WO2023035527A1 - 用于生产橄榄醇和橄榄醇酸的工程化微生物 - Google Patents
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/52—Genes encoding for enzymes or proenzymes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/001—Oxidoreductases (1.) acting on the CH-CH group of donors (1.3)
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/1025—Acyltransferases (2.3)
- C12N9/1029—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/88—Lyases (4.)
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y203/00—Acyltransferases (2.3)
- C12Y203/01—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
- C12Y203/01086—Fatty-acyl-CoA synthase (2.3.1.86)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y203/00—Acyltransferases (2.3)
- C12Y203/01—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
- C12Y203/0118—Beta-ketoacyl-acyl-carrier-protein synthase III (2.3.1.180)
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y203/00—Acyltransferases (2.3)
- C12Y203/01—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
- C12Y203/01206—3,5,7-Trioxododecanoyl-CoA synthase (2.3.1.206)
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y404/00—Carbon-sulfur lyases (4.4)
- C12Y404/01—Carbon-sulfur lyases (4.4.1)
- C12Y404/01026—Olivetolic acid cyclase (4.4.1.26)
Definitions
- the invention relates to the technical field of microorganisms, in particular to engineered microorganisms for producing olivetol (olivetol, OL) and olivetolic acid (olivetolic acid, OA).
- Cannabinoids refer to a large class of chemical molecules derived from the plant cannabis (Cannabis sativa), with more than 150 species. Cannabinoids currently used internationally include cannabidiol (CBD), tetrahydrocannabinol (THC) and cannabigerol (CBG). CBD is one of the main chemical components in plant cannabis and is a non-addictive component of cannabinoids. THC is the main psychoactive substance in marijuana, which can be addictive and is strictly controlled in various countries around the world. Because of its low content in plant cannabis, CBG is usually classified as a trace cannabinoid or a rare cannabinoid.
- Olive alcohol and olive oleic acid are type III polyketides derived from Cannabis plants, which have antibacterial, antitumor and antiultraviolet activities, and are also key biosynthetic precursors of cannabinoids. There are few reports on the natural extraction of olivetol and olivelic acid from plants. Moreover, plant cultivation is limited by many factors. For example, compared with biosynthesis, it is more difficult to obtain high-purity products, strict cultivation regulations, limited production capacity, high investment in plant cultivation and downstream extraction, and low production stability. The chemical synthesis of olive alcohol and olive oil is expensive, and there are problems such as environmental pollution and harsh conditions.
- the present invention fulfills the aforementioned needs in the art by providing novel engineered microorganisms that biosynthesize olivetol and olivetolic acid in high yields.
- the engineered microorganism according to the present invention can produce high-concentration olivetol and olivetol acid under small-scale cultivation, and because the cell concentration (OD600) of the fermentation liquid during small-scale cultivation is far lower than that of the fermentation liquid during industrialized cultivation.
- the cell concentration in industrial culture is about ten times higher than that in small-scale culture, so the yield will be more than ten times. It can be expected that the engineered microorganisms of the present invention have a strong industrial production potential of olive alcohol and olive acid, which is beneficial to the production of cannabinoids. industrial biosynthesis.
- the present invention provides an engineered Escherichia coli, wherein the engineered Escherichia coli is modified to express olivetolic acid synthase (olivetolic acid synthase, OLS), wherein the genome fabH of the engineered Escherichia coli Genes are deleted.
- OLS olivetolic acid synthase
- the engineered E. coli is modified to express olivetolic acid cyclase (OAC).
- OAC olivetolic acid cyclase
- the engineered E. coli has a genomic fadE gene deleted.
- the engineered E. coli is modified to overexpress long-chain acyl-CoA synthetase (fadD).
- the engineered E. coli is modified from BW25113.
- the modification is by introduction of a plasmid.
- the OLS and OAC are co-expressed from the same plasmid.
- the fadD is overexpressed by a plasmid.
- the method provides a method for preparing engineered Escherichia coli, which comprises modifying Escherichia coli to express olivetol synthase (OLS), and deleting the fabH gene in the genome of the Escherichia coli.
- OLS olivetol synthase
- the method comprises modifying the E. coli to express olivate cyclase (OAC).
- OAC olivate cyclase
- the method comprises deleting the genomic fadE gene of the E. coli.
- the method comprises modifying the E. coli to overexpress a long chain acyl-CoA synthetase.
- the engineered E. coli is modified from BW25113.
- the modification is by introduction of a plasmid.
- the OLS and OAC are co-expressed from the same plasmid.
- the fadD is overexpressed by a plasmid.
- the term "about” or “approximately” refers to a change of up to 15%, 10%, or 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% in quantity, level, value, quantity, frequency, percentage, dimension, size, volume, weight or length.
- the term "about” or “approximately” refers to ⁇ 15%, ⁇ 10%, ⁇ 9% around a reference amount, level, value, quantity, frequency, percentage, dimension, size, amount, weight or length , ⁇ 8%, ⁇ 7%, ⁇ 6%, ⁇ 5%, ⁇ 4%, ⁇ 3%, ⁇ 2%, or ⁇ 1% amount, level, value, amount, frequency, percentage, scale, size, amount , weight or length range.
- the term “substantially/essentially” means about 90%, 91%, compared to a reference amount, level, value, amount, frequency, percentage, dimension, size, amount, weight, or length , 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or greater in quantity, level, value, amount, frequency, percentage, dimension, size, amount, weight, or length.
- the term “substantially the same” refers to a quantity, level, value, amount, frequency, A percentage, measure, size, amount, weight, or length range.
- the term "substantially free” when used to describe a composition such as a cell population or a culture medium means free of a specified substance, for example 95% free, 96% free, 97% free, 98% free A composition that is free, 99% free of the specified substance, or is undetectable as measured by conventional means.
- a similar meaning applies to the term “absent” when referring to the absence of a particular substance or component of the composition.
- Consisting of means including, but limited to, anything following the phrase “consisting of”. Thus, the phrase “consisting of” indicates that the listed elements are required or mandatory, and that no other elements may be present.
- Consisting essentially of is meant to include any of the elements listed after the phrase “consisting essentially of” and is limited to activities or actions specified in the disclosure that do not interfere with or contribute to the listed elements other elements. Thus, the phrase “consisting essentially of” is to indicate that the listed elements are required or mandatory, but that no other elements are optional, and depending on whether they affect the activities or actions of the listed elements and may or may not exist.
- the present invention is based, at least in part, on the discovery that host microorganisms expressing oliveol synthase (OLS) and/or olivet acid cyclase (OAC) can be improved by deleting the genomic fabH coding sequence in the host microorganism (e.g. E. Can produce high concentrations of olivetol and/or olivetolic acid.
- OLS oliveol synthase
- OAC olivet acid cyclase
- Coenzyme A (Malonyl-CoA) and hexanoyl-CoA (Hexanoyl-CoA) are substrates for the synthesis of olivetol and/or olivetolic acid.
- the engineered microorganisms could be used to produce olivetol or olivine acid on demand, thereby surpassing cumbersome and costly existing technologies that still rely on complex synthetic chemistry.
- the present invention provides an engineered microorganism, wherein the engineered microorganism is modified to express olivetol synthase (OLS), wherein the genomic fabH gene of the engineered microorganism is deleted.
- OLS olivetol synthase
- the microorganism engineered to biosynthesize olivetol and olivetolic acid is Escherichia coli (E. coli) or a relative thereof.
- the microorganism engineered to biosynthesize olivetol and olivetolic acid is Escherichia coli BW25113 (ATCC accession number; available from the American Type Culture Collection).
- the BW25113 strain is derived from E.coli K-12W1485, which is a derivative strain of K-12W1485. It is similar to MG1655 and is an engineering strain of E. coli that has undergone less modification and is closer to the "wild type". Improved production of malonyl-CoA and hexanoyl-CoA compared to WT.
- the olivetol synthase used herein may be derived from Cannabis sativa.
- Olivetol synthase may be a variant optimized for expression in E. coli.
- the olive alcohol synthase may comprise, consist essentially of, or consist of the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 2 or the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 2 At least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86 %, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% or any two of the preceding values
- the amino acid sequence encoded by the nucleotide sequence constitutes the range of identity.
- the olivetol synthase may comprise, consist essentially of, or consist of: the amino acid sequence shown in SEQ ID NO:3 or at least 70%, 71%, 72% of SEQ ID NO:3 %, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any two of the preceding values constitute the range of identity amino acid sequence.
- Exemplary oliveol synthases used herein may include full-length olivetol synthase, fragments of olivetol synthase, variants of olivetol synthase, truncated olivetol synthase, or at least one of olivetol synthase active fusion enzyme.
- the olivetol synthase used in the present invention has the activity of catalyzing the synthesis of olivetol from malonyl-CoA and hexanoyl-CoA.
- the olivate cyclase used herein may be derived from Cannabis sativa. Olivate cyclase may be a variant optimized for expression in E. coli.
- the olivet acid cyclase may comprise, consist essentially of, or consist of: an amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 4 or composed of the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 4 have at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85% , 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% or any of the preceding values An amino acid sequence encoded by a nucleotide sequence within the range of identity between the two.
- the olivate cyclase may comprise, consist essentially of, or consist of the amino acid sequence shown in SEQ ID NO:5 or at least 70%, 71% of SEQ ID NO:5 , 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88 %, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, or any two of the preceding values constitute the same range Sexual amino acid sequence.
- Exemplary olivate cyclases as used herein may include full-length olivate cyclases, fragments of olivate cyclases, variants of olivate cyclases, truncated olivate cyclases Or a fusion enzyme having at least one activity of olivet acid cyclase.
- the olivate cyclase used in the invention has activity to carboxylate olivetol to olivate.
- long-chain acyl-CoA synthetase (fadD), acyl-CoA dehydrogenase (fadE) and ⁇ -ketoacyl-acyl carrier protein synthase (fabH) are Escherichia coli inherent components.
- fadD as used herein can be derived from E. coli.
- fadD may be a variant optimized for expression in E. coli.
- fadD may comprise, consist essentially of, or consist of the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 13 or have at least 70% of the amino acid sequence of SEQ ID NO: 13 , 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87 %, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, or any two of the foregoing values The range of identity of the nucleotide sequence encoded by the amino acid sequence.
- fadD may comprise, consist essentially of, or consist of the amino acid sequence shown in SEQ ID NO: 14 or at least 70%, 71%, 72%, 73% of SEQ ID NO: 14 %, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, An amino acid sequence having an identity of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any two of the aforementioned values.
- Exemplary fadDs used herein can include full-length fadD, a fragment of fadD, a variant of fadD, a truncated fadD, or a fusion enzyme having at least one activity of fadD.
- fadD used in the present invention has the activity of catalyzing the production of hexanoyl-CoA from hexanoic acid by acylation.
- one or more enzymes used in the invention may be mutants or variants of the enzymes described herein.
- mutant and variant refer to a molecule that retains the same or substantially the same biological activity as that of the original sequence.
- the mutant or variant may be from the same or different species, or may be based on a natural molecule or a synthetic sequence of an existing molecule.
- the terms "mutant” and “variant” refer to a polypeptide having an amino acid sequence that differs from a corresponding wild-type polypeptide by at least one amino acid.
- mutants and variants may contain conservative amino acid substitutions: that is, amino acids with similar properties are substituted for the original corresponding amino acids.
- Conservative substitutions can be polar to polar amino acids (glycine (G, Gly), serine (S, Ser), threonine (T, Thr), tyrosine (Y, Tyr), cysteine (C, Cys), asparagine (N, Asn) and glutamine (Q, Gln)); non-polar to non-polar amino acids (alanine (A, Ala), valine (V, Val), color amino acid (W, Trp), leucine (L, Leu), proline (P, Pro), methionine (M, Met), phenylalanine (F, Phe)); acid to acid Amino acids (aspartic acid (D, Asp), glutamic acid (E, Glu)); basic-to-basic amino acids (arginine (R, Arg), histidine (H, His), lysine (K, Lys)); charged amino acid pair charged amino acid (aspartic acid (D, Asp), glutamic acid (E, Glu), histidine (H
- a mutant or variant polypeptide may have about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80 , 90, 100 or more, or amino acid substitutions, additions, insertions or deletions within the range of any two of the aforementioned values.
- the mutant or variant may have at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, compared to the unaltered enzyme %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, or any two of the aforementioned values constitute the range of activity.
- Enzyme activity can be determined by conventional techniques known in the art, such as colorimetric enzymatic assays.
- one or more coding nucleotides (i.e. codons) in a nucleotide sequence encoding a polypeptide such as an enzyme can be replaced by another codon that is better expressed in the host. Improved expression of heterologous nucleic acids in the host (ie codon optimization). One reason for this effect is due to the fact that different organisms display preferences for different codons.
- a nucleotide sequence encoding a polypeptide, such as an enzyme, disclosed herein is modified or optimized such that the resulting nucleotide sequence reflects the codon bias for a particular host.
- a nucleotide sequence encoding a polypeptide, such as an enzyme is modified or optimized for E. coli codon bias. See eg Gouy M, Gautier C. Codon usage in bacteria: correlation with gene expressivity [J]. Nucleic acids research, 1982, 10(22): 7055-7074.; Eyre-Walker A. Synonymous codon bias is related to gene length in Escherichia coli: selection for translational accuracy[J]. Molecular biology and evolution, 1996,13(6):864-872.; Nakamura Y, Gojobori T, Ikemura T. Codon usage tabulated from international DNA sequence databases: status for the year 2000[J]. Nucleic acids research, 2000, 28(1): 292-292.
- Some embodiments of the present invention relate to expression constructs comprising one or more nucleotide sequences encoding OLS and/or OAC, such as vectors, such as plasmids, preferably comprising one or more nucleotide sequences encoding OLS and OAC Expression constructs for sequences.
- the nucleotide sequence encoding OLS or OAC is as described above.
- said expression construct is a plasmid.
- the expression construct may be used to express OLS and/or OAC, more preferably co-express both OLS and OAC, in E. coli, preferably E. coli BW25113.
- Some embodiments of the invention relate to expression constructs, such as vectors, such as plasmids, comprising a nucleotide sequence encoding fadD.
- the nucleotide sequence encoding fadD is as described above.
- said expression construct is a plasmid.
- the expression construct can be used to express fadD in E. coli, preferably E. coli BW25113.
- the host microorganism itself expresses fadD, so an expression construct encoding fadD is introduced into the host microorganism to overexpress fadD.
- Engineering a microorganism can include expressing an enzyme of interest in the microorganism.
- an expression construct described herein is introduced into a host microorganism by transformation to express an enzyme of interest.
- the conversion can be performed by methods known in the art.
- a plasmid comprising a nucleotide sequence encoding fadD described herein can be introduced into E. coli by transformation to overexpress fadD. Transformation can be, but is not limited to, Agrobacterium-mediated transformation, electroporation with plasmid DNA, DNA uptake, biolistic transformation, virus-mediated transformation, or protoplast transformation. Transformation may be any other transformation method suitable for a particular host.
- Expressing the enzyme of interest in the host microorganism to achieve the desired purpose can be achieved by transforming an expression construct encoding the enzyme into the host microorganism as described above, or by converting the enzyme encoding the enzyme into the host microorganism in a variety of ways.
- the expression construct of the enzyme is integrated into the genome sequence of the host microorganism, and it can also be achieved by enhancing the transcription and/or expression of the original enzyme-encoding gene in the host microorganism such as the fadD encoding gene in various ways. This is achieved, for example, by using stronger regulatory elements such as promoters. Such methods are generally well known to those skilled in the art.
- the plasmid used herein is shown in SEQ ID NO: 1.
- the plasmid used herein is shown in SEQ ID NO: 12.
- the plasmid used herein is shown in SEQ ID NO: 15.
- Engineering a microorganism can include interfering with the function of a protein of interest in the microorganism, e.g., reducing or eliminating expression of the protein, which can be achieved, for example, by deleting a genomic sequence of interest in the microorganism.
- the genomic fabH gene in the microorganisms described herein is deleted such that 3-oxoacyl-[acyl carrier protein] synthase 3 (3-oxoacyl-[acyl carrier protein] synthase 3) is not present in the described expressed in microorganisms.
- the amino acid sequence of 3-oxoacyl-[acyl carrier protein] synthetase 3 is shown in NCBI ACCESSION NO: NP_415609 (https://www.ncbi.nlm.nih.gov/protein/16129054) .
- the genomic fadE gene in a microorganism described herein is deleted such that acyl-CoA dehydrogenase is not expressed in the microorganism.
- the amino acid sequence of an acyl-CoA dehydrogenase is set forth in NCBI ACCESSION NO: NP_414756 (https://www.ncbi.nlm.nih.gov/protein/90111100).
- both the genomic fabH and fadE genes in the microorganisms described herein are deleted such that both 3-oxoacyl-[acyl carrier protein] synthetase 3 and acyl-CoA dehydrogenase are absent from the expressed in microorganisms.
- the fabH gene sequence that is deleted is as shown in SEQ ID NO: 8, or the deletion of SEQ ID NO: 8 can make it have the same as 3-oxoacyl-[acyl carrier protein] synthetase 3 Or a variant sequence in which a protein of similar function is not expressed.
- the deleted fadE gene sequence is shown in SEQ ID NO: 11, or the deletion of SEQ ID NO: 11 can make the protein with the same or similar function as acyl-CoA dehydrogenase not be expressed variant sequence.
- genomic sequences of interest in microorganisms can be performed by methods known in the art.
- the genome sequence can be deleted by designing an artificial sequence for ⁇ -Red homologous recombination with respect to the genome sequence, and integrating the artificial sequence into a target position in the genome by using ⁇ -Red homologous recombination.
- the artificial sequence used to delete the fabH gene is shown in SEQ ID NO:6.
- the partial sequence upstream and downstream of the original fabH gene site in the genome sequence of Escherichia coli in which the fabH gene is deleted, such as BW25113, is SEQ ID NO: 7.
- the artificial sequence used to delete the fadE gene is shown in SEQ ID NO:9.
- the partial sequence upstream and downstream of the original fadE gene site in the Escherichia coli lacking the fadE gene, such as BW25113 genome sequence is SEQ ID NO: 10.
- the function of the protein of interest can also be intervened by other methods known in the art, including, but not limited to, interfering with the transcription of the genomic sequence encoding the protein of interest, Interfering with the expression of mRNA encoding a protein of interest, intervening in the delivery of a protein of interest, for example to extracellular delivery; more specifically, including, but not limited to, making the genomic sequence encoding the protein of interest or its regulatory elements such as promoters
- Suitable media for host culture may include standard media (eg, Luria-Bertani broth, optionally supplemented with one or more other agents, such as inducers; standard yeast media; etc.).
- the medium can be supplemented with fermentable sugars (eg, hexoses, eg, glucose, xylose, etc.).
- a suitable medium comprises an inducing agent.
- the inducing agent comprises rhamnose.
- Carbon sources in suitable media for host culture can vary from simple sugars such as glucose to more complex hydrolysates of other biomass such as yeast extracts.
- the addition of salt often provides essential elements such as magnesium, nitrogen, phosphorus, and sulfur to allow cells to synthesize polypeptides and nucleic acids.
- Suitable media may also be supplemented with selective agents, such as antibiotics, to selectively maintain certain plasmids and the like. For example, if a microbe is resistant to an antibiotic, such as ampicillin, tetracycline, or kanamycin, that antibiotic can be added to the culture medium to prevent the growth of cells that lack the resistance.
- Appropriate media can be supplemented with other compounds as needed to select for desired physiological or biochemical properties, such as specific amino acids, etc.
- Exemplary techniques can be found in WO2009/076676; US12/335,071 (US2009/0203102); WO2010/003007; US2010/0048964; WO2009/132220; of methods for general bacteriology[J].1981.; Crueger W, Crueger A, Brock T D, et al.Biotechnology: a textbook of industrial microbiology[J].1990., which describes the standard culture conditions and fermentation that can be used Mode, such as batch, fed-batch or continuous fermentation, the entire contents of which are incorporated herein by reference in their entirety.
- engineered microorganisms can be grown in batches, for example, on a scale of about 100 mL, 500 mL, 1 L, 5 L, or 10 L, fermented, and induced to express desired nucleotide sequences, such as encoding OLS, OAC, and/or fadD nucleotide sequences, and/or synthesize desired fermentation products, such as olivetol and/or olivetolic acid.
- desired nucleotide sequences such as encoding OLS, OAC, and/or fadD nucleotide sequences
- desired fermentation products such as olivetol and/or olivetolic acid.
- engineered microorganisms can be grown in batches of about 10L, 100L, 1000L, 10,000L, 100,000L or larger, fermented, and induced to express desired nucleotide sequences, such as encoding OLS, OAC and/or the nucleotide sequence of fadD, and/or synthesize desired fermentation products, such as olivetol and/or olivetolic acid.
- Analysis of the fermentation product may be performed by chromatographically, preferably HPLC, separation of the fermentation product of interest to determine the concentration at one or more times during the cultivation.
- Microbial cultures and fermentation products can also be detected photometrically (absorption, fluorescence).
- the engineered microorganisms described herein achieve improved production of olivetol and/or olivetolic acid.
- the engineered microorganisms described herein achieve at least about 1, 2, 3, 4 , 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150 . Higher yield.
- the engineered microorganisms described herein achieve at least about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2 ,1.4,1.6,1.8,2.0,2.2,2.4,2.6,2.8,3.0,3.2,3.4,3.6,3.8,4.0,4.2,4.4,4.6,4.8,5.0,5.2,5.4,5.6,5.8,6.0,6.2 , 6.4, 6.6, 6.8, 7.0, 7.2, 7.4, 7.6, 7.8, 8.0, 8.2, 8.4, 8.6, 8.8, 9.0, 9.2, 9.4, 9.6, 9.8, 10, 11, 12, 13, 14, 15, 16 , 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 ,42,43,44,45,46,47,48,49,50,55,60,65,70,75,80,85,90,95,100,150,200,250,300,350,400 , 450, 500, 550,
- Item 1 An engineered Escherichia coli, wherein the engineered Escherichia coli is modified to express olivetol synthase (OLS), wherein the genome fabH gene of the engineered Escherichia coli is deleted.
- OLS olivetol synthase
- Item 2 The engineered E. coli according to any one of the preceding items, wherein the engineered E. coli is modified to express olivet acid cyclase (OAC).
- OAC olivet acid cyclase
- Item 3 The engineered Escherichia coli according to any one of the preceding items, wherein the genome fadE gene of the engineered Escherichia coli is deleted.
- Item 4 The engineered Escherichia coli according to any one of the preceding items, wherein the engineered Escherichia coli is modified to overexpress long-chain acyl-CoA synthetase (fadD).
- Item 5 The engineered Escherichia coli according to any one of the preceding items, wherein the engineered Escherichia coli is modified from BW25113.
- Item 6 The engineered Escherichia coli according to any one of the preceding items, wherein the modification is performed by introducing a plasmid.
- Item 7 The engineered Escherichia coli according to any one of the preceding items, wherein the OLS and OAC are co-expressed by the same plasmid.
- Item 8 The engineered Escherichia coli according to any one of the preceding items, wherein the fadD is overexpressed by a plasmid.
- Item 9 A method for preparing engineered Escherichia coli, comprising modifying Escherichia coli to express olivetol synthase (OLS), and deleting the fabH gene of the Escherichia coli genome.
- OLS olivetol synthase
- Item 10 The method according to any one of the preceding items, comprising modifying the E. coli to express olivate cyclase (OAC).
- OAC olivate cyclase
- Item 11 The method according to any one of the preceding items, comprising deleting the genomic fadE gene of the Escherichia coli.
- Item 12 The method according to any one of the preceding items, comprising modifying the E. coli to overexpress long-chain acyl-CoA synthetase (fadD).
- Item 13 The method according to any one of the preceding items, wherein the engineered E. coli is modified from BW25113.
- Item 14 The method according to any one of the preceding items, wherein said modification is performed by introducing a plasmid.
- Item 15 The method according to any one of the preceding items, wherein the OLS and OAC are co-expressed by the same plasmid.
- Item 16 The method according to any one of the preceding items, wherein said fadD is overexpressed by a plasmid.
- the enzyme reagents used were purchased from ThermoFisher Company and New England Biolabs (NEB) Company, the small molecule standard used was purchased from Sigma Company, the kit used to extract the plasmid was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd., and the reagents for recovering DNA fragments
- the box was purchased from Omega Corporation in the United States, and the corresponding operation steps were strictly followed the product instructions. All media were prepared with deionized water unless otherwise specified.
- the yeast extract and peptone in the media were purchased from OXID Company in the UK, and other reagents were purchased from Sinopharm Chemical Reagent Company. Gene synthesis service is provided by BGI Institute.
- E. coli media that can be used here:
- LB medium 5g/L yeast extract, 10g/L peptone, 10g/L NaCl. Adjust the pH value to 7.0-7.2, and autoclave for 30 minutes.
- ⁇ SOB medium 5g/L yeast extract, 20g/L peptone, 0.5g/L NaCl, 2.5mL of 1M KCl. Adjust the pH value to 7.0-7.2, and sterilize with high pressure steam.
- ⁇ ZY medium peptone 10g/L, yeast extract 5g/L, dissolved in distilled water, adjust the pH to 7.0. Autoclave for 30 minutes.
- ⁇ 50 ⁇ 5052 25% glycerol, 2.5% glucose, autoclaved for 30 minutes.
- ⁇ 1000 ⁇ trace elements 50mmol/L FeCl 3 , 20mmol/L CaCl 2 , 10mmol/L MnCl 2 , 10mmol/L ZnSO 4 , CoCl 2 , NiCl 2 , Na 2 MO 4 , Na 2 SeO 3 , H 3 BO 3 Each 2mmol/L.
- ⁇ ZYM medium add 2 mL 50 ⁇ 5052, 2 mL 50 ⁇ M, 200 ⁇ L 1M MgSO 4 , 100 ⁇ L 1000 ⁇ trace elements to ZY medium.
- the fabH gene in the genome was deleted to reduce the flow of intracellular malonyl-CoA to the bypass metabolism, thereby increasing the accumulation of intracellular malonyl-CoA to further increase the synthesis of the target product OA.
- the electric shock cup into the electric shock instrument and give an electric shock.
- the electric shock conditions are 200 ⁇ , 25 ⁇ F, and 2.5KV;
- the OLS&OAC expression plasmid P15A-Prha-OLS-OAC was introduced into the obtained BW25113 lacking the fabH gene as described in Example 1 to obtain an engineered E. coli strain CZ-2 expressing both OLS and OAC therein and lacking fabH .
- the partial sequence upstream and downstream of the original fabH gene site in the BW25113 genome sequence in which the fabH gene is deleted is SEQ ID NO: 7.
- the fadE gene in the genome of Escherichia coli BW25113 was deleted to reduce the flow of intracellular hexanoyl-CoA to the bypass metabolism, thereby increasing the accumulation of intracellular hexanoyl-CoA to further increase the synthesis of the target product OA.
- the OLS&OAC expression plasmid P15A-Prha-OLS-OAC was introduced as described in Example 1 into the obtained BW25113 lacking the fadE gene to obtain an engineered E. coli strain CZ-3 expressing both OLS and OAC therein and lacking fadE .
- the partial sequence upstream and downstream of the original fadE gene site in the BW25113 genome sequence in which the fadE gene is deleted is SEQ ID NO: 10.
- both the fabH and fadE genes in the genome were deleted to reduce the influx of intracellular malonyl-CoA and hexanoyl-CoA to the branch metabolism, thereby increasing intracellular malonyl-CoA and hexanoyl-CoA Accumulation of both A to further increase the synthesis of the target product OA.
- Both the fabH and fadE genes were deleted in the genome of BW25113 as described in Examples 2 and 3.
- the OLS&OAC expression plasmid P15A-Prha-OLS-OAC was introduced as described in Example 1 to obtain expression therein of both OLS and OAC, and deletion of both fabH and fadE genes
- the long-chain acyl-CoA synthetase (fadD) was overexpressed in the engineered BW25113 to convert hexanoate to hexanoyl-CoA, thereby increasing the accumulation of intracellular hexanoyl-CoA to further increase the synthesis of the target product OA.
- fadD long-chain acyl-CoA synthetase expression plasmid pL-Prha-fadD shown in SEQ ID NO: 12, transform it into CZ-4 to obtain both OLS and OAC expressed therein, and pass
- the engineered Escherichia coli strain CZ-5 which expresses fadD and deletes both fabH and fadE genes, utilizes intracellular background levels of malonyl-CoA and further increased hexanoyl-CoA as substrates to synthesize OA.
- engineered strains were made in the same manner as in Example 5, except that the OLS&OAC expression plasmid p15A-Prha-OLS-OAC was replaced by the OLS expression plasmid p15A-Prha-OLS shown in SEQ ID NO:15 , to obtain an engineered E. coli strain CZ-6 in which OLS was expressed, fadD was overexpressed, and both fabH and fadE genes were deleted to synthesize OL.
- Embodiment 7 Performance test of engineered bacterial strain CZ1-6
- the OL production in the fermentation broth extract was analyzed using the HPLC detection method described above.
- the OA production in the fermentation broth extract was analyzed by the HPLC detection method described above.
- the OA production in the fermentation broth extract was calculated according to the peak area.
- the results show that the highest yield of OA is 224.64mg/L, which is much higher than that of the existing technology (references: Tan Z, Clomburg J M, Gonzalez R.Synthetic pathway for the production of olivetolic acid in Escherichia coli[J].ACS synthetic biology , 2018,7(8):1886-1896.) achieved yield (only 80mg/L).
- CZ-6 is CZ-5 without OAC expression.
- Corresponding versions of the strains used herein to produce OA without expression of OAC i.e., producing OL instead of OA
- were also obtained relative to the corresponding versions of the OA producing control strains herein without expression of OAC i.e., producing OL instead of OA
- beneficial OL yield e.g., OL yield of OAC
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Abstract
提供了用于生产橄榄醇和橄榄醇酸的工程化大肠杆菌,是将大肠杆菌fabH基因进行缺失并表达橄榄醇合成酶,从而改善了橄榄醇和橄榄醇酸的收率。
Description
本发明涉及微生物技术领域,具体涉及用于生产橄榄醇(olivetol,OL)和橄榄醇酸(olivetolic acid,OA)的工程化微生物。
大麻素(cannabinoids)是指来源于植物大麻(Cannabis sativa)的一大类化学分子,种类超过150种。目前国际上使用较多的大麻素包括大麻二酚(cannabidiol,CBD),四氢大麻酚(tetrahydrocannabinol,THC)和大麻萜酚(cannabigerol,CBG)。CBD是植物大麻中的主要化学成分之一,是大麻素中的非成瘾性成分。THC是大麻中的主要精神活性物质,可成瘾,是在世界各国被严格管制的物质。CBG因其在植物大麻中含量较低,通常被归类为微量大麻素或稀有大麻素,因其为其它大麻素的共同前体,被称为是“大麻素之母”。随着植物大麻的生长,大部分CBG转化为CBD和THC,只有微量的CBG残留在植物体内,供应严重受限,使得CBG应用的发展受到了较大的限制。
橄榄醇和橄榄醇酸是来源于大麻属(Cannabis)植物的III型聚酮类化合物,具有抗菌,抗肿瘤和抗紫外线等活性,同时也是大麻素的关键生物合成前体。很少有报道从植物天然提取橄榄醇和橄榄醇酸。而且植物种植受诸多因素限制,例如,相比于生物合成更难获得高纯度产品,种植管制严格,产能有限,需要高昂的植物种植和下游提取的投入,生产稳定性较低。橄榄醇和橄榄醇酸的化学合成成本高昂,并且存在环境污染和条件苛刻等问题。而对于橄榄醇和橄榄醇酸的生物合成,根据发明人所知,以大肠杆菌为底盘菌来生物合成橄榄醇和橄榄醇酸的产量尚不足以达到产业化的要求。Tan Z 等(Tan Z,Clomburg J M,Gonzalez R.Synthetic pathway for the production of olivetolic acid in Escherichia coli[J].ACS synthetic biology,2018,7(8):1886-1896.)报道了在500mL生物反应器中橄榄醇酸的产量仅为80mg/L。2020年,专利号为WO2020176547A1的专利报道了,通过筛选来源于Cymbidium hybrid cultivar的橄榄醇合酶,测试发现橄榄醇的产量为40.7mg/L。
本领域仍然需要以高收率来生物合成橄榄醇和橄榄醇酸的新型微生物。
发明内容
本发明通过提供以高收率来生物合成橄榄醇和橄榄醇酸的新型工程化微生物而满足了本领域的上述需要。根据本发明的工程化微生物能够在小规模培养下产生高浓度的橄榄醇和橄榄醇酸,而且由于小规模培养时发酵液的细胞浓度(OD600)远低于工业化培养时发酵液的细胞浓度。工业化培养时细胞浓度约为小规模培养时的十倍以上,因此产量也会为十倍以上,可以预期本发明的工程化微生物具有强大的橄榄醇和橄榄醇酸工业化生产潜力,有利于大麻素的工业化生物合成。
因此,在一个方面,本发明提供了一种工程化大肠杆菌,其中所述工程化大肠杆菌被修饰以表达橄榄醇合成酶(olivetolic acid synthase,OLS),其中所述工程化大肠杆菌的基因组fabH基因被缺失。
在所述方面的一些实施方式中,所述工程化大肠杆菌被修饰以表达橄榄醇酸环化酶(olivetolic acid cyclase,OAC)。
在所述方面的一些实施方式中,所述工程化大肠杆菌的基因组fadE基因被缺失。
在所述方面的一些实施方式中,所述工程化大肠杆菌被修饰以过表达长链酯酰辅酶A合成酶(fadD)。
在所述方面的一些实施方式中,所述工程化大肠杆菌是从 BW25113修饰而来。
在所述方面的一些实施方式中,所述修饰是通过引入质粒来进行。
在所述方面的一些实施方式中,所述OLS和OAC通过同一质粒来共表达。
在所述方面的一些实施方式中,所述fadD通过质粒来过表达。
在另一个方面,本方法提供了一种用于制备工程化大肠杆菌的方法,其包括修饰大肠杆菌以表达橄榄醇合成酶(OLS),且使所述大肠杆菌的基因组fabH基因缺失。
在所述方面的一些实施方式中,所述方法包括修饰所述大肠杆菌以表达橄榄醇酸环化酶(OAC)。
在所述方面的一些实施方式中,所述方法包括使所述大肠杆菌的基因组fadE基因缺失。
在所述方面的一些实施方式中,所述方法包括修饰所述大肠杆菌以过表达长链酯酰辅酶A合成酶。
在所述方面的一些实施方式中,所述工程化大肠杆菌是从BW25113修饰而来。
在所述方面的一些实施方式中,所述修饰是通过引入质粒来进行。
在所述方面的一些实施方式中,所述OLS和OAC通过同一质粒来共表达。
在所述方面的一些实施方式中,所述fadD通过质粒来过表达。
除非另外指出,否则本发明的实践将采用分子生物学(包括重组技术)、微生物学、细胞生物学、生物化学和合成生物学等等的常规技术,其在本领域技术范围内。这样的技术在文献中有充分的解释:“Molecular Cloning:A Laboratory Manual,”第二版(Sambrook等人,1989);“Oligonucleotide Synthesis”(M.J.Gait编,1984);“Animal Cell Culture”(R.I.Freshney编,1987);“Methods in Enzymology”(Academic Press,Inc.);“Current Protocols in Molecular Biology”(F.M.Ausubel等人编,1987,以及定期更新);“PCR:The Polymerase Chain Reaction,”(Mullis等人编,1994);Singleton等人,Dictionary of Microbiology and Molecular Biology第二版,J.Wiley&Sons(New York,N.Y.1994)和March’s Advanced Organic Chemistry Reactions,Mechanisms and Structure第四版,John Wiley&Sons(New York,N.Y.1992),为本领域技术人员提供了本申请中使用的许多术语的通用指南。
除非另有定义,否则本文使用的所有技术和科学术语具有与本发明所属领域普通技术人员通常理解的相同的含义。为了本发明的目的,下文定义了下述术语。
冠词"一个/一种(a/an)"和"该/所述(the)"在本文中用于指一个/一种或超过一个/一种(即至少一个/一种)所述冠词的语法对象。例如,"要素"意指一个/一种要素或超过一个/一种要素。
替代(例如"或")的使用应理解为意指替代方案中任一、两者或其任何组合。
术语"和/或"应理解为意指替代方案中任一或两者。
如本文使用的,术语"约"或"大约”是指与参考数量、水平、值、数量、频率、百分比、尺度、大小、量、重量或长度相比较,改变多达15%、10%、9%、8%、7%、6%、5%、4%、3%、2%或1%的数量、水平、值、数量、频率、百分比、尺度、大小、量、重量或长度。在一个实施方式中,术语"约"或"大约”是指围绕参考数量、水平、值、数量、频率、百分比、尺度、大小、量、重量或长度±15%、±10%、±9%、±8%、±7%、±6%、±5%、±4%、±3%、±2%或±1%的数量、水平、值、数量、频率、百分比、尺度、大小、量、重量或长度范围。
如本文使用的,术语"基本上(substantially/essentially)”是指与参考数量、水平、值、数量、频率、百分比、尺度、大小、量、重 量或长度相比较,是约90%、91%、92%、93%、94%、95%、96%、97%、98%或99%或更高的数量、水平、值、数量、频率、百分比、尺度、大小、量、重量或长度。在一个实施方式中,术语"基本上相同”是指与参考数量、水平、值、数量、频率、百分比、尺度、大小、量、重量或长度大约相同的数量、水平、值、数量、频率、百分比、尺度、大小、量、重量或长度范围。
如本文使用的,术语"基本上不含"当用于描述组合物例如细胞群或培养基时,指不含指定物质,例如95%不含、96%不含、97%不含、98%不含、99%不含指定物质的组合物,或如通过常规手段测量是无法检测的。类似含义可应用于术语"不存在",当指不存在组合物的特定物质或组分时。
在本说明书全文,除非上下文另有要求,否则术语"包含",“包括”、“含有”和“具有”应理解为暗示包括所述步骤或要素或者步骤组或要素组,但不排除任何其他步骤或要素或者步骤组或要素组。在特定实施方式中,术语"术语"包含",“包括”、“含有”和“具有”同义使用。
"由……组成"意指包括但限于在短语"由……组成"后的任何。因此,短语"由……组成”是指示所列出的要素是需要的或强制性的,并且没有其他要素是可以存在的。
"基本上由……组成"意指包括在短语"基本上由……组成"后列出的任何要素,并且限于不干扰或贡献于所列出的要素的公开内容中指定的活动或动作的其他要素。因此,短语"基本上由……组成”是指示所列出的要素是需要的或强制性的,但没有其他要素是任选的,并且取决于它们是否影响所列出的要素的活动或动作而可以存在或不存在。
在本说明书全文,提到"一个实施方式"、"一些实施方式"、"一个具体的实施方式"等类似表述意指与所述实施方式结合描述的特定特征、结构或特性被包括在本发明的至少一个实施方式中。因此,前述短语在本说明书全文的各个地方的出现不一定全部指相同实施 方式。此外,特定特征、结构或特性可以以任何合适方式在一个或多个实施方式中组合。
在公开关于本发明的特定方面(例如本发明的产品)的特征时,这样的公开也被认为适用于本发明的任何其他方面(例如本发明的方法和用途),并作出必要的修正。
本发明至少部分地基于这样的发现:通过使宿主微生物(例如大肠杆菌)中的基因组fabH编码序列缺失,表达橄榄醇合成酶(OLS)和/或橄榄醇酸环化酶(OAC)的宿主微生物可产生高浓度的橄榄醇和/或橄榄醇酸。不希望受到理论的限制,据信通过使fabH基因缺失,促进工程化微生物中的橄榄醇合成酶(OLS)和/或橄榄醇酸环化酶(OAC)利用胞内增加的水平的丙二酰辅酶A(Malonyl-CoA)和己酰辅酶A(Hexanoyl-CoA)为底物来合成橄榄醇和/或橄榄醇酸。这样,可将该工程化微生物用于按需生产橄榄醇或橄榄醇酸,由此超越了仍然依赖于复杂合成化学的繁琐高成本的现有技术。
因此,在一个方面,本发明提供了一种工程化微生物,其中所述工程化微生物被修饰以表达橄榄醇合成酶(OLS),其中所述工程化微生物的基因组fabH基因被缺失。
在一些实施方式中,被工程化来生物合成橄榄醇和橄榄醇酸的微生物是大肠杆菌(E.coli)或其亲缘菌。在一个具体实施方式中,被工程化来生物合成橄榄醇和橄榄醇酸的微生物是大肠杆菌BW25113(ATCC编号;购自美国菌种保藏中心American Type Culture Collection)。BW25113菌株来源于E.coli K-12W1485,是K-12W1485的衍生菌株,与MG1655类似,是一种经过较少改造,比较接近于“野生型”的大肠杆菌工程菌株。与WT相比在丙二酰辅酶A和己酰辅酶A的产量方面有所改善。
在一些实施方式中,本文所用的橄榄醇合成酶可以来源于大麻(Cannabis sativa)。橄榄醇合成酶可以是优化的用于在大肠杆菌中表达的变体。在一些实施方式中,橄榄醇合成酶可以包含,基本上由以下组成,或者由以下组成:由SEQ ID NO:2所示的核苷酸序列 编码的氨基酸序列或由与SEQ ID NO:2具有至少70%、71%、72%、73%、74%、75%、76%、77%、78%、79%、80%、81%、82%、83%、84%、85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%或前述数值中的任意两者构成的范围的同一性的核苷酸序列编码的氨基酸序列。在一些实施方式中,橄榄醇合成酶可以包含,基本上由以下组成,或者由以下组成:SEQ ID NO:3所示的氨基酸序列或与SEQ ID NO:3具有至少70%、71%、72%、73%、74%、75%、76%、77%、78%、79%、80%、81%、82%、83%、84%、85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%或前述数值中的任意两者构成的范围的同一性的氨基酸序列。
本文所用的示例性橄榄醇合成酶可以包括全长橄榄醇合成酶、橄榄醇合成酶的片段、橄榄醇合成酶的变体、截短的橄榄醇合成酶或具有橄榄醇合成酶的至少一种活性的融合酶。在一些实施方式中,在本发明中使用的橄榄醇合成酶具有催化从丙二酰辅酶A和己酰辅酶A合成橄榄醇的活性。
在一些实施方式中,本文所用的橄榄醇酸环化酶可以来源于大麻(Cannabis sativa)。橄榄醇酸环化酶可以是优化的用于在大肠杆菌中表达的变体。在一些实施方式中,橄榄醇酸环化酶可以包含,基本上由以下组成,或者由以下组成:由SEQ ID NO:4所示的核苷酸序列编码的氨基酸序列或由与SEQ ID NO:4具有至少70%、71%、72%、73%、74%、75%、76%、77%、78%、79%、80%、81%、82%、83%、84%、85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%或前述数值中的任意两者构成的范围的同一性的核苷酸序列编码的氨基酸序列。在一些实施方式中,橄榄醇酸环化酶可以包含,基本上由以下组成,或者由以下组成:SEQ ID NO:5所示的氨基酸序列或与SEQ ID NO:5具有至少70%、71%、72%、73%、74%、75%、76%、77%、78%、79%、80%、81%、82%、83%、84%、85%、86%、87%、88%、89%、90%、 91%、92%、93%、94%、95%、96%、97%、98%、99%或100%或前述数值中的任意两者构成的范围的同一性的氨基酸序列。
本文所用的示例性橄榄醇酸环化酶可以包括全长橄榄醇酸环化酶、橄榄醇酸环化酶的片段、橄榄醇酸环化酶的变体、截短的橄榄醇酸环化酶或具有橄榄醇酸环化酶的至少一种活性的融合酶。在一些实施方式中,在本发明中使用的橄榄醇酸环化酶具有将橄榄醇羧化成橄榄醇酸的活性。
在本发明的一些实施方式中,长链酯酰辅酶A合成酶(fadD)、脂酰辅酶A脱氢酶(fadE)和β-酮脂酰-酰基载体蛋白合酶(fabH)是大肠杆菌中的固有组分。
在一些实施方式中,本文所用的fadD可以来源于大肠杆菌。fadD可以是优化的用于在大肠杆菌中表达的变体。在一些实施方式中,fadD可以包含,基本上由以下组成,或者由以下组成:由SEQ ID NO:13所示的核苷酸序列编码的氨基酸序列或由与SEQ ID NO:13具有至少70%、71%、72%、73%、74%、75%、76%、77%、78%、79%、80%、81%、82%、83%、84%、85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%或前述数值中的任意两者构成的范围的同一性的核苷酸序列编码的氨基酸序列。在一些实施方式中,fadD可以包含,基本上由以下组成,或者由以下组成:SEQ ID NO:14所示的氨基酸序列或与SEQ ID NO:14具有至少70%、71%、72%、73%、74%、75%、76%、77%、78%、79%、80%、81%、82%、83%、84%、85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%或前述数值中的任意两者构成的范围的同一性的氨基酸序列。
本文所用的示例性fadD可以包括全长fadD、fadD的片段、fadD的变体、截短的fadD或具有fadD的至少一种活性的融合酶。在一些实施方式中,在本发明中使用的fadD具有催化从己酸通过酰化产生己酰辅酶A的活性。
在一些实施方式中,本发明所用的一种或多种酶可以是本文描 述的酶的突变体(mutant)或变体(variant)。如本文所用的,“突变体”和“变体”是指保留与原始序列的生物学活性相同或基本上相同的生物学活性的分子。该突变体或变体可以来自相同或不同的物种,或者可以是基于天然的分子或现有的分子的合成序列。在一些实施方式中,术语“突变体”和“变体”是指多肽具有的氨基酸序列与对应的野生型多肽至少相差一个氨基酸。例如,突变体和变体可以包含保守氨基酸取代:即用具有相似性质的氨基酸取代原有的对应氨基酸。保守取代可以是极性对极性氨基酸(甘氨酸(G,Gly)、丝氨酸(S,Ser)、苏氨酸(T,Thr)、酪氨酸(Y,Tyr)、半胱氨酸(C,Cys)、天冬酰胺(N,Asn)和谷氨酰胺(Q,Gln));非极性对非极性氨基酸(丙氨酸(A,Ala)、缬氨酸(V,Val)、色氨酸(W,Trp)、亮氨酸(L,Leu)、脯氨酸(P,Pro)、甲硫氨酸(Μ,Met)、苯丙氨酸(F,Phe));酸性对酸性氨基酸(天冬氨酸(D,Asp)、谷氨酸(E,Glu));碱性对碱性氨基酸(精氨酸(R,Arg)、组氨酸(H,His)、赖氨酸(K,Lys));带电荷氨基酸对带电荷氨基酸(天冬氨酸(D,Asp)、谷氨酸(E,Glu)、组氨酸(H,His)、赖氨酸(K,Lys)和精氨酸(R,Arg));和疏水对疏水性氨基酸(丙氨酸(A,Ala)、亮氨酸(ULeu)、异亮氨酸(I,Ile)、缬氨酸(V,Val)、脯氨酸(P,Pro)、苯丙氨酸(F,Phe)、色氨酸(W,Trp)和甲硫氨酸(M,Met))。在一些其他实施方式中,突变体或变体也可以包含非保守性取代。
在一些实施方式中,突变体或变体多肽可以具有约1、2、3、4、5、6、7、8、9、10、15、20、30、40、50、60、70、80、90、100个或更多个或前述数值中的任意两者构成的范围的氨基酸的置换、添加、插入或缺失。与未改变的酶相比,突变体或变体可以具有至少40%、45%、50%、55%、60%、65%、70%、75%、80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或100%或前述数值中的任意两者构成的范围的活性。酶活性可以通过本领域已知的常规技术,例如比色酶学测定来测定。
如本领域技术人员众所周知的,可以通过使编码多肽如酶的核苷酸序列中的一个或多个编码核苷酸(即密码子)被在宿主中更好表达的另一种密码子替代来改善在宿主中异源核酸的表达(即密码子优化)。产生这种效应的一种原因是由于不同的生物体显示出对不同密码子的偏好。在一些实施方式中,本文公开的编码多肽如酶的核苷酸序列被修饰或优化,使得所得核苷酸序列反映针对特定宿主的密码子偏好。例如,在一些实施方式中,编码多肽如酶的核苷酸序列被针对大肠杆菌密码子偏好进行修饰或优化。参见例如Gouy M,Gautier C.Codon usage in bacteria:correlation with gene expressivity[J].Nucleic acids research,1982,10(22):7055-7074.;Eyre-Walker A.Synonymous codon bias is related to gene length in Escherichia coli:selection for translational accuracy[J].Molecular biology and evolution,1996,13(6):864-872.;Nakamura Y,Gojobori T,Ikemura T.Codon usage tabulated from international DNA sequence databases:status for the year 2000[J].Nucleic acids research,2000,28(1):292-292.。
多核苷酸或多肽与另一个多核苷酸或多肽具有一定的“序列同一性”或“同一性”百分比,意味着当比对两条序列时,该百分比的碱基或氨基酸相同并且在相同的相对位置。确定两个氨基酸序列或两个核苷酸序列的百分比同一性可以包括比对和比较两个序列中相应位置处的氨基酸残基或核苷酸。如果两个序列中的所有位置被相同的氨基酸残基或核苷酸占据,那么所述序列被认为是100%相同的。序列同一性可以以多种不同方式确定,例如,可以使用各种方法和计算机程序(例如,BLAST、T-COFFEE、MUSCLE、MAFFT等)对序列进行比对。
本发明的一些实施方式涉及包含一个或多个编码OLS和/或OAC的核苷酸序列的表达构建体,例如载体,如质粒,优选地是包含一个或多个编码OLS和OAC的核苷酸序列的表达构建体。所述编码OLS或OAC的核苷酸序列如上所述。优选地,所述表达构建 体是质粒。优选地,所述表达构建体可以用于在大肠杆菌,优选地在大肠杆菌BW25113中表达OLS和/或OAC,更优选共表达OLS和OAC两者。
本发明的一些实施方式涉及包含编码fadD的核苷酸序列的表达构建体,例如载体,如质粒。所述编码fadD的核苷酸序列如上所述。优选地,所述表达构建体是质粒。优选地,所述表达构建体可以用于在大肠杆菌,优选地在大肠杆菌BW25113中表达fadD。在一些实施方式中,宿主微生物本身表达fadD,因此在所述宿主微生物中引入编码fadD的表达构建体以过表达fadD。
对微生物进行工程化可以包括在所述微生物中表达感兴趣的酶。在一些实施方式中,本文所述的表达构建体通过转化(transformation)而引入到宿主微生物中以表达感兴趣的酶。所述转化可以通过本领域公知的方法进行。例如,本文所述的包含编码fadD的核苷酸序列的质粒可以通过转化而引入到大肠杆菌中以过表达fadD。转化可以是,但不限于,农杆菌介导的转化、用质粒DNA的电穿孔、DNA摄取、基因枪转化、病毒介导的转化或原生质体转化。转化可以是适用于特定宿主的任意其他转化方法。
在宿主微生物中表达感兴趣的酶以实现预期目的可以如上所述通过向所述宿主微生物中转化编码所述酶的表达构建体来实现,也可以通过采用各种各样的方式将编码所述酶的表达构建体整合到所述宿主微生物的基因组序列中来实现,也可以通过采用各种各样的方式增强所述宿主微生物中原有的酶编码基因如fadD编码基因的转录和/或表达来实现,例如通过使用更强的调节元件如启动子。这样的方式大体上都是本领域技术人员熟知的。在一些实施方式中,本文所用的质粒如SEQ ID NO:1所示。在一些实施方式中,本文所用的质粒如SEQ ID NO:12所示。在一些实施方式中,本文所用的质粒如SEQ ID NO:15所示。
对微生物进行工程化可以包括干预所述微生物中的感兴趣的蛋白的功能,例如,减少或消除所述蛋白的表达,这可以例如通过在 所述微生物中使感兴趣的基因组序列缺失来实现。在一些实施方式中,本文所述的微生物中的基因组fabH基因被缺失,使得3-氧代酰基-[酰基载体蛋白]合成酶3(3-oxoacyl-[acyl carrier protein]synthase 3)不在所述微生物中被表达。在一些实施方式中,3-氧代酰基-[酰基载体蛋白]合成酶3的氨基酸序列以NCBI ACCESSION NO:NP_415609(https://www.ncbi.nlm.nih.gov/protein/16129054)示出。在一些实施方式中,本文所述的微生物中的基因组fadE基因被缺失,使得酰基-CoA脱氢酶(acyl-CoA dehydrogenase)不在所述微生物中被表达。在一些实施方式中,酰基-CoA脱氢酶的氨基酸序列以NCBI ACCESSION NO:NP_414756(https://www.ncbi.nlm.nih.gov/protein/90111100)示出。在一些实施方式中,本文所述的微生物中的基因组fabH和fadE基因两者被缺失,使得3-氧代酰基-[酰基载体蛋白]合成酶3和酰基-CoA脱氢酶两者不在所述微生物中被表达。在一些实施方式中,被缺失的fabH基因序列如SEQ ID NO:8所示,或者是SEQ ID NO:8的被缺失可以使得与3-氧代酰基-[酰基载体蛋白]合成酶3具有相同或相似功能的蛋白不被表达的变体序列。在一些实施方式中,被缺失的fadE基因序列如SEQ ID NO:11所示,或者是SEQ ID NO:11的被缺失可以使得与酰基-CoA脱氢酶具有相同或相似功能的蛋白不被表达的变体序列。
微生物中的感兴趣的基因组序列的缺失可以通过本领域已知的方法进行。例如可以通过针对所述基因组序列设计用于λ-Red同源重组的人工序列,利用λ-Red同源重组将所述人工序列整合到基因组中的目标位置来使所述基因组序列缺失。具体实验方案可以参见本文描述的实施例。在一些实施方式中,用于缺失fabH基因的人工序列如SEQ ID NO:6所示。在一些实施方式中,其中缺失fabH基因的大肠杆菌如BW25113基因组序列中在原fabH基因位点处上下游的部分序列为SEQ ID NO:7。在一些实施方式中,用于缺失fadE基因的人工序列如SEQ ID NO:9所示。在一些实施方式中,其中缺 失fadE基因的大肠杆菌如BW25113基因组序列中在原fadE基因位点处上下游的部分序列为SEQ ID NO:10。
除了使微生物中的感兴趣的基因组序列缺失以外,还可以通过本领域已知的其他方法来干预感兴趣的蛋白的功能,包括,但不限于,干预编码感兴趣的蛋白的基因组序列的转录、干预编码感兴趣的蛋白的mRNA的表达、干预感兴趣的蛋白的递送,例如向细胞外递送;更具体地,包括,但不限于,使编码感兴趣的蛋白的基因组序列或其调控元件如启动子的全部或部分缺失、在编码感兴趣的蛋白的基因组序列或其调控元件如启动子的中间插入影响其转录的一个或多个核苷酸例如终止密码子或使其一个或多个核苷酸突变至所述基因组序列无法正常转录的程度的方法、引入干预或沉默编码感兴趣的蛋白的mRNA的试剂如siRNA或dsRNAi试剂、或者抑制或停止递送感兴趣的蛋白例如至细胞外的系统(例如伴侣蛋白、信号序列、转运蛋白)的功能的方法。
用于宿主培养的合适的培养基可以包括标准培养基(例如Luria-Bertani肉汤,任选地补充一种或多种其他试剂,例如诱导剂;标准酵母培养基;等等)。在一些实施方式中,培养基可以补充有可发酵糖(例如己糖,例如葡萄糖、木糖等)。在一些实施方式中,合适的培养基包含诱导剂。在某些这样的实施方式中,诱导剂包括鼠李糖。
用于宿主培养的合适的培养基中的碳源可以不同,从简单的糖如葡萄糖到其他生物质的更复杂的水解产物,如酵母提取物。盐的添加通常提供必需的元素,例如镁、氮、磷和硫,以允许细胞合成多肽和核酸。合适的培养基还可以补充有选择性试剂,例如抗生素,以选择维持某些质粒等。例如,如果微生物对某种抗生素具有抗性,如氨苄青霉素、四环素或卡那霉素,则可以将该抗生素添加到培养基中,以阻止缺乏抗性的细胞生长。合适的培养基可以根据需要补充其他化合物以选择期望的生理或生化特性,如特定的氨基酸等。
本文例如在实施例部分中描述了适用于本发明的微生物的维持 和生长的材料和方法。适用于微生物(例如大肠杆菌)的维持和生长的其他材料和方法在本领域中是众所周知的。示例性技术可以见于WO2009/076676;US12/335,071(US2009/0203102);WO2010/003007;US2010/0048964;WO2009/132220;US2010/0003716;Gerhardt P,Murray R G E,Costilow R N,et al.Manual of methods for general bacteriology[J].1981.;Crueger W,Crueger A,Brock T D,et al.Biotechnology:a textbook of industrial microbiology[J].1990.,其中描述了可以使用的标准培养条件和发酵模式,例如分批、补料分批或连续发酵,其全部内容通过引用全文并入本文。
对于小规模生产,可以使工程化微生物以例如约100mL、500mL、1L、5L或10L的规模进行批量生长,发酵,并诱导其表达期望的核苷酸序列,如编码OLS、OAC和/或fadD的核苷酸序列,和/或合成期望的发酵产物,如橄榄醇和/或橄榄醇酸。对于大规模生产,可以使工程化微生物以约10L、100L、1000L、10,000L、100,000L或更大的规模进行批量生长,发酵,并诱导其表达期望的核苷酸序列,如编码OLS、OAC和/或fadD的核苷酸序列,和/或合成期望的发酵产物,如橄榄醇和/或橄榄醇酸。
可以通过色谱法优选HPLC分离感兴趣的发酵产物来进行发酵产物的分析,以确定培养过程中的一个或多个时间处的浓度。也可以通过光度测定方式(吸收、荧光)检测微生物培养物和发酵产物。
本文所述的工程化微生物实现了改善的橄榄醇和/或橄榄醇酸的产量。在一些实施方式中,本文所述的工程化微生物与适当的未工程化或部分工程化的微生物对照相比,在橄榄醇和/或橄榄醇酸生产方面实现了至少约1、2、3、4、5、6、7、8、9、10、15、20、25、30、35、40、45、50、55、60、65、70、75、80、85、90、95、100、150、200、250、300、350、400、450、500、550、600、650、700、750、800、850、900、950或1000倍或更高或前述数值中的任意两者构成的范围的更高产量。在一些实施方式中,本文所述的工程化 微生物在橄榄醇和/或橄榄醇酸生产方面实现了至少约0.1、0.2、0.3、0.4、0.5、0.6、0.7、0.8、0.9、1.0、1.1、1.2、1.4、1.6、1.8、2.0、2.2、2.4、2.6、2.8、3.0、3.2、3.4、3.6、3.8、4.0、4.2、4.4、4.6、4.8、5.0、5.2、5.4、5.6、5.8、6.0、6.2、6.4、6.6、6.8、7.0、7.2、7.4、7.6、7.8、8.0、8.2、8.4、8.6、8.8、9.0、9.2、9.4、9.6、9.8、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、55、60、65、70、75、80、85、90、95、100、150、200、250、300、350、400、450、500、550、600、650、700、750、800、850、900、950、1000mg/L或更高或前述数值中的任意两者构成的范围的产量。
本发明还提供了以下一些优选实施方式:
项目1.一种工程化大肠杆菌,其中所述工程化大肠杆菌被修饰以表达橄榄醇合成酶(OLS),其中所述工程化大肠杆菌的基因组fabH基因被缺失。
项目2.根据前述项目中任一项所述的工程化大肠杆菌,其中所述工程化大肠杆菌被修饰以表达橄榄醇酸环化酶(OAC)。
项目3.根据前述项目中任一项所述的工程化大肠杆菌,其中所述工程化大肠杆菌的基因组fadE基因被缺失。
项目4.根据前述项目中任一项所述的工程化大肠杆菌,其中所述工程化大肠杆菌被修饰以过表达长链酯酰辅酶A合成酶(fadD)。
项目5.根据前述项目中任一项所述的工程化大肠杆菌,其中所述工程化大肠杆菌是从BW25113修饰而来。
项目6.根据前述项目中任一项所述的工程化大肠杆菌,其中所述修饰是通过引入质粒来进行。
项目7.根据前述项目中任一项所述的工程化大肠杆菌,其中所述OLS和OAC通过同一质粒来共表达。
项目8.根据前述项目中任一项所述的工程化大肠杆菌,其中 所述fadD通过质粒来过表达。
项目9.一种用于制备工程化大肠杆菌的方法,其包括修饰大肠杆菌以表达橄榄醇合成酶(OLS),且使所述大肠杆菌的基因组fabH基因缺失。
项目10.根据前述项目中任一项所述的方法,其包括修饰所述大肠杆菌以表达橄榄醇酸环化酶(OAC)。
项目11.根据前述项目中任一项所述的方法,其包括使所述大肠杆菌的基因组fadE基因缺失。
项目12.根据前述项目中任一项所述的方法,其包括修饰所述大肠杆菌以过表达长链酯酰辅酶A合成酶(fadD)。
项目13.根据前述项目中任一项所述的方法,其中所述工程化大肠杆菌是从BW25113修饰而来。
项目14.根据前述项目中任一项所述的方法,其中所述修饰是通过引入质粒来进行。
项目15.根据前述项目中任一项所述的方法,其中所述OLS和OAC通过同一质粒来共表达。
项目16.根据前述项目中任一项所述的方法,其中所述fadD通过质粒来过表达。
实施例
在下文中,将通过实施例详细描述本发明。然而,在此提供的实施例仅用于说明目的,并不用于限制本发明。
下述实施例所用的实验方法如无特殊说明,均为常规方法。
下述实施例所用的材料、试剂等,如无特殊说明,均可从商业途径得到。
所用酶试剂采购自ThermoFisher公司和New England Biolabs(NEB)公司,所用的小分子标准品采购自Sigma公司,提取质粒所用的试剂盒购自天根生化科技(北京)有限公司,回收DNA片段的试剂盒购自美国Omega公司,相应的操作步骤严格按照产品说明 书进行。所有培养基如无特殊说明均用去离子水配制,培养基中酵母提取物和蛋白胨购自英国OXID公司,其它试剂购自国药集团化学试剂公司。基因合成服务由华大基因研究院提供。
本文中可使用的大肠杆菌培养基:
·LB培养基:5g/L酵母提取物,10g/L蛋白胨,10g/L NaCl。调pH值至7.0-7.2,高压蒸汽灭菌30分钟。
·SOB培养基:5g/L酵母提取物,20g/L蛋白胨,0.5g/L NaCl,浓度为1M的KCl 2.5mL。调pH值至7.0-7.2,高压蒸汽灭菌。
·ZY培养基:蛋白胨10g/L,酵母提取物5g/L,蒸馏水溶解后,调pH至7.0。高压蒸汽灭菌30分钟。
·50×M:1.25mol/L Na
2HPO
4,1.25mol/L KH
2PO
4,2.5mol/L NH
4Cl,0.25mol/L Na
2SO
4,高压蒸汽灭菌30分钟。
·50×5052:25%甘油,2.5%葡萄糖,高压蒸汽灭菌30分钟。
·1M MgSO
4:称取24.6g MgSO
4·7H
2O加H
2O溶解,定容至100mL,然后高压蒸汽灭菌30分钟。
·1000×微量元素:50mmol/L FeCl
3,20mmol/L CaCl
2,10mmol/L MnCl
2,10mmol/L ZnSO
4,CoCl
2,NiCl
2,Na
2MO
4,Na
2SeO
3,H
3BO
3各2mmol/L。
·ZYM培养基:在ZY培养基中加入2mL 50×5052,2mL 50×M,200μL 1M的MgSO
4,100μL 1000×微量元素。
实施例1:导入OLS&OAC表达质粒以获得CZ-1
设计合成SEQ ID NO:1所示的OLS&OAC表达质粒p15A-Prha-OLS-OAC,将其通过大肠杆菌化学转化的方式转到作为底盘菌的大肠杆菌BW25113中以获得在其中表达OLS和OAC两者的工程化大肠杆菌菌株CZ-1,从而利用胞内本底水平的丙二酰辅酶A和己酰辅酶A为底物来合成OA。
实施例2:缺失fabH基因以获得CZ-2
在大肠杆菌BW25113中使其基因组中的fabH基因缺失,以减少胞内丙二酰辅酶A流向支路代谢,从而提高胞内丙二酰辅酶A的积累以进一步增加目标产物OA的合成。
设计合成SEQ ID NO:6所示的H1-kana-H2,根据文献(Datsenko K A,Wanner B L.One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products[J].Proceedings of the National Academy of Sciences,2000,97(12):6640-6645.)提供的方法通过λ-Red同源重组将SEQ ID NO:6整合至BW25113的基因组的fabH基因位置处以使fabH基因缺失,步骤如下:
1.制备BW25113的感受态;
2.导入质粒pKD46;
3.将BW25113(pKD46)接种到3mL含有氨苄青霉素的LB,氨苄青霉素的浓度为100μg/L,30℃摇床过夜培养;
4.取过夜培养的BW25113(pKD46)细胞悬液100μL,加入到10mL SOB培养基中,再加入浓度为1M的阿拉伯糖100μL和10μL浓度为100mg/L的氨苄青霉素。30℃摇床培养至OD600=0.4-0.6;
5. 4℃条件下,离心收集菌体,并用10mL预冷的超纯水重悬菌体,同样的方法再洗两次菌体,最后用50μL浓度为10%的甘油溶液重悬菌体;
6.加入50ng的SEQ ID NO:6核苷酸序列片段,混匀后将混合液加入电击杯;
7.将电击杯放进电击仪,进行一次电击,电击条件为200Ω、25μF、2.5KV;
8.加入1mL预冷的SOB培养基,混合液转入无菌的EP管中,30℃摇床培养1小时;
9.将菌液均匀涂布于含有氨苄青霉素和卡那霉素的LB平板培养基上,30℃静置培养16-20小时;
10.验证平板上长出的转化子。
在整合SEQ ID NO:6后,根据文献(Datsenko K A,Wanner B L., 同上)提供的方法使KanR抗性基因缺失,步骤如下:
1.制备整合有SEQ ID NO:6的菌株的感受态;
2.转化pCP20进入感受态,培养温度为30度;
3.挑取单克隆至3mL SOB培养基,30度过夜培养;
4.取100μL转接至10mL SOB,30度摇床培养3-4小时,OD600=0.4;
5. 4度条件下离心收集菌体,用冰浴的无菌水洗菌体两次;
6.重悬于50μL-100μL无菌水中,用于电击转化;
7.加入100ng的pCP20,电击转化(电击仪设置:1.8KV,5.5ms),30度复苏1小时;
8.均匀涂布于含有氨苄青霉素的LB平板上,30度培养2-3天;
9.挑取单克隆在没有抗生素的LB平板上划线,42度培养过夜;
10.挑20-30个克隆分别至卡那霉素抗性平板和无抗平板,在卡那霉素平板上不能生长而同时在无抗平板上生长的克隆为目标克隆,进行PCR验证。
在所得到的缺失fabH基因的BW25113中如实施例1所述导入OLS&OAC表达质粒P15A-Prha-OLS-OAC以获得在其中表达OLS和OAC两者、且缺失fabH的工程化大肠杆菌菌株CZ-2。其中缺失fabH基因的BW25113基因组序列中在原fabH基因位点处上下游的部分序列为SEQ ID NO:7。
实施例3:缺失fadE基因以获得CZ-3
在大肠杆菌BW25113中使其基因组中的fadE基因缺失,以减少胞内己酰辅酶A流向支路代谢,从而提高胞内己酰辅酶A的积累以进一步增加目标产物OA的合成。
设计合成SEQ ID NO:9所述的H3-kana-H4,如实施例2所述将SEQ ID NO:9整合至BW25113的基因组的fadE基因位置处以使fadE基因缺失和在整合SEQ ID NO:4后使KanR抗性基因缺失。
在所得到的缺失fadE基因的BW25113中如实施例1所述导入 OLS&OAC表达质粒P15A-Prha-OLS-OAC以获得在其中表达OLS和OAC两者、且缺失fadE的工程化大肠杆菌菌株CZ-3。其中缺失fadE基因的BW25113基因组序列中在原fadE基因位点处上下游的部分序列为SEQ ID NO:10。
实施例4:缺失fabH基因和fadE基因两者以获得CZ-4
在大肠杆菌BW25113中使其基因组中的fabH和fadE基因两者缺失,以减少胞内丙二酰辅酶A和己酰辅酶A两者流向支路代谢,从而提高胞内丙二酰辅酶A和己酰辅酶A两者的积累以进一步增加目标产物OA的合成。
如实施例2和3所述使BW25113的基因组中的fabH和fadE基因两者缺失。
在所得到的缺失fabH和fadE基因两者的BW25113中如实施例1所述导入OLS&OAC表达质粒P15A-Prha-OLS-OAC以获得在其中表达OLS和OAC两者、且缺失fabH和fadE基因两者的工程化大肠杆菌菌株CZ-4。
实施例5:导入fadD表达质粒以获得CZ-5
在工程化BW25113中过表达长链酯酰辅酶A合成酶(fadD)以将己酸转化为己酰辅酶A,从而增加胞内己酰辅酶A积累,以进一步增加目标产物OA的合成。
设计合成SEQ ID NO:12所示的fadD(长链酯酰辅酶A合成酶)表达质粒pL-Prha-fadD,将其转化到CZ-4中以获得在其中表达OLS和OAC两者、且过表达fadD、且缺失fabH和fadE基因两者的工程化大肠杆菌菌株CZ-5,从而利用胞内本底水平的丙二酰辅酶A和进一步增加的己酰辅酶A为底物来合成OA。
实施例6:取消OAC以获得CZ-6
为了制造OL而非OA,以与实施例5相同的方式制造工程化菌 株,除了其中OLS&OAC表达质粒p15A-Prha-OLS-OAC替换为SEQ ID NO:15所示的OLS表达质粒p15A-Prha-OLS,以获得在其中表达OLS、且过表达fadD、且缺失fabH和fadE基因两者的工程化大肠杆菌菌株CZ-6来合成OL。
实施例7:工程化菌株CZ1-6的性能测试
实施例1-6中获得的工程化菌株CZ-1、CZ-2、CZ-3、CA-4、CZ-5和CZ-6按照下文所示程序一式三份进行测试。
发酵和样品制备:
1.重组菌接种至3mL LB液体培养基中,37度,220转/分钟,过夜培养,约14小时,终OD600值达到2-3;
2.在24深孔板中加入ZYM培养基,每个孔加入2mL;
3.将步骤1中菌液转接至步骤2中的ZYM培养基,转接后OD600为0.01;
4.待菌液OD600生长至0.2,加入诱导剂(添加量:鼠李糖0.2%)和前体己酸(1mM),从接种至发酵结束总共24小时;
5.取1mL发酵液,加入3mL乙酸乙酯,震荡混匀10min,后离心收集上层有机相;
6.重复操作5,两次离心获得有机相合并在一起约6mL转移至10mL试管中。
7.利用真空浓缩仪将试管中的有机相全部蒸干,加入1mL甲醇重悬试管中所有样品。
8.用0.22μM滤器过滤第7步的样品,然后转移至HPLC样品瓶中。
停止发酵时发酵液中的细胞浓度:
用上述HPLC检测方法分析发酵液萃取物中的OL产量。
用上述HPLC检测方法分析发酵液萃取物中的OA产量。
根据峰面积计算发酵液萃取物中的OA产量。结果显示,OA最高产量为224.64mg/L,远高于现有技术(参考文献:Tan Z,Clomburg J M,Gonzalez R.Synthetic pathway for the production of olivetolic acid in Escherichia coli[J].ACS synthetic biology,2018,7(8):1886-1896.)实现的产量(仅为80mg/L)。
CZ-1、CZ-2、CZ-3、CZ-4和CZ-5发酵液样品中OA产量
另外,CZ-6的平均OL产量为116.02mg/L。CZ-6即为没有OAC表达的CZ-5。本文中用于产生OA的菌株的没有OAC表达的对应版本(即,产生OL而非OA)也获得相对于本文的OA生产对照菌株的没有OAC表达的对应版本(即,产生OL而非OA)的有益OL产量。
虽然已经在此示出并描述了本发明的优选实施方式,但是对本领域技术人员而言应该显而易见是这样的实施方式仅以举例方式提供。在不偏离本发明的情况下众多变化、改变和取代现在将被本领域的普通技术人员想到。应该理解的是,在实践本发明中可以采用在此描述的本发明的实施方式的不同替代方案。
Claims (16)
- 一种工程化大肠杆菌,其中所述工程化大肠杆菌被修饰以表达橄榄醇合成酶,其特征在于所述工程化大肠杆菌的SEQ ID NO:8所示的基因组fabH基因被缺失。
- 根据权利要求1所述的工程化大肠杆菌,其特征在于所述工程化大肠杆菌被修饰以表达橄榄醇酸环化酶。
- 根据权利要求1或2所述的工程化大肠杆菌,其特征在于所述工程化大肠杆菌的SEQ ID NO:11所示的基因组fadE基因被缺失。
- 根据权利要求1或2所述的工程化大肠杆菌,其特征在于所述工程化大肠杆菌被修饰以过表达长链酯酰辅酶A合成酶。
- 根据权利要求1或2所述的工程化大肠杆菌,其特征在于所述工程化大肠杆菌是从BW25113修饰而来。
- 根据权利要求1或2所述的工程化大肠杆菌,其特征在于所述修饰是通过引入质粒来进行。
- 根据权利要求1或2所述的工程化大肠杆菌,其特征在于所述橄榄醇合成酶和橄榄醇酸环化酶通过同一质粒来共表达。
- 根据权利要求1或2所述的工程化大肠杆菌,其特征在于所述长链酯酰辅酶A合成酶通过质粒来过表达。
- 一种用于制备工程化大肠杆菌的方法,其包括修饰大肠杆菌以表达橄榄醇合成酶,其特征在于所述方法包括使所述大肠杆菌的SEQ ID NO:8所示的基因组fabH基因缺失。
- 根据权利要求9所述的方法,其特征在于所述方法包括修饰所述大肠杆菌以表达橄榄醇酸环化酶。
- 根据权利要求9或10所述的方法,其特征在于所述方法包括使所述大肠杆菌的SEQ ID NO:11所示的基因组fadE基因缺失。
- 根据权利要求9或10所述的方法,其特征在于所述方法包括修饰所述大肠杆菌以过表达长链酯酰辅酶A合成酶。
- 根据权利要求9或10所述的方法,其特征在于所述工程化大肠杆菌是从BW25113修饰而来。
- 根据权利要求9或10所述的方法,其特征在于所述修饰是通过引入质粒来进行。
- 根据权利要求9或10所述的方法,其特征在于所述橄榄醇合成酶和橄榄醇酸环化酶通过同一质粒来共表达。
- 根据权利要求9或10所述的方法,其特征在于所述长链酯酰辅酶A合成酶通过质粒来过表达。
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