium was chloramphenicol 34 ⁇ g / mL.
  • Extraction of fatty acids Take 0.5 mL of medium fermented at 30 °C for 30 h after induction, add 50 ⁇ L of hydrochloric acid and 25 ⁇ g of heptadecanoic acid as internal standard; add 0.5 mL of ethyl acetate, vortex for 5 min, centrifuge at 15000 rpm for 2 min (the same below) The upper organic phase was aspirated, 0.5 mL of ethyl acetate was added to the lower layer solution, vortexed for 5 min, and the upper organic phase was centrifuged; the two extracts were combined, 20 ⁇ L of diazomethane, 1 ⁇ L of hydrochloric acid, and 9 ⁇ L of methanol were added.
  • the extracted free fatty acid was methylated, and after reacting for two hours, it was dried with nitrogen; the product was evaporated (0.5 mL of FAME) by dissolving with 0.5 mL of n-hexane, and filtered through a 0.22 ⁇ m nylon membrane. Samples were stored in a -80 °C freezer prior to injection.
  • the YX135, YX104, YX105 plasmids were heat shock transformed into E. coli BL21 (DE3) strains, respectively, and screened on LB solid plates.
  • the cells were cultured in an incubator at 30 ° C, and the content of each antibiotic in the solid and liquid medium was chloramphenicol 34 ⁇ g / mL.
  • Escherichia coli containing the YX101, YX102 or YX103 plasmid was fermented, and the results are shown in Fig. 5.
  • Engineered strains overexpressing TesA', BTE or BnFatA synthesize C 14 /C 16 (39%/36%), C 12 (75%), and C 16 /C 18 (75%/24%) fatty acids, respectively.
  • the main product When ⁇ DOX is overexpressed with thioesterase, different engineered strains synthesize C 13 /C 15 (57%/30%), C 11 (95%), and C 15 (93%) fatty aldehyde + fatty alcohol As the main product.
  • Example 9 Adjusting the expression intensity of the upstream fatty acid synthesis pathway to increase the synthesis ability of odd-chain mid-chain fatty alcohols
  • the vector is pTrcHis2A, digested with NcoI and BamHI, purified
  • the fragment template is the E. coli MG1655 genome, and the different gene fragments (*** represents the gene name) are amplified by the primers NcoI-GCG-***-fwd and BamHI-SpeI-***-rev.
  • the primer sequences are shown in the table. 12, the fragment name, the length after PCR is shown in Table 13, wherein FabD needs to replace the XhoI restriction site, FabG needs to replace the NcoI restriction site, the gel is recovered, digested with NcoI and BamHI, purified, and ligated to the carrier;
  • the pKD46 plasmid was transferred into E. coli BL21 (DE3), cultured in LB liquid medium at 30 ° C overnight, and the activated cells were inoculated to a liquid medium containing 10 mmol/L L-arabinose at a ratio of 1:100.
  • Culture at 30 ° C when the OD 600 of the cells grows to 0.5-0.6, pre-cool for 10 min on ice, centrifuge at 4 ° C, 4000 rpm for 5 min (the same below), then centrifuge three times with ice-cold 10% glycerol, concentrate 100 times.
  • Electroporation competent cells 100 ⁇ L per tube, stored in a -80 ° C refrigerator for use;
  • PCR gel-receiving product (10-100 ng) in step 1) was added to competent BL-46 cells using an electrotransformer Electroroporator 2170 (Eppendorf, Germany) (0.1-cm chambers), after 1800V shock for 5-6 ms.
  • step 3 Pick a single colony on the plate in step 3), and colony PCR verifies whether chloramphenicol replaces the fadE gene in the genome.
  • step 4) The correct transformant in step 4) was ligated into 2 mL of chloramphenicol LB medium, and cultured at 43 ° C for 12 h, and the pKD46 plasmid was deleted. After scribing, the same single colony was picked and applied to the plates of ampicillin and chloramphenicol, and cultured at 30 ° C for 24 h. If the same single colony grew in the chloramphenicol plate and did not grow in the ampicillin plate, the pKD46 was completely deleted.
  • *50-nt is a sequence homologous to both ends of the gene in the genome that need to be knocked out. Identification, bold, italic sequence is the start codon or stop codon
  • the CYX144 plasmid and one of the plasmids constructed in Method 1 were heat shock transformed into E. coli BL21 (DE3) strain and screened on LB solid plates. The cells were cultured in an incubator at 30 ° C. The content of each antibiotic in the solid and liquid medium was chloramphenicol 34 ⁇ g/mL and ampicillin 100 ⁇ g/mL.
  • E. coli BL21 (DE3) strain transformed with each plasmid was subjected to fermentation in the same manner as in Example 1.
  • the IPTG concentration was divided into three concentrations of 1 mM, 0.1 mM and 0.01 mM.
  • Each plasmid was transferred to E. coli BL21 (DE3), and fermentation was induced at 30 ° C for 40 h, and the fermentation product was subjected to GC-MS detection, and the results are shown in Fig. 6.
  • IPTG concentration 1 mM
  • any gene in the overexpression fatty acid synthesis pathway cannot enhance the synthesis ability of fatty alcohol; blocking the fatty acid ⁇ -oxidation pathway by knocking out the fadD or fadE gene also does not promote the synthesis of fatty alcohol.
  • the concentration of the inducer IPTG was lowered to 0.1 mM, overexpression of acetyl-CoA carboxylase (ACC), FabD, FabI and FadR significantly increased the production of fatty alcohol.
  • ACC acetyl-CoA carboxylase
  • FabD FabI
  • FadR FadR significantly increased the production of fatty alcohol.
  • the yield of odd-chain fatty alcohol can be increased from 65.1 mg/L to 100.8 mg/L or 101.5 mg/L.
  • the concentration of IPTG is 0.01 mM
  • the same expression of ACC, FabD, FabI and FadR can promote the production of fatty alcohol.
  • FadR and CYX144 were simultaneously overexpressed, the yield of fatty alcohol increased by 77.1% compared to CYX144 alone.
  • the CYX144 and FadR plasmids were heat-transformed into E. coli BL21 (DE3) strain, and cultured on LB solid plate at 30 ° C overnight.
  • the recombinant single colony was inoculated into 2 mL of LB medium and cultured at 30 ° C until the OD was 2.5. -4, and transferred to 20 mL of M9 medium at a ratio of 1:100, cultured at 30 ° C to an OD of 2.5-4, and again transferred to 800 mL of M9 medium at a ratio of 1:100.
  • the culture solution was centrifuged to 50 mL, and inoculated with a 2.5 L fermentor for fed-batch fermentation.
  • Cell density was measured using a TU-1810 UV-Vis spectrophotometer (Beijing General Instrument Co., Ltd.) at a wavelength of 600.

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Abstract

提供了基因、其编码的蛋白质及用途、基因元件、基因及用途、基因元件、合成奇数中链脂肪醛、合成奇数中链脂肪醇以及合成偶数中链脂肪烃的方法。提供了在大肠肝菌内构建奇数脂肪醇的方法。采用来源于大米的α－加双氧酶，无需细胞提供额外的还原力和能量。并且能够与不同的硫脂酶配合，合成从C11到C15的不同比例的脂肪醇。

Description

基因及用途、基因元件、合成奇数中链脂肪醛的方法以及合成偶数中链脂肪烃的方法
本申请要求于2014年10月22日提交中国专利局、申请号为201410566258.8、发明名称为“基因及用途、基因元件、合成奇数中链脂肪醛的方法以及合成偶数中链脂肪烃的方法”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明涉及生物技术领域，特别涉及基因、其编码的蛋白质及用途、基因元件、合成奇数中链脂肪醛的方法以及合成偶数中链脂肪烃的方法。
背景技术
中链脂肪醇因具有两亲的特性而在工业中具有非常重要的应用，可用于表面活性剂、医药、化妆品以及能源领域，具有1-1.2亿美元的市值。而碳链长度在6-16的脂肪烃类分子是航空煤油的主要成分，具有热值高、蒸汽压低、凝固点低以及吸湿性低等优点。50％的商用脂肪醇从植物种子或者动物脂肪中提取，剩余脂肪醇以及全部烃类都从石油中精炼而来。无论是哪一种方法，都不能满足现代社会对可持续性和环境友好的生产要求。相反的，在合成生物学手段快速发展的条件下，基因工程菌株却可以利用糖、木聚糖或者甘油等可再生能源，特异性的合成所需产品。
在工程改造的大肠杆菌中，脂肪醇和烃类物质主要从脂肪酸合成途径进行衍生合成。分别可以利用脂酰-ACP/CoA和自由脂肪酸三种分子作为合成前体物。在烃醇的合成中，将脂酰-ACP/CoA或脂肪酸转化为脂肪醛是一个关键性步骤，随后脂肪醛再被还原为脂肪醇或经脱羰基反应成为少一分子碳的烃类物质。以脂酰ACP/CoA为前体物的脂肪醇/烃类物质的微生物合成自2010年起出现报到。而以自由脂肪酸为底物合成中链烃醇类物质的人工合成体系直到2013年才出现两篇报道。Howard等人在大肠杆菌中过表达来自香樟树(Cinnamomum camphora)中的硫酯酶，将特定长度 的自由脂肪酸从脂酰-ACP中释放出来，并同时表达发光杆菌(Photorhabdus luminescens)的luxC,luxD,luxE基因编码的脂肪酸还原酶(fatty acid reductase,FAR)和来自念珠藻(Nostoc punctiforme PCC73102)中的脂肪醛脱羰基酶，将自由脂肪酸还原成脂肪醛并脱羧成减一个碳原子的烃类分子，构建了能够合成长度相对可控的以自由脂肪酸为底物的烃类合成系统。Akhtar等人发现来自海鱼分枝杆菌(Mycobacterium marinum)中的羧酸还原酶(carboxylic acid reductase,CAR)能够将链长范围从C6到C18的自由脂肪酸转化为相应的脂肪醛。将这个酶与脂肪醛还原酶或者脂肪醛脱羧酶结合，可以在体外构建生产偶数链长(C8-C16)的脂肪醇和奇数链长(C7-C15)的烃类化合物。当在胞内将该路径与能产生特定链长自由脂肪酸的硫酯酶相结合后，E.coli BL21(DE3)菌株在以葡萄糖为碳源的最小培养基中能够合成出产量高达350mg/L的脂肪醇。
以上两种以自由脂肪酸为底物的烃类合成系统因都采用还原酶进行成醛反应，故称之为还原型烃类合成系统。因为在相同的底物条件下，还原酶需要细胞为其提供还原力(NAD(P)H)和能量(ATP)才能发生反应，而氧化酶则是由氧分子来提供反应动力，因此氧化型合成体系是更为经济的微生物合成体系。目前还没有氧化型烃类人工合成构建的相关工作被发表出来。
另外一方面，在现有报道的工作中，无论是以脂酰-ACP/CoA还是以自由脂肪酸为前体物的烃醇人工合成体系，因为第一步的还原反应均不涉及脱碳反应，因此所合成的脂肪醇均为偶数碳链，而烃类分子因为一步脱羰基反应而均为奇数碳链。实际上，石油基的化学品和燃料结构都具有多样性，同时包含直链和支链、奇数链和偶数链的分子，一个理想的生物燃料应该是在结构和化学特性上都与现行的石油基燃料相类似。曾有工作通过改变上游脂肪酸合成路径用于下游合成支链和偶数链烷烃。但还没有任何工作直接调控下游合成路径。
发明内容
有鉴于此，本发明提供基因、其编码的蛋白质及用途、基因元件、合成奇数中链脂肪醛的方法以及合成偶数中链脂肪烃的方法。α-加双氧酶将前体物脂肪酸分子转化为重要中间代谢物——脂肪醛的过程为氧化反应，不需要细胞提供额外的还原力和能量，降低了细胞生产的负担；填补了脂肪醇产物链长仅为偶数，脂肪烃产物链长大多为奇数的技术局限，可以使生物基大宗化学品以及生物燃料的分子与石油基相关产品更为匹配。
为了实现上述发明目的，本发明提供以下技术方案：
本发明提供了一种基因，其具有：
(Ⅰ)如SEQ ID No.1所示的核苷酸序列；或
(Ⅱ)如SEQ ID No.1所示的核苷酸序列的互补序列；或
(Ⅲ)与(Ⅰ)或(Ⅱ)的核苷酸序列编码相同蛋白质，但因遗传密码的简并性而与(Ⅰ)或(Ⅱ)的核苷酸序列不同的序列；或
(Ⅳ)与(Ⅰ)或(Ⅱ)或(Ⅲ)所述序列至少有80％同源性的序列。
本发明还提供了上述基因用于合成脂肪醛、奇数中链脂肪酸、奇数中链脂肪醇、偶数中链脂肪烃的用途。
在本发明的一些实施例中，所述脂肪醛为奇数中链脂肪醛。本发明所述中链脂肪酸、中链脂肪醇、中链脂肪烃分别表示含有8-14个碳原子的脂肪酸、脂肪醇和脂肪烃分子。
在本发明的一些实施例中，所述脂肪醛为奇数中链脂肪醛。
在本发明的一些实施例中，所述脂肪醛为1-十一醛、十三醛或十五烯醛。
在本发明的一些实施例中，所述奇数中链脂肪醇为1-十一醇、1-十三醇或1-十五醇。
在本发明的一些实施例中，所述偶数中链脂肪烃为链长为C12和C14的脂肪烃。
本发明还提供了一种含有SEQ ID NO:1所示的基因(α-dox)的载体。
本发明还提供了一种含有上述载体的宿主细胞。
在本发明的一些实施例中，所述宿主细胞为大肠杆菌。
本发明还提供了一种用于合成脂肪醛的基因元件，其含有SEQ ID NO:1所示的基因(α-dox)。
本发明还提供了一种用于合成奇数中链脂肪醛的基因元件，其含有SEQ ID NO:1所示的基因(α-dox)和硫酯酶基因。
本发明提供的用于合成奇数中链脂肪醛的基因元件，为pACYC-(T7-Dox-tesA')质粒(本发明中编号为YX135)、pACYC-(T7-Dox-BTE)质粒(本发明中编号为YX104)、pACYC-(T7-Dox-BnFatA)质粒(本发明中编号为YX105)。
本发明还提供了一种用于合成奇数中链脂肪醛的基因元件的构建的方法，包括如下内容：
1)将大肠杆菌内源硫酯酶基因(tesA’)连接到pTrcHis2A载体中，形成pHisTrc-tesA质粒；
2)将含有PTrc启动子的硫酯酶序列连接到pACYCDuet-1质粒中，形成pACYC-Trc-tesA质粒；
3)将SEQ ID NO:1所示的α-加双氧酶基因(α-dox)连接到pET21a质粒中，形成pET21a-Dox质粒；
4)以pACYC-Trc-tesA为载体，用SpeI和BamHI酶切，纯化；片段模板为21A-Dox，用XbaI和BamHI酶切pET21a-Dox质粒，切胶回收，与所述载体连接，构建pACYC-Trc-tesA-Dox(CYX134)质粒。
本发明还提供了一种用于合成脂肪醇的基因元件，其含有SEQ ID NO:1所示的基因(α-dox)。
本发明还提供了一种用于合成奇数中链脂肪醇的基因元件，其含有SEQ ID NO:1所示的α-加双氧酶基因(α-dox)、硫酯酶基因和醛基还原酶基因。
在本发明的一些实施例中，所述醛基还原酶基因选自具有如SEQ ID No.2所示的核苷酸序列的基因、adhP、yjgB、yqhD或adhE。
本发明提供的用于合成奇数中链脂肪醇的基因元件，为 pACYC-Trc-tesA-Dox质粒(本发明中编号为CYX134)、pACYC-(T7-Dox)-(T7-tesA’)质粒(本发明中编号为YX220)、pACYC-(T5-Dox)-(T7-tesA’)质粒(本发明中编号为YX232)、pACYC-(Trc-Dox)-(T7-tesA’)质粒(本发明中编号为YX233)、pACYC-(LacUV5-Dox)-(T7-tesA’)质粒(本发明中编号为YX234)、pACYC-(BAD-Dox)-(T7-tesA’)质粒(本发明中编号为YX235)、pACYC-(T7-Doxhis)-(T7-tesA’)质粒(本发明中编号为YX221)、pACYC-(T5-Doxhis)-(T7-tesA’)质粒(本发明中编号为YX222)、pACYC-(Trc-Doxhis)-(T7-tesA’)质粒(本发明中编号为YX223)、pACYC-(LacUV5-Doxhis)-(T7-tesA’)质粒(本发明中编号为YX224)、pACYC-(BAD-Doxhis)-(T7-tesA’)质粒(本发明中编号为YX225)、pACYC-(T7-Dox-tesA')质粒(本发明中编号为YX135)、pACYC-(T5-Dox-tesA')质粒(本发明中编号为YX136)、pACYC-(LacUV5-Dox-tesA')质粒(本发明中编号为YX137)、pACYC-(Trc-Dox-tesA')质粒(本发明中编号为YX138)、pBAD33-Dox-tesA'质粒(本发明中编号为YX140)、pACYC-(T7-tesA'-Dox)(本发明中编号为YX131)、pACYC-(T5-tesA'-Dox)质粒(本发明中编号为YX132)、pACYC-(LacUV5-tesA'-Dox)质粒(本发明中编号为YX133)、pACYC-(Trc-tesA'-Dox)质粒(本发明中编号为YX134)、pBAD33-tesA'-Dox质粒(本发明中编号为YX130)、pACYC-(T7-Dox-BTE)质粒(本发明中编号为YX104)、pACYC-(T7-Dox-BnFatA)质粒(本发明中编号为YX105)、pACYC-(Trc-tesA’-Dox-AdhP)质粒(本发明中编号为CYX143)、pACYC-(Trc-tesA’-Dox-yjgB)质粒(本发明中编号为CYX144)、pACYC-(Trc-tesA’-Dox-yqhD)质粒(本发明中编号为CYX145)、pACYC-(Trc-tesA’-Dox-AdhE)质粒(本发明中编号为CYX146)、pACYC-(Trc-tesA’-Dox-slr1192)质粒(本发明中编号为CYX147)。
一种用于合成奇数中链脂肪醇的基因元件的构建的方法，其特征是：将不同类型或来源的醛基脱氢酶基因(adhP、yjgB、yqhD、adhE和SEQ  ID NO:2所示的slr1192)连接到pET28a质粒中，形成28a-AdhP、28a-YjgB、pET28a-YqhD、pET28a-AdhE、pET28a-Slr1192质粒；
以CYX134(pACYC-Trc-tesA-Dox)为载体，用SpeI和BamHI进行酶切，纯化。分别以28a-AdhP、28a-YjgB、pET28a-YqhD、pET28a-AdhE、pET28a-Slr1192为模板，用XbaI和BamHI进行酶切，切胶回收，与载体连接。
本发明还提供了一种用于合成偶数中链脂肪烃的基因元件，其含有如权利要求1所述的基因、硫酯酶基因和羰基脱羰酶基因。
在本发明的一些实施例中，所述羰基脱羰酶基因选自具有如SEQ ID No.3、4或5所示的核苷酸序列的基因或ad73102。
本发明提供的用于合成偶数中链脂肪烃的基因元件，为pACYC-(Trc-tesA’-Dox-CER1)质粒(本发明中编号为CYX148)、pACYC-(Trc-tesA’-Dox-AD9313)质粒(本发明中编号为CYX149)、pACYC-(Trc-tesA’-Dox-AD7942)质粒(本发明中编号为CYX150)、pACYC-(Trc-tesA’-Dox-AD73102)质粒(本发明中编号为CYX151)。
本发明还提供了一种用于合成偶数中链脂肪烃的基因元件的构建的方法将不同类型或来源的醛基脱羰酶基因(SEQ ID NO:3所示的cer1、SEQ ID NO:4所示的ad9313、SEQ ID NO:5所示的ad7942、ad73102)连接到pET28a质粒中，形成pET28a-CER1，pET28a-AD9313，pET28a-AD7942和pET28a-AD73102质粒；
以CYX134为载体。分别以pET28a-CER1，pET28a-AD9313，pET28a-AD7942和pET28a-AD73102为模板，酶切，切胶回收，与载体连接。
本发明提供了一种合成奇数中链脂肪醛的方法，包括如下步骤：
步骤1：将上述的基因连接到载体中，构建表达载体；
步骤2：将所述表达载体转化宿主细胞，表达，收集表达产物，即得。
具体的，包括如下步骤：
1)将α-加双氧酶基因从pET21a-Dox质粒中带RBS的dox基因连接到pACYC-Trc-tesA质粒中，形成CYX134质粒
2)将CYX134质粒转化进入E.coli BL21(DE3)菌株中，发酵，收集产物，即得。
本发明还提供了一种合成奇数中链脂肪醇的方法，包括如下步骤：
步骤1：构建含有硫酯酶基因及所述硫酯酶基因启动子的第一载体；
步骤2：将上述的基因经酶切连接到所述第一载体，构建第二载体；
步骤3：将醛基还原酶基因经酶切连接到所述第二载体，构建表达载体；
步骤4：取所述表达载体转化宿主细胞，表达，收集表达产物，即得。
具体的，包括如下步骤：
1)将不同来源的醛基还原酶基因从28a-AdhP、28a-YjgB、pET28a-YqhD、pET28a-AdhE、pET28a-Slr1192的质粒中用XbaI和BamHI酶切后分别连接到用SpeI和BamHI酶切的CYX134质粒中，形成CYX143、CYX144、CYX145、CYX146、CYX147质粒；
将各质粒转化进入E.coli BL21(DE3)菌株中，进行发酵，收集产物，即得。
本发明还提供了一种合成偶数中链脂肪烃的方法，包括如下步骤：
步骤1：构建含有硫酯酶基因及所述硫酯酶基因启动子的第一载体；
步骤2：将如权利要求1所述的基因经酶切连接到所述第一载体，构建第二载体；
步骤3：将羰基脱羰酶基因经酶切连接到所述第二载体，构建表达载体；
步骤4：取所述表达载体转化宿主细胞，表达，收集表达产物，即得。
具体的，包括如下步骤：
1)将不同来源的醛基脱羰酶基因从pET28a-CER1，pET28a-AD9313，pET28a-AD7942和pET28a-AD73102的质粒中分别连接到CYX134质粒中，形成CYX148、CYX149、CYX150、CYX151质粒；
2)将各质粒转化进入E.coli BL21(DE3)菌株中，进行发酵，收 集产物，即得。
本发明提供了一种分批补料发酵合成奇数中联脂肪醇的方法，将CYX144质粒和FadR质粒热激转化入宿主细胞，分批补料发酵；
所述CYX144质粒为pACYC-(Trc-tesA’-Dox-yjgB)；
所述FadR质粒pTrcHis2A-fadR。
具体的，本发明提供了一种分批补料发酵合成奇数中联脂肪醇的方法，将CYX144和FadR质粒热激转化进入E.coli BL21(DE3)菌株中，并在LB固体平板上30℃过夜培养，挑重组子单菌落接种于2mL LB培养基中30℃培养至OD为2.5-4，并按照1:100的比例转接于20mL M9培养基中，30℃培养至OD为2.5-4，并按照1:100的比例再次转接于800mL M9培养基中。当OD涨至2.5-4的时候，将培养液离心浓缩至50mL，并接种与2.5L的发酵罐中进行分批补料发酵。当OD升至15的时候，用10μM的IPTG进行诱导。每隔4h取样，每次取15mL用于细胞密度、甘油、乙酸、脂肪醇浓度的分析。固体及液体培养基中各抗生素的含量为氯霉素34μg/mL、氨苄100μg/mL。
细胞密度采用TU-1810紫外可见分光光度计(北京普析通用仪器有限公司)，在波长600的条件下进行测量。
甘油和乙酸浓度的测量：取1mL发酵液12,000rpm离心10min，取上清用0.22μm滤膜过滤，根据情况进行稀释，或者直接进样到HPLC中进行分离检测。HPLC为Waters e2695，检测器为2414RI示差检测器，色谱柱为Aminex HPX-87H column(BioRad,CA)，柱温保持在65℃，流动相为5mM的稀硫酸水溶液，流速为0.6mL/min。
脂肪醇的提取；
脂肪醇提取样品的检测；
如图7所示，诱导18.5h后，脂肪醇产量达到1.95g/L，OD值达到124.5，生产率为0.105g/L/h。发酵过程中甘油消耗和添加速率几乎持平，无乙酸生成。在发酵过程中，不同链长的脂肪醇比例随时间基本不变，在发酵结束是，C11，C13和C15脂肪醇的比例分别为18.6％，66.2％和15.2％。
本发明提供了一种基因，其具有：
(Ⅰ)如SEQ ID No.1所示的核苷酸序列；或
(Ⅱ)如SEQ ID No.1所示的核苷酸序列的互补序列；或
(Ⅲ)与(Ⅰ)或(Ⅱ)的核苷酸序列编码相同蛋白质，但因遗传密码的简并性而与(Ⅰ)或(Ⅱ)的核苷酸序列不同的序列；或
(Ⅳ)与(Ⅰ)或(Ⅱ)或(Ⅲ)所述序列至少有80％同源性的序列。
与现有的脂肪醇和脂肪烃的微生物合成路径相比，本发明具有如下两个优点：(1)α-加双氧酶将前体物脂肪酸分子转化为重要中间代谢物——脂肪醛的过程为氧化反应，不需要细胞提供额外的还原力和能量，降低了细胞生产的负担；(2)填补了脂肪醇产物链长仅为偶数，脂肪烃产物链长大多为奇数的技术局限，可以使生物基大宗化学品以及生物燃料的分子与石油基相关产品更为匹配。
本发明利用合成生物学手段，提供了一种奇数中链脂肪醇和偶数中链烃的微生物合成路径及构建方法，本发明同时提供了用上述路径制备奇数中链脂肪醇和偶数中链烃的工程大肠杆菌。
附图说明
图1示实施例4中含pACYC-Trc-tesA(图1A)或CYX134质粒(图1B)的E.coli BL21(DE3)30℃诱导发酵40h后的产物气相色谱谱图；其中，1：C11醛；2：C11醇；3：C13醛；4：C13醇；5：C15:1醛；6：C15醇；7：C16醇(内标，internal standard,IS)；(图1C)C11醛质谱图；(图1D)C11醇质谱图；(图1E)C13醛质谱图；(图1F)C13醇质谱图；(图1G)C15:1醛质谱图；(图1H)C15醇质谱图；括号中数字为(匹配因子，反向匹配因子)，匹配因子(match factors)和反向匹配因子(reverse match factors)(reverse match factors)能够定量地描述产品质谱谱图与数据库谱图的匹配程度，值大于900表明非常优异的匹配，800-900为优异匹配，700-800为良好匹配；
图2示实施例5中含CYX143、CYX144、CYX145、CYX146、CYX147质粒的E.coli BL21(DE3)30℃诱导发酵40h后的脂肪醇产出结果；
图3示实施例6中含CYX148、CYX149、CYX150、CYX151质粒的E.coli BL21(DE3)30℃诱导发酵40h后的脂肪烃产出结果；(图3A)不同工程菌株的脂肪烃产量对比；(图3B)含CYX148质粒的E.coli BL21(DE3)30℃诱导发酵40h后的产物气相色谱谱图；(图3C)含CYX151质粒的E.coli BL21(DE3)30℃诱导发酵40h后的产物气相色谱谱图。8：C12烃；9：C14烃；(图3D)C12烃质谱图；(图3E)C14烃质谱图；
图4示实施例7中α-加双氧酶和内源硫酯酶之间的代谢流优化的结果；
图5示实施例8证实α-加双氧酶在胞内具有广泛的底物选择性和可调控能力。图5A示实施例8中含YX101、YX102、YX103质粒的E.coli BL21(DE3)30℃诱导发酵40h后的不同链长的脂肪酸产出比例结果；图5B示实施例8中含YX135、YX104、YX105质粒的E.coli BL21(DE3)30℃诱导发酵40h后的不同链长的脂肪醛加脂肪醇产出比例结果；图5C示实施例8中含YX101、YX102、YX103质粒的E.coli BL21(DE3)30℃诱导发酵40h后的脂肪酸产出结果；图5D示实施例8中含YX135、YX104、YX105质粒的E.coli BL21(DE3)30℃诱导发酵40h后的脂肪醛和脂肪醇产出结果；
图6示实施例9调节上游脂肪酸合成路径表达强度以提高奇数中链脂肪醇的合成能力的结果；
图7示实施例10中分批补料发酵的结果。图7A示实施例10中分批补料发酵过程中生物量(OD600)、甘油剩余量、乙酸含量、脂肪醇含量随时间变化曲线；图7B示实施例10中，分批补料发酵9小时、17小时、27.5小时后，发酵液中不同链长脂肪醇含量的比例。
具体实施方式
本发明公开了基因、其编码的蛋白质及用途、基因原件、合成奇数中链脂肪醛、合成奇数中链脂肪醇的方法以及合成偶数中链脂肪烃的方法，本领域技术人员可以借鉴本文内容，适当改进工艺参数实现。特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它 们都被视为包括在本发明。本发明的方法及应用已经通过较佳实施例进行了描述，相关人员明显能在不脱离本发明内容、精神和范围内对本文所述的方法和应用进行改动或适当变更与组合，来实现和应用本发明技术。
本发明提供的基因、其编码的蛋白质及用途、基因原件、合成奇数中链脂肪醛、合成奇数中链脂肪醇的方法以及合成偶数中链脂肪烃的方法中所用原料及试剂均可由市场购得。
质粒编号及信息
Figure PCTCN2015089232-appb-000001
Figure PCTCN2015089232-appb-000002
质粒具体信息
Figure PCTCN2015089232-appb-000003
Figure PCTCN2015089232-appb-000004
Figure PCTCN2015089232-appb-000005
Figure PCTCN2015089232-appb-000006
下面结合实施例，进一步阐述本发明：
实施例1：用于合成奇数中链脂肪醛的基因元件的构建
实验材料：
硫酯酶(TesA’)：大肠杆菌内源硫酯酶基因I(tesA’)从addgene购买(Plasmid 24636)，该基因被至于一个复制子为p15A、启动子为placUV5的质粒上，命名为pKS1，并去掉了起始密码子ATG之后75bp的核苷酸。被去掉的核苷酸编码的氨基酸序列是一段信号肽，用于将该酶定位于细胞间质。将这一段信号肽序列去掉，可是将硫酯酶富集在细胞内，并使大肠杆菌产生大量的自由脂肪酸。
α-加双氧酶(Dox)：根据已报导的大米(Oryza sativa)中α-加双氧酶(NCBI Reference Sequence:NP_001066718.1)的蛋白序列，按照大肠 杆菌进行密码子优化，优化之后的编码α-加双氧酶的DNA分子具有SEQ ID NO:1所示的核苷酸序列，在金唯智公司合成基因。
质粒pTrcHis2A购买自Invitrogen公司。
质粒pACYCDuet-1购买自Novagen公司。
质粒pET21a购买自Novagen公司。
实验方法：
1.构建pHisTrc-tesA质粒
1)载体为pTrcHis2A，用NcoI和BamHI酶切，长度为4400，纯化
2)模板为pKS1，用引物NcoI-tesA-fwd和BamHI-SpeI-tesA-rev进行扩增，长度为575，切胶回收，用NcoI和BamHI酶切，纯化，与载体连接。
3)菌落PCR用引物pTrcHis2A-F和引物pTrcHis2A-R正确长度为894
2.构建pACYC-Trc-tesA质粒
1)用引物AflII-pACYC-fwd和PstI-pACYC-rev扩增pACYCDuet-1，长度为3810，切胶回收，用PstI和AflII酶切，纯化。
2)片段模板为pHisTrc-tesA，用PstI-Gibson-pHisTrc-fwd和AflII-Gibson-rrnBT1-rev进行PCR，长度为1190，切胶回收，与载体进行Gibson连接
3)菌落PCR用Duet-seq-F和pACYCDuet-R，长度1443。
3.构建pET21a-Dox质粒
1)载体为pET21a，用NdeI和BamHI酶切，长度为5350
2)片段模板为合成的dox基因，用引物NdeI-Dox-fwd和BamHI-SpeI-Dox-rev扩增，长度为1885，切胶回收，用NdeI和BamHI酶切，纯化，与载体连接
3)菌落PCR用引物pET-fwd和引物pET-rev，正确长度为2401.
4.构建pET28a-Dox质粒
1)载体为pET28a，用NdeI和BamHI酶切；
2)片段模板为合成的dox基因，用引物NdeI-Dox-fwd和BamHI-SpeI-Dox-rev扩增，长度为1885，切胶回收，用NdeI和BamHI酶切，纯化，与载体连接；
3)菌落PCR用引物pET-fwd和引物pET-rev。
5.构建pET21a-tesA质粒
1)载体为pET21a，用NdeI和BamHI酶切；
2)片段模板为pKS1，用引物NdeI-tesA-fwd和BamHI-SpeI-tesA-rev扩增，切胶回收，用NdeI和BamHI酶切，纯化，与载体连接；
3)菌落PCR用引物pET-fwd和引物pET-rev。
表1用于合成奇数中链脂肪醛的基因元件的构建所需引物列表
Figure PCTCN2015089232-appb-000007
Figure PCTCN2015089232-appb-000008
实施例2：用于合成奇数中链脂肪醇的基因元件的构建
实验材料：
AdhE：乙醛辅酶A还原酶/铁离子依赖型乙醇脱氢酶，来自于E.coli BL21(DE3)基因组(NCBI-GeneID:8180074)，单点突变替换了序列中NcoI酶切位点。
AdhP：乙醇活性脱氢酶/乙醛活性还原酶，来自于E.coli BL21(DE3)基因组(NCBI-GeneID:8181169)。
YqdD：NADPH依赖型乙醛还原酶，来自于E.coli BL21(DE3)基因组(NCBI-GeneID:8180496)，单点突变替换了序列中NdeI酶切位点。
YjgB：乙醇脱氢酶(非典型的锌型乙醇脱氢酶相似蛋白，锌和NADPH依赖型)，来自于E.coli BL21(DE3)基因组(NCBI-GeneID:8182107)。
Slr1192：包含锌的乙醇脱氢酶，来源于集胞藻属PCC6803菌株(Synechocystis sp.PCC 6803)，蛋白序列为NCBI Reference Sequence:NP_443028.1，按照大肠杆菌进行密码子优化，优化之后的编码Slr1192的DNA分子具有SEQ ID NO:2所示的核苷酸序列，在本实验室内合成基 因。
质粒pET28a购买自Novagen公司。
实验方法：
1.构建pHisTrc-tesA质粒
1)载体为pTrcHis2A，用NcoI和BamHI酶切，长度为4400，纯化
2)模板为pKS1，用引物NcoI-tesA-fwd和BamHI-SpeI-tesA-rev进行扩增，长度为575，切胶回收，用NcoI和BamHI酶切，纯化，与载体连接。
3)菌落PCR用引物pTrcHis2A-F和引物pTrcHis2A-R正确长度为894
2.构建pACYC-Trc-tesA质粒
1)用引物AflII-pACYC-fwd和PstI-pACYC-rev扩增pACYCDuet-1，长度为3810，切胶回收，用PstI和AflII酶切，纯化。
2)片段模板为pHisTrc-tesA，用PstI-Gibson-pHisTrc-fwd和AflII-Gibson-rrnBT1-rev进行PCR，长度为1190，切胶回收，与载体进行Gibson连接
3)菌落PCR用Duet-seq-F和pACYCDuet-R，长度1443。
3.构建pET21a-Dox质粒
1)载体为pET21a，用NdeI和BamHI酶切，长度为5350
2)片段模板为合成的dox基因，用引物NdeI-Dox-fwd和BamHI-SpeI-Dox-rev扩增，长度为1885，切胶回收，用NdeI和BamHI酶切，纯化，与载体连接；
3)菌落PCR用引物pET-fwd和引物pET-rev，正确长度为2401.
4.构建pET28a-AdhP质粒
1)载体为pET28a，用NdeI和BamHI酶切，长度为5400
2)片段模板为E.coli BL21(DE3)基因组，用引物NdeI-AdhP-fwd和BamHI-SpeI-AdhP-rev扩增，长度为1036
3)菌落PCR用引物pET-fwd和引物pET-rev，正确长度为1552.
5.构建pET28a-YjgB，pET28a-YqhD，pET28a-AdhE，pET28a-Slr1192质粒
1)载体为pET28a-AdhP，用NdeI和SpeI酶切，切胶回收长度为5350的片段
2)片段模板为E.coli BL21(DE3)基因组，用引物NdeI-***-fwd和SpeI-***-rev扩增不同的基因片段(***代表基因名称)，引物序列见表3，片段名称、PCR后长度如表2所示，其中YqhD需要替换NdeI酶切位点，AdhE需要替换NcoI酶切位点，因此需要以突变点为中心左右PCR扩增两部分，片段切胶回收后再进行overlap，最后带了NdeI和SpeI酶切位点的片段切胶回收，用NdeI和SpeI酶切，纯化，与载体连接；
3)菌落PCR用引物pET-fwd和引物pET-rev，正确长度如表2所示
表2片段详情
Figure PCTCN2015089232-appb-000009
表3用于合成奇数中链脂肪醇的基因元件的构建所需引物列表
Figure PCTCN2015089232-appb-000010
Figure PCTCN2015089232-appb-000011
实施例3：用于合成偶数中链脂肪烃的基因元件的构建
实验材料：
CER1：脂肪醛脱羰酶，来源于拟南芥(Arabidopsis thaliana)，蛋白序列为UniProtKB/Swiss-Prot:F4HVY0.1，按照大肠杆菌进行密码子优化，优化之后的编码CER1的DNA分子具有SEQ ID NO:3所示的核苷酸序列，在genscript合成基因。
AD9313：脂肪醛脱羰酶，来源于原绿球藻(Prochlorococcus marinus MIT9313)，蛋白序列为NCBI Reference Sequence:NP_895059.1，按照大肠杆菌进行密码子优化，优化之后的编码AD9313的DNA分子具有SEQ ID NO:4所示的核苷酸序列，在本实验室内合成基因。
AD7942：脂肪醛脱羰酶，来源于聚球藻(Synechococcus elongatus PCC7942)，蛋白序列为accession number：YP_400610，按照大肠杆菌进行密码子优化，优化之后的编码AD7942的DNA分子具有SEQ ID NO:5所示的核苷酸序列，在GENEART合成基因。
AD73102：脂肪醛脱羰酶，来源于念珠藻(Nostoc punctiforme PCC73102)，蛋白序列为accession number：YP_001865325，按照大肠杆菌进行密码子优化，优化之后的编码AD73102的DNA分子，由宾夕法尼亚州立大学(The Pennsylvania State University，USA)的Squire J.Booker课题组赠与。
实验方法：
1.构建pET28a-CER1，pET28a-AD9313，pET28a-AD7942和pET28a-AD73102和质粒
1)载体为pET28a-AdhP，用NdeI和SpeI酶切，切胶回收长度为5350的片段
2)用引物NdeI-***-fwd和SpeI-***-rev扩增不同的基因片段(***代表基因名称)，引物序列见表5，片段名称、PCR后 长度如表4所示，切胶回收，用NdeI和SpeI酶切，纯化，与载体连接
3)菌落PCR用引物pET-fwd和引物pET-rev，正确长度如表4所示.
表4片段详情
Figure PCTCN2015089232-appb-000012
表5用于合成偶数中链脂肪烃的基因元件的构建所需引物列表
Figure PCTCN2015089232-appb-000013
实施例4：α-加双氧酶用于合成奇数中链脂肪醇和偶数中链脂肪烃的可行性验证
实验方法：
1.构建pACYC-Trc-tesA-Dox(CYX134)质粒
1)载体为pACYC-Trc-tesA，用SpeI和BamHI酶切，纯化
2)片段模板为21A-Dox，用XbaI和BamHI酶切，长度为1911，切胶回收，与载体连接。
2.将质粒CYX134热激转化进入E.coli BL21(DE3)菌株中，并在LB固体平板上进行筛选。细胞均在30℃的培养箱中进行培养，固体及液体培养基中各抗生素的含量为氯霉素34μg/mL。
3.将转化了质粒pACYC-Dox-tesA’的E.coli BL21(DE3)菌株进行发酵。挑重组子单菌落接种于LB培养基中30℃过夜培养，并按照1:100的比例接种于5mL M9培养基中，在30℃，220rpm的摇床中进行进行发酵。当生物量长到OD600＝1.0-1.2之间时，加入1mM的IPTG，诱导表达40h之后，提取脂肪醇样品进行检测。
4.脂肪醇的提取，具体做法如下：
1)取诱导后30℃发酵40h的培养基0.5mL，加入25mg/L的十六醇作为内标；
2)加入0.5mL的乙酸乙酯，漩涡震荡5min，15000rpm离心2min；
3)吸取上层有机相，用0.22μm尼龙膜过滤。进样前将样品保存在-80℃冰箱中。
5.脂肪醇提取样品的检测。本实验中涉及的气质联用系统为Waters GCT Premier MICROMASS系统，其中包括：
1)Agilent 7683自动进样器
2)Agilent 6890气相色谱(GC，Agilent Technologies，USA)
3)飞行时间质谱仪(TOF-MS，Waters Corp.，USA)
4)J&W DB-5毛细管石英柱(30m length，I.D.0.25mm，Film0.25μm，Agilent Technologies，USA)
GC条件如下：采用DB-5气相色谱柱，进样量为1μL，采用柱后分流技术，分流比为2:1。进样口温度为260℃，GC interface温度为280℃。高纯氦为载气，91Kpa恒压。色谱分离的升温程序如下：初始温度70℃，保持2min，以8℃·min-1的速度升到290℃，并在290℃保持6min。
TOF/MS.
质谱条件如下：质谱电离方式为正离子模式的电轰击电离(EI+)，其电离电压为70eV，源温保持在250℃。质谱的扫描范围为50-800m/z，扫描速度为2scan·s-1。
产物的定性和定量分析采用Masslynx软件(Version 4.1，Waters Corp.，USA)对GC-TOF/MS数据进行定性定量分析。采用NIST数据库(National Institute of Standard and Technology library，NIST，2005，Gaithersburg，MD)识别色谱峰，并用QuanLynx软件对各代谢物峰面积进行自动积分。通过各种物质的总离子流图的峰面积与同一张谱图上内标的峰面积的比值以获得标准化的FAME和烃类物质的相对浓度值。
实验结果：
将pACYC-Trc-tesA和CYX134质粒转入E.coli BL21(DE3)中，在30℃诱导发酵40h，对发酵产物进行GC-MS检测，结果如图2所示。转入了pACYC-Trc-tesA模块的菌株只能检测到脂肪酸的生成，这是由硫酯酶(TesA’)水解脂酰-ACP所得。而在pACYC-Trc-tesA质粒中加入dox基因后(CYX134)，在保留时间(RT)为11.20min、14.73min和17.59min处分别检测到了1-十一醛、十三醛和十五烯醛三种脂肪醛，这三种脂肪醛是由大肠杆菌细胞内C12、C14和C16:1的自由脂肪酸经α-Dox氧化得来，该结果证实了本专利申请的一种奇数中链脂肪醇和偶数中链烃合成的可行性，即α-加双氧酶可以在大肠杆菌中合成奇数中链脂肪醛，能够为合成奇数中链脂肪醇和偶数中链脂肪烃提供前体物。
此外，转入了CYX134模块的菌株在保留时间(RT)为12.37min、15.75min、和18.80min处还分别检测到了1-十一醇、1-十三醇和1-十五醇三种脂肪醇(如图2B所示)。这三种脂肪醇是对应碳链的脂肪醛在细胞内被自发还原的产物。各产物的质谱图如图1C-H所示。各物质的匹配因子和反向匹配因子均为850以上，证实了本发明奇数中链脂肪醛/醇分子定性的准确性。
实施例5：醛基还原酶的选择
实验方法：
1.质粒的构建：将不同的醛基还原酶(AdhP、YjgB、YqhD、AdhE、Slr1192)连接到CYX134质粒中。具体做法为，以CYX134为载体，用SpeI和BamHI进行酶切，纯化。分别以28a-AdhP、28a-YjgB、pET28a-YqhD、pET28a-AdhE、pET28a-Slr1192为模板，用XbaI和BamHI进行酶切，切胶回收，与载体连接。具体片段长度详见表6。
表6质粒的构建
Figure PCTCN2015089232-appb-000014
2.将各质粒热激转化进入E.coli BL21(DE3)菌株中，并在LB固体平板上进行筛选。细胞均在30℃的培养箱中进行培养，固体及液体培养基中各抗生素的含量为氯霉素34μg/mL。
3.将转化了各质粒的E.coli BL21(DE3)菌株进行发酵，方法过程同实施例1。
4.脂肪醇的提取，方法过程同实施例1。
5.脂肪醇提取样品的检测，方法过程同实施例1。
实验结果：
将各质粒转入E.coli BL21(DE3)中，在30℃诱导发酵40h，对发酵 产物进行GC-MS检测，结果如图2所示。可以看出，AdhE和AdhP对于奇数中链脂肪醇的产出没有促进作用，而YqdD、Slr1192和YjgB对本专利提出的一种奇数中链脂肪醇的大肠杆菌合成具有进一步的促进作用，这其中以YjgB对产量的促进作用最大，将总脂肪醇的产量从35.2mg/L提升到68.3mg/L。
实施例6：醛基脱羰酶的选择
实验方法：
1.质粒的构建：将不同的醛基脱羰酶(CER1、AD9313、AD7942、AD7310)连接到CYX134质粒中。具体做法为，以CYX134为载体。分别以pET28a-CER1，pET28a-AD9313，pET28a-AD7942和pET28a-AD73102为模板，酶切，切胶回收，与载体连接。载体酶切位点、载体长度、片段酶切位点、片段长度等具体信息详见表7。
表7质粒构建信息
Figure PCTCN2015089232-appb-000015
2.将各质粒热激转化进入E.coli BL21(DE3)菌株中，并在LB固体平板上进行筛选。细胞均在30℃的培养箱中进行培养，固体及液体培养基中各抗生素的含量为氯霉素34μg/mL。
3.将转化了各质粒的E.coli BL21(DE3)菌株进行发酵，方法过程同实施例1。
4.脂肪醇的提取，方法过程同实施例1。
5.脂肪醇提取样品的检测，方法过程同实施例1。
实验结果：
将各质粒转入E.coli BL21(DE3)中，在30℃诱导发酵40h，对发酵产物进行GC-MS检测，结果如图3所示。可以看出，来源于拟南芥的脱羰酶CER1并不能让工程大肠杆菌合成偶数链脂肪烃，而表达了来源于蓝细菌的三个脱羰酶之后，工程大肠杆菌都合成了链长为C12和C14的脂肪烃，证实了本专利一种用大肠杆菌合成中链脂肪烃的方法。其中，当表达来源于念珠藻的脱羰酶AD7942之后，中链烃具有最高产出，为5.2mg/L。图3B为转入了CYX151质粒后，产物产出气相色谱图中脂肪烃的位置。其中8为十二烃，9为十四烃。各产物的质谱图如图3D和E所示。各物质的匹配因子和反向匹配因子均为850以上，证实了本发明偶数中链脂肪烃产物定性的准确性。
综上所述，本发明利用合成生物学手段，提供了一种奇数中链脂肪醇和偶数中链烃的微生物合成路径及构建方法，本发明同时提供了用上述路径制备奇数中链脂肪醇和偶数中链烃的工程大肠杆菌。
实施例7：优化α-加双氧酶和硫酯酶之间的代谢流
实验材料：
质粒pBAD33购买自ATCC公司。
实验方法：
1.构建含不同启动子的表达质粒(YX210、YX211、YX212、YX213)
1)载体为pACYCDute-1，用表8中的引物AflII-pACYC-fwd和引物PstI-pACYC-rev进行扩增
2)片段模板为pQE-80L，pKS1，pTrcHis2A和pBAD33，用表8中的其余引物，进行扩增，与载体连接，构建出YX210、YX211、YX212和YX213质粒，这些质粒同时具有两个启动子，一个启动子为T7，另外一个启动子分别为T5，pLacUV5，Trc和BAD。这些质粒的具体的信息如表9所示。
2.构建由T7启动子调控的大肠杆菌内源硫酯酶基因的质粒
1)载体为pACYCDute-1、YX210、YX211、YX212和YX213，用NdeI和KpnI酶切，纯化；
2)片段模板为pKS1质粒，用引物NdeI-tesA-fwd和KpnI-tesA-rev扩增，引物序列见表8，PCR后酶切，纯化，与载体连接，挑取正确的转化子。
3.构建不含6*His标签的双启动子控制的α-加双氧酶和内源硫酯酶
质粒(YX220，YX232，YX233，YX234，YX235)
1)载体为步骤2中产生的5个质粒，用NcoI和BamHI酶切开第一个启动子中的多克隆酶切位点，纯化
2)片段模板为合成的dox基因，用引物NcoI-Dox-fwd和BamHI-SpeI-Dox-rev扩增，引物序列见表8，PCR后长度为1874，酶切，纯化，与载体连接，挑取正确的转化子。YX220，YX232，YX233，YX234，YX235中第一个启动子(T7，T5，pLacUV5，Trc和BAD)控制不含6*His标签的dox基因，第二个启动子(T7)控制tesA’基因
4.构建含6*His标签的双启动子控制的α-加双氧酶和内源硫酯酶质粒(YX221，YX222，YX223，YX224，YX225)
1)载体为步骤2中产生的5个质粒，用NcoI和BamHI酶切开第一个启动子中的多克隆酶切位点，纯化
2)片段模板为21a-dox质粒，用NcoI和BamHI酶切，切胶回收，与载体连接，挑取正确的转化子。YX221，YX222，YX223，YX224，YX225中第一个启动子(T7，T5，pLacUV5，Trc和BAD)控制含6*His标签的dox基因，第二个启动子(T7)控制tesA’基因
5.构建含由T7，T5，pLacUV5，Trc和BAD启动子控制的α-加双氧酶的质粒
1)载体为pACYCDute-1、YX210、YX211、YX212和YX213，用NcoI和BamHI酶切，纯化
2)片段模板为合成的dox基因，用引物NcoI-Dox-fwd和BamHI-SpeI-Dox-rev扩增，引物序列见表8，PCR后酶切，纯化，与载体连接，挑取正确的转化子
6.构建含由T7，T5，pLacUV5，Trc和BAD启动子控制的dox-tesA’质粒(YX135、YX136、YX137、YX138和YX140)
1)载体为步骤5中产生的5个质粒，用SpeI和BamHI酶切，纯化
2)片段模板21a-tesA，用XbaI和BamHI进行酶切，切胶回收，与载体连接，挑取正确的转化子。YX135、YX136、YX137、YX138和YX140中第一个启动子(T7，T5，pLacUV5，Trc和BAD)控制dox和tesA’两个基因
7.构建含由T7，T5，pLacUV5，Trc和BAD启动子控制的大肠杆菌内源硫酯酶基因的质粒
1)载体为YX210、YX211、YX212和YX213，用NcoI和BamHI酶切，纯化
2)片段模板为pKS1，用引物NcoI-tesA-fwd和BamHI-SpeI-tesA-rev扩增，引物序列见表8，PCR后酶切，纯化，与载体连接，挑取正确的转化子
8.构建含由T7，T5，pLacUV5，Trc和BAD启动子控制的tesA’-dox质粒(YX131、YX132、YX133、YX134和YX130)
1)载体为步骤7中产生的5个质粒，用SpeI和BamHI酶切，纯化
2)片段模板21a-Dox，用XbaI和BamHI进行酶切，切胶回收，与载体连接，挑取正确的转化子。YX131、YX132、YX133、YX134和YX130中第一个启动子(T7，T5，pLacUV5，Trc和BAD)控制tesA’和dox两个基因
表8优化α-加双氧酶和硫酯酶之间的代谢流所需引物
Figure PCTCN2015089232-appb-000016
*直线下划线处序列为酶切位点，加粗、斜体处序列为起始密码子或终止密码子。
表9含不同启动子的表达质粒信息
Figure PCTCN2015089232-appb-000017
9.将各质粒热激转化进入E.coli BL21(DE3)菌株中，并在LB固体平板上进行筛选。细胞均在30℃的培养箱中进行培养，固体及液体培养基中各抗生素的含量为氯霉素34μg/mL。
10.将转化了各质粒的E.coli BL21(DE3)菌株进行发酵，方法过程同实施例1。
11.脂肪醇的提取，方法过程同实施例1。
12.脂肪醇提取样品的检测，方法过程同实施例1。
实验结果：
将各质粒转入E.coli BL21(DE3)中，在30℃诱导发酵40h，对发酵产物进行GC-MS检测，结果如图4所示。针对TesA’和α-Dox之间代谢流分为两部分进行优化。
首先，将TesA’和α-Dox在两个开放阅读框(Open reading frame，ORF)中进行表达。TesA’用T7启动子控制，α-Dox用五种不同的启动子进行表达。当α-Dox在表达强度最强的T7或者表达强度最弱的BAD启动子的控制下，脂肪醇的产出量均比较低。而当α-Dox的表达强度适中时(由T5，LacUV5和Trc启动子控制)，脂肪醇的产出相对较高。这说明TesA’和α-Dox两者之间的表达强度相差太大，α-Dox的表达量略小于TesA’时，代谢流较为平衡。此外，在α-Dox蛋白N端加上6*His标签不能够提高相同表达强度下脂肪醇的产出，这说明α-Dox在(后)转录或/和(后)翻译阶段比较稳定。
其次，将TesA’和α-Dox在一个开放阅读框中进行表达，优化两个基 因整体的表达强度。在质粒的构建中，分别构建了dox-tesA’和tesA’-dox两种表达结构。当两个基因同时在一个开放阅读框内进行表达时，靠近启动子的基因会相对的具有更强一点的表达强度。从图4中可以看出，当tesA’更靠近启动子时，脂肪醇的产出更高一些。这也与第一种优化的结论相符，即TesA’的表达强度需要略比α-Dox高。另外，tesA’-dox两者的表达强度不宜过高或者过低，当在Trc启动子控制下，脂肪醇的产出最高(35.2mg/L)。
实施例4中的CYX134质粒即为通过此实施例中的tesA’和α-Dox之间代谢流最优化的质粒(CYX等同于YX)。
实施例8：证实α-加双氧酶在胞内具有广泛的底物选择性和可调控能力
实验方法：
1.构建含不同硫酯酶的脂肪酸路径过表达质粒(YX101、YX102、YX103)
1)载体为pACYCDute-1，用EcoRI和SacI酶切，或者用SalI和HindIII酶切，纯化
2)片段模板为三种不同的硫酯酶基因：pKS1中的tesA’基因(Escherichia coli，大肠杆菌来源)，或者合成的bte(Umbellularia californica，加州月桂来源)和BnFatA(Brassica napus，欧洲油菜来源)基因，用对应引物扩增不同的基因片段，引物序列见表10，片段名称、PCR后长度如表11所示，酶切，纯化，与载体连接；
3)菌落PCR用引物Duet-seq-F和引物pACYCDuet-R，正确长度如表11所示
2.构建含不同硫酯酶的脂肪醇合成路径过表达质粒(YX135、YX104、YX105)
1)载体为YX101，YX102或YX103，用NcoI和BamHI酶切，纯化
2)片段模板为合成的dox基因，用引物NcoI-Dox-fwd和BamHI-SpeI-Dox-rev扩增，PCR后长度为1874，酶切，纯化，与载体连接，挑取正确的转化子
表10用于证实α-加双氧酶在胞内具有广泛的底物选择性和可调控能力所需引物列表
Figure PCTCN2015089232-appb-000018
*直线下划线处序列为酶切位点，加粗、斜体处序列为起始密码子或终止密码子，
Figure PCTCN2015089232-appb-000019
Figure PCTCN2015089232-appb-000020
处序列为RBS。。
表11片段详情
Figure PCTCN2015089232-appb-000021
3.将YX101，YX102，YX103质粒分别热激转化进入E.coli BL21(DE3)菌株中，并在LB固体平板上进行筛选。细胞均在30℃的培养箱中进行培养，固体及液体培养基中各抗生素的含量为氯霉素34μg/mL。
4.将转化了各质粒的E.coli BL21(DE3)菌株进行发酵，方法过程同实施例1。
5.脂肪酸的提取：取诱导后30℃发酵40h的培养基0.5mL，加入50μL盐酸和25μg的十七酸作为内标；加入0.5mL乙酸乙酯后漩涡震荡5min，15000rpm离心2min(下同)；吸取上层有机相，向下层溶液再加0.5mL乙酸乙酯，漩涡震荡5min，离心取上层有机相；将两部分提取液结合，加入20μL的重氮甲烷，1μL的盐酸，9μL的甲醇，以对提取的自由脂肪酸进行甲基化，反应两小时后用氮气吹干；用0.5mL的正己烷溶解蒸干产物(脂肪酸甲酯，FAME)，用0.22μm尼龙膜过滤。进样前将样品保存在-80℃冰箱中。
6.脂肪酸提取样品的检测，方法过程与实施例1中脂肪醇的检测相同。
7.将YX135，YX104，YX105质粒分别热激转化进入E.coli BL21(DE3)菌株中，并在LB固体平板上进行筛选。细胞均在30℃的培养箱中进行培养，固体及液体培养基中各抗生素的含量为氯霉素34μg/mL。
8.将转化了各质粒的E.coli BL21(DE3)菌株进行发酵，方法过程同实施例1。
9.脂肪醇的提取，方法过程同实施例1。
10.脂肪醇提取样品的检测，方法过程同实施例1。
实验结果：
将含有YX101，YX102或YX103质粒的大肠杆菌进行发酵，结果如图5所示。过表达了TesA’，BTE或者BnFatA的工程菌株分别合成C14/C16(39％/36％)，C12(75％)，以及C16/C18(75％/24％)的脂肪酸作为主要产物。当αDOX与硫酯酶共同过表达的时候，不同的工程菌株合成C13/C15(57％/30％)，C11(95％)，以及C15(93％)的脂肪醛+脂肪醇作为主要产物。这说明，当过表达了αDOX后，脂肪醛+脂肪醇的产物比例，与其前体物脂肪酸的产物比例相匹配。唯一例外的是当BnFatA过表达时，有24％ 的C18脂肪酸，但是当αDOX过表达后却不产生对应的C17脂肪醛/醇。这些结果说明αDOX在胞内能够氧化链长为C12-C16的脂肪酸，在此范围内，能够将不同链长的脂肪酸底物转化为对应的脂肪醛，说明αDOX和本体系具有可调控的能力。
实施例9：调节上游脂肪酸合成路径表达强度以提高奇数中链脂肪醇的合成能力
实验方法：
1.构建上游脂肪酸路径过表达质粒
1)载体为pTrcHis2A，用NcoI和BamHI酶切，纯化
2)片段模板为大肠杆菌MG1655基因组，用引物NcoI-GCG-***-fwd和BamHI-SpeI-***-rev扩增不同的基因片段(***代表基因名称)，引物序列见表12，片段名称、PCR后长度如表13所示，其中FabD需要替换XhoI酶切位点，FabG需要替换NcoI酶切位点，切胶回收，用NcoI和BamHI酶切，纯化，与载体连接；
3)菌落PCR用引物pTrcHis2A-F和引物pTrcHis2A-R，正确长度如表13所示
表12用于过表达脂肪酸路径基因所需引物列表
Figure PCTCN2015089232-appb-000022
Figure PCTCN2015089232-appb-000023
*直线下划线处序列为酶切位点，加粗、斜体处序列为起始密码子或终止密码子。
表13片段详情
构建的质粒名称 片段名称 片段长度 菌落PCR长度
FabA fabA 534 863
FabI fabI 814 1133
FabG fabG 761 1082
FabD fabD 955 1277
FabB fabB 1246 1568
FadR fadR 735 1055
2.β-氧化途径中fadD和fadE基因的敲除
1)用于同源重组的引物序列如表14所示。采用这两个将氯霉素从pKD3质粒中扩增下来，产物经琼脂糖凝胶电泳鉴定，并用琼脂糖凝胶DNA回收试剂盒回收PCR切胶产物；
2)将pKD46质粒转入大肠杆菌BL21(DE3)中，LB液体培养基中30℃过夜培养，活化后的菌体按照1:100的比例接种到含有10mmol/L L-阿拉伯糖的液体培养基中30℃培养，当菌体OD600长到0.5-0.6时冰上预冷10min，4℃，4000rpm离心5min(下同)，随后用冰冷的10％甘油离心洗涤三次，浓缩100倍制成的电转感受态细胞,每管100μL,存放于-80℃冰箱待用；
3)采用电转化仪Electroporator 2170(Eppendorf，Germany)(0.1-cm chambers)将步骤1)中的PCR胶收产物(10-100ng)加入到感受态BL-46细胞中，1800V电击5-6ms后加入1mL无抗LB培养基于37℃，150rpm复苏3-4h，随后一半的细胞涂布在含有25μg/mL氯霉素的LB平板培养基中，剩余细胞室温放置过夜，如果24h后氯霉素平板中依然没有菌株生长，则将这些剩余细胞重新涂板。
4)对步骤3)中的平板挑单菌落，菌落PCR验证氯霉素是否将基因组中的fadE基因替换下来。
5)将步骤4)中验证正确的转化子接到2mL氯霉素LB培养基中，43℃培养12h，将pKD46质粒缺失。划线后挑同一单菌落同时涂在氨苄和氯霉素的平板中，30℃培养24h，若同一单菌落在氯霉素平板中生长，在氨苄平板中不生长，则说明pKD46缺失完全。
6)将缺失过pKD46的转化子接菌制作电转感受态细胞，转入pCP20质粒，1mL无抗培养基30度复苏3-4小时后，吸出100μl接到2ml氨苄和氯霉素双抗培养基中30℃过夜培养，随后按照1:200的比例转接至无抗培养基中，43℃培养至稳定器后在无抗LB平板中划线，取单菌落在氨苄和氯霉素平 板中分别划线验证染色体中氯霉素的弹出和pCP20质粒的丢失。并进行PCR和测序验证。
表14用于大肠杆菌BL21(DE3)基因组中fadD和fadE基因敲除所需引物列表
Figure PCTCN2015089232-appb-000024
*50-nt与基因组中需要敲除的基因两端同源的序列用
Figure PCTCN2015089232-appb-000025
标识，加粗、斜体处序列为起始密码子或终止密码子
3.将CYX144质粒和1个方法1中所构建的质粒热激转化进入E.coliBL21(DE3)菌株中，并在LB固体平板上进行筛选。细胞均在30℃的培养箱中进行培养，固体及液体培养基中各抗生素的含量为氯霉素34μg/mL，氨苄100μg/mL。
4.将转化了各质粒的E.coli BL21(DE3)菌株进行发酵，方法过程同实施例1。当CYX144与fab系列基因共同转化进入E.coli BL21(DE3)菌株发酵时，IPTG浓度分成1mM，0.1mM和0.01mM三种浓度进行诱导。
5.脂肪醇的提取，方法过程同实施例1。
6.脂肪醇提取样品的检测，方法过程同实施例1。
实验结果：
将各质粒转入E.coli BL21(DE3)中，在30℃诱导发酵40h，对发酵产物进行GC-MS检测，结果如图6所示。当IPTG浓度为1mM的时候，过表达脂肪酸合成路径中的任何基因都无法增强脂肪醇的合成能力；通过敲除fadD或fadE基因阻断脂肪酸β-氧化途径也不能促进脂肪醇的合成。但是当将诱导物IPTG的浓度降低到0.1mM的时候，过表达乙酰辅酶A羧化酶(acetyl-CoA carboxylase,ACC)，FabD，FabI和FadR能够显著的提高脂肪醇的产出。比如当FabD或FadR与CYX144同时过表达的时候，奇数链脂肪醇的产出能够从65.1mg/L提高到100.8mg/L或101.5mg/L。当IPTG的浓度为0.01mM的时候，同样是过表达ACC，FabD，FabI和FadR能够促进脂肪醇的产出。当FadR与CYX144同时过表达的时候，脂肪醇的产出比单独表达CYX144时提高了77.1％。
实施例10：分批补料发酵
实验方法：
1.将CYX144和FadR质粒热激转化进入E.coli BL21(DE3)菌株中，并在LB固体平板上30℃过夜培养，挑重组子单菌落接种于2mLLB培养基中30℃培养至OD为2.5-4，并按照1:100的比例转接于20mL M9培养基中，30℃培养至OD为2.5-4，并按照1:100的比例再次转接于800mL M9培养基中。当OD涨至2.5-4的时候，将培养液离心浓缩至50mL，并接种与2.5L的发酵罐中进行分批补料发酵。当OD升至15的时候，用10μM的IPTG进行诱导。每隔4h取样，每次取15mL用于细胞密度、甘油、乙酸、脂肪醇浓度的分析。固体及液体培养基中各抗生素的含量为氯霉素34μg/mL、氨苄100μg/mL。
2.细胞密度采用TU-1810紫外可见分光光度计(北京普析通用仪器有限公司)，在波长600的条件下进行测量。
3.甘油和乙酸浓度的测量：取1mL发酵液12,000rpm离心10min，取上清用0.22μm滤膜过滤，根据情况进行稀释，或者直接进样到HPLC中进行分离检测。HPLC为Waters e2695，检测器为2414RI 示差检测器，色谱柱为Aminex HPX-87H column(BioRad,CA)，柱温保持在65℃，流动相为5mM的稀硫酸水溶液，流速为0.6mL/min。
4.脂肪醇的提取，方法过程同实施例1。
5.脂肪醇提取样品的检测，方法过程同实施例1。
实验结果：
如图7所示，诱导18.5h后，脂肪醇产量达到1.95g/L，OD值达到124.5，生产率为0.105g/L/h。发酵过程中甘油消耗和添加速率几乎持平，无乙酸生成。在发酵过程中，不同链长的脂肪醇比例随时间基本不变，在发酵结束是，C11，C13和C15脂肪醇的比例分别为18.6％，66.2％和15.2％。
以上对本发明所提供的基因、其编码的蛋白质及用途、基因元件、合成奇数中链脂肪醛的方法以及合成偶数中链脂肪烃的方法进行了详细介绍。本文应用了具体个例对本发明的原理及实施方式进行了阐述，以上实施例的说明只是用于帮助理解本发明的方法及其核心思想。应当指出，对于本技术领域技术人员来说，在不脱离本发明原理的前提下，还可以对本发明进行若干改进和修饰，这些改进和修饰也落入本发明权利要求的保护范围内。
Figure PCTCN2015089232-appb-000026
Figure PCTCN2015089232-appb-000027
Figure PCTCN2015089232-appb-000028
Figure PCTCN2015089232-appb-000029
Figure PCTCN2015089232-appb-000030
Figure PCTCN2015089232-appb-000031
Figure PCTCN2015089232-appb-000032
Figure PCTCN2015089232-appb-000033
Figure PCTCN2015089232-appb-000034
Figure PCTCN2015089232-appb-000035
Figure PCTCN2015089232-appb-000036
Figure PCTCN2015089232-appb-000037
Figure PCTCN2015089232-appb-000038
Figure PCTCN2015089232-appb-000039
Figure PCTCN2015089232-appb-000040
Figure PCTCN2015089232-appb-000041
Figure PCTCN2015089232-appb-000042
Figure PCTCN2015089232-appb-000043
Figure PCTCN2015089232-appb-000044
Figure PCTCN2015089232-appb-000045
Figure PCTCN2015089232-appb-000046
Figure PCTCN2015089232-appb-000047
Figure PCTCN2015089232-appb-000048

Claims (17)

  1. 一种基因，其特征在于，其具有：
    (Ⅰ)如SEQ ID No.1所示的核苷酸序列；或
    (Ⅱ)如SEQ ID No.1所示的核苷酸序列的互补序列；或
    (Ⅲ)与(Ⅰ)或(Ⅱ)的核苷酸序列编码相同蛋白质，但因遗传密码的简并性而与(Ⅰ)或(Ⅱ)的核苷酸序列不同的序列；或
    (Ⅳ)与(Ⅰ)或(Ⅱ)或(Ⅲ)所述序列至少有80％同源性的序列。
  2. 根据权利要求1所述的基因用于合成脂肪醛、奇数中链脂肪酸、奇数中链脂肪醇、偶数中链脂肪烃的用途。
  3. 根据权利要求2所述的用途，其特征在于，所述脂肪醛为奇数中链脂肪醛。
  4. 一种含有如权利要求1所述基因的载体。
  5. 一种含有如权利要求4所述载体的宿主细胞。
  6. 根据权利要求5所述的宿主细胞，其特征在于，所述宿主细胞为大肠杆菌。
  7. 一种用于合成奇数中链脂肪醛的基因元件，其特征在于，其含有如权利要求1所述的基因。
  8. 根据权利要求7所述的用于合成奇数中链脂肪醛的基因元件，其特征在于，还包括硫酯酶。
  9. 一种用于合成奇数中链脂肪醇的基因元件，其特征在于，其含有如权利要求1所述的基因。
  10. 根据权利要求9所述的用于合成奇数中链脂肪醇的基因元件，其特征在于，还包括硫酯酶和醛基还原酶基因。
  11. 根据权利要求10所述的基因元件，其特征在于，所述醛基还原酶基因选自具有如SEQ ID No.2所示的核苷酸序列的基因、adhP、yjgB、yqhD或adhE。
  12. 一种用于合成偶数中链脂肪烃的基因元件，其特征在于，其含有 如权利要求1所述的基因。
  13. 根据权利要求12所述的用于合成偶数中链脂肪烃的基因元件，其特征在于，还包括硫酯酶和羰基脱羰酶基因。
  14. 根据权利要求13所述的基因元件，其特征在于，所述羰基脱羰酶基因选自具有如SEQ ID No.3、4或5所示的核苷酸序列的基因或ad73102。
  15. 一种合成奇数中链脂肪醛的方法，其特征在于，包括如下步骤：
    步骤1：将如权利要求1所述的基因连接到载体中，构建表达载体；
    步骤2：将所述表达载体转化宿主细胞，表达，收集表达产物，即得。
  16. 一种合成奇数中链脂肪醇的方法，其特征在于，包括如下步骤：
    步骤1：构建含有硫酯酶基因及所述硫酯酶基因启动子的第一载体；
    步骤2：将如权利要求1所述的基因经酶切连接到所述第一载体，构建第二载体；
    步骤3：将醛基还原酶基因经酶切连接到所述第二载体，构建表达载体；
    步骤4：取所述表达载体转化宿主细胞，表达，收集表达产物，即得。
  17. 一种合成偶数中链脂肪烃的方法，其特征在于，包括如下步骤：
    步骤1：构建含有硫酯酶基因及所述硫酯酶基因启动子的第一载体；
    步骤2：将如权利要求1所述的基因经酶切连接到所述第一载体，构建第二载体；
    步骤3：将羰基脱羰酶基因经酶切连接到所述第二载体，构建表达载体；
    步骤4：取所述表达载体转化宿主细胞，表达，收集表达产物，即得。
PCT/CN2015/089232 2014-10-22 2015-09-09 基因及其用途、合成奇数中链脂肪醛的方法以及合成偶数中链脂肪烃的方法 WO2016062171A1 (zh)

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