WO2023035396A1 - Variant d'olivétol synthétase et micro-organisme génétiquement modifié exprimant celui-ci - Google Patents

Variant d'olivétol synthétase et micro-organisme génétiquement modifié exprimant celui-ci Download PDF

Info

Publication number
WO2023035396A1
WO2023035396A1 PCT/CN2021/129656 CN2021129656W WO2023035396A1 WO 2023035396 A1 WO2023035396 A1 WO 2023035396A1 CN 2021129656 W CN2021129656 W CN 2021129656W WO 2023035396 A1 WO2023035396 A1 WO 2023035396A1
Authority
WO
WIPO (PCT)
Prior art keywords
escherichia coli
engineered
seq
olivetol
coli
Prior art date
Application number
PCT/CN2021/129656
Other languages
English (en)
Chinese (zh)
Inventor
杜德尧
王高艳
张倩
宗朕
李明月
邱悦悦
尹进
张浩千
Original Assignee
北京蓝晶微生物科技有限公司
深圳蓝晶生物科技有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 北京蓝晶微生物科技有限公司, 深圳蓝晶生物科技有限公司 filed Critical 北京蓝晶微生物科技有限公司
Publication of WO2023035396A1 publication Critical patent/WO2023035396A1/fr

Links

Classifications

    • 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
    • C12N9/10—Transferases (2.)
    • C12N9/1025—Acyltransferases (2.3)
    • C12N9/1029—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
    • 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
    • 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)
    • 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.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • 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
    • C12P7/22—Preparation of oxygen-containing organic compounds containing a hydroxy group aromatic
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • 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
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C12P7/42—Hydroxy-carboxylic acids
    • 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)
    • C12Y203/01086—Fatty-acyl-CoA synthase (2.3.1.86)
    • 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)
    • 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)
    • C12Y203/01206—3,5,7-Trioxododecanoyl-CoA synthase (2.3.1.206)
    • C—CHEMISTRY; METALLURGY
    • 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)
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E50/00—Technologies for the production of fuel of non-fossil origin
    • Y02E50/10—Biofuels, e.g. bio-diesel

Definitions

  • the invention relates to the field of microorganisms and enzymes, in particular to olive alcohol synthase (OLS) variants and engineered microorganisms expressing the same for producing olive alcohol (olivetol, OL) and olivetolic acid (olivetolic acid, OA).
  • OLS olive alcohol synthase
  • 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 olivetol synthase (OLS) variants that biosynthesize olivetol and olivetolic acid in high yields.
  • OLS olivetol synthase
  • the engineered microorganisms according to the present invention can produce high concentrations of olivetol and olivine acid under small-scale cultivation, and since the cell concentration of the fermentation broth in small-scale cultivation is much lower than that in industrialized cultivation, it is expected that the present invention
  • the engineered microorganisms have strong potential for industrial production of olive alcohol and olive acid, which is conducive to the industrial biosynthesis of cannabinoids.
  • the present invention provides an engineered Escherichia coli, wherein the engineered Escherichia coli is modified to express an olivetol synthase (OLS) variant, wherein the OLS variant comprises, compared to its wild type One or more selected from the following mutations: I303T, I52L, S56A, H262M and K263R.
  • OLS olivetol synthase
  • the engineered E. coli is modified to express olivate cyclase (OAC).
  • OAC olivate cyclase
  • the engineered E. coli has a genomic fabH gene deleted.
  • the engineered E. coli has a genomic fadE gene deleted.
  • both the genomic fabH and fadE genes of the engineered E. coli are deleted.
  • the engineered E. coli is modified to overexpress long-chain acyl-CoA synthetase (fadD).
  • the engineered E. coli is modified from wild-type E. coli, BW25113 or BL21.
  • the modification is by introduction of a plasmid.
  • the OLS and OAC are expressed from the same plasmid.
  • the fadD is overexpressed by a plasmid.
  • the present invention provides a method for preparing engineered Escherichia coli, comprising modifying Escherichia coli to express an olivetol synthase (OLS) variant, wherein the OLS variant comprises, compared with its wild type One or more selected from the following mutations: I303T, I52L, S56A, H262M and K263R.
  • OLS olivetol synthase
  • the method comprises modifying the rod to express olivate cyclase (OAC).
  • OAC olivate cyclase
  • the method comprises deleting the genomic fabH gene of the E. coli.
  • the method comprises deleting the genomic fadE gene of the E. coli.
  • the method comprises deleting both the fabH and fadE genes of the E. coli genome.
  • the method comprises modifying the E. coli to overexpress long-chain acyl-CoA synthetase (fadD).
  • the E. coli is wild type E. coli, BW25113 or BL21.
  • the modification is by introduction of a plasmid.
  • the OLS and OAC are 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: 12 or the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 12 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: 13 or at least 70%, 71%, 72% of SEQ ID NO: 13 %, 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 the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 14 or by the amino acid sequence encoded by the sequence of SEQ ID NO: 14 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: 15 or at least 70%, 71% of SEQ ID NO: 15 , 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: an amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 8 or having at least 70% of the same sequence as SEQ ID NO: 8 , 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:9 or at least 70%, 71%, 72%, 73% of SEQ ID NO:9 %, 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 means are generally well known to those skilled in the art.
  • the plasmid used herein is shown in SEQ ID NO:7.
  • the plasmid used herein is shown in SEQ ID NO: 10.
  • the plasmid used herein is shown in SEQ ID NO: 11.
  • Engineering a microorganism can include interfering with the function of a protein of interest in the microorganism, eg, 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: 3, or the deletion of SEQ ID NO: 3 can make it have the same 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: 6, or the deletion of SEQ ID NO: 6 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:1.
  • 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: 2.
  • the artificial sequence used to delete the fadE gene is as shown in SEQ ID NO:4.
  • the partial sequence upstream and downstream of the original fadE gene site in the genome sequence of Escherichia coli in which the fadE gene is deleted, such as BW25113, is SEQ ID NO:5.
  • 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.
  • 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 an olivetol synthase (OLS) variant, wherein the OLS variant comprises one selected from the following mutations compared to its wild type One or more species: I303T, I52L, S56A, H262M and K263R.
  • 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 genomic fabH gene of the engineered Escherichia coli is deleted.
  • Item 4 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 5 The engineered E. coli according to any one of the preceding items, wherein both the fabH and fadE genes of the engineered E. coli genome are deleted.
  • Item 6 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 7 The engineered Escherichia coli according to any one of the preceding items, wherein the engineered Escherichia coli is modified from wild-type Escherichia coli, BW25113 or BL21.
  • Item 8 The engineered Escherichia coli according to any one of the preceding items, wherein the modification is performed by introducing a plasmid.
  • Item 9 The engineered Escherichia coli according to any one of the preceding items, wherein the OLS and OAC are expressed by the same plasmid.
  • Item 10 The engineered Escherichia coli according to any one of the preceding items, wherein the fadD is overexpressed by a plasmid.
  • Item 11 A method for preparing engineered Escherichia coli, comprising modifying Escherichia coli to express an olivetol synthase (OLS) variant, wherein the OLS variant comprises a mutation selected from the following mutations compared to its wild type One or more of: I303T, I52L, S56A, H262M and K263R.
  • OLS olivetol synthase
  • Item 12 The method according to any one of the preceding items, comprising modifying the coliform rod to express olivate cyclase (OAC).
  • OAC olivate cyclase
  • Item 13 The method according to any one of the preceding items, comprising deleting the genomic fabH gene of the Escherichia coli.
  • Item 14 The method according to any one of the preceding items, comprising deleting the genomic fadE gene of the Escherichia coli.
  • Item 15 The method according to any one of the preceding items, comprising deleting both the genomic fabH and fadE genes of the E. coli.
  • Item 16 The method according to any one of the preceding items, comprising modifying the E. coli to overexpress long-chain acyl-CoA synthetase (fadD).
  • Item 17 The method according to any one of the preceding items, wherein the E. coli is wild-type E. coli, BW25113 or BL21.
  • Item 18 The method according to any one of the preceding items, wherein said modification is performed by introducing a plasmid.
  • Item 19 The method according to any one of the preceding items, wherein the OLS and OAC are expressed by the same plasmid.
  • Item 20 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, after dissolving in distilled water, adjust the pH to 7.0. Autoclave 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.
  • Example 1 Deletion of the fabH gene
  • the fabH gene in the genome of Escherichia coli BW25113 was deleted to reduce the flow of intracellular Malonyl-CoA to the branch 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 KanR resistance gene was deleted according to the method provided in the literature (Datsenko K A, Wanner BL., supra), and the steps were as follows:
  • the partial sequence upstream and downstream of the original fabH gene site in the obtained genome sequence of the engineered BW25113 lacking the fabH gene is SEQ ID NO: 2.
  • Example 2 Deletion of the fadE gene
  • the fadE gene in the genome was further deleted to reduce the flow of intracellular hexanoyl-CoA to the branch metabolism, thereby increasing the accumulation of intracellular hexanoyl-CoA to further increase the synthesis of the target product OA .
  • Embodiment 3 import fadD expression plasmid
  • fadD long-chain ester acyl-CoA synthetase expression plasmid pL-Prha-fadD shown in SEQ ID NO: 7, which is transformed into BW25113 that lacks both fabH and fadE genes to obtain overexpression of fadD therein , and an engineered E. coli strain lacking both the fabH and fadE genes.
  • Embodiment 4 import OLS expression plasmid or OLS&OAC expression plasmid
  • Embodiment 6 the performance test that contains OLS variant engineered bacterial strain
  • 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 in the above-mentioned engineered strains, for OLS (I52L), the output of OA is 260.49mg/L; for OLS (S56A), the output of OA is 320.31mg/L; for OLS (H262M), the output of OA is 311.60mg/L; for OLS (K263R), the yield of OA was 300.89mg/L, both much higher than the prior art (Tan Z, Clomburg J M, Gonzalez R.Synthetic pathway for the production of olivetolic acid in Escherichia coli[ J].
  • the yield is 80mg/L.
  • the OL yield of CZ-OL is 116.02 mg/L, which is higher than the yield reported in the patent No. WO2020176547A1 of the prior art.
  • the patent screens the olive alcohol synthase derived from Cymbidium hybrid cultivar and finds that olive alcohol The yield was 40.7 mg/L.
  • Embodiment 7 the performance test that contains OLS variant bacterial strain
  • the p15A-Prha-OLS(WT)-OAC plasmid and the four p15A-Prha-OLS(mutated)-OAC plasmids were transformed into these two E. coli strains to obtain strains BW(WT), BW(I52L), BW( S56A), BW(H262M), BW(K263R), BL21(WT), BL21(I52L), BL21(S56A), BL21(H262M), BL21(K263R).
  • the strains were tested for OA production as described in Example 6.

Abstract

L'invention concerne un variant d'olivétol synthétase et une Escherichia coli génétiquement modifiée exprimant celui-ci pour la production d'olivétol et d'acide olivétolique. L'Escherichia coli modifiée est modifiée pour exprimer un variant d'olivétol synthétase qui comprend un ou plusieurs des mutants suivants par comparaison avec un variant d'olivétol synthétase de type sauvage : I303T, I52L, S56A, H262M et K263R. L'Escherichia coli modifiée permet d'obtenir des rendements améliorés d'olivétol et d'acide olivétolique.
PCT/CN2021/129656 2021-09-10 2021-11-09 Variant d'olivétol synthétase et micro-organisme génétiquement modifié exprimant celui-ci WO2023035396A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202111058663.5 2021-09-10
CN202111058663.5A CN113502254B (zh) 2021-09-10 2021-09-10 橄榄醇合成酶变体和表达其的工程化微生物

Publications (1)

Publication Number Publication Date
WO2023035396A1 true WO2023035396A1 (fr) 2023-03-16

Family

ID=78016556

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2021/129656 WO2023035396A1 (fr) 2021-09-10 2021-11-09 Variant d'olivétol synthétase et micro-organisme génétiquement modifié exprimant celui-ci

Country Status (2)

Country Link
CN (6) CN114196647B (fr)
WO (1) WO2023035396A1 (fr)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114196647B (zh) * 2021-09-10 2022-08-12 北京蓝晶微生物科技有限公司 一种橄榄醇合成酶变体r及其用途
CN114703171B (zh) * 2022-06-06 2022-09-13 深圳蓝晶生物科技有限公司 酯酰辅酶a合成酶变体及其工程化微生物

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20190352679A1 (en) * 2016-03-16 2019-11-21 William Marsh Rice University Microbial synthesis of isoprenoid precursors, isoprenoids and derivatives including prenylated aromatic compounds
CN110914416A (zh) * 2017-04-27 2020-03-24 加州大学董事会 产生大麻素和大麻素衍生物的微生物和方法
WO2020160289A1 (fr) * 2019-01-30 2020-08-06 Genomatica, Inc. Cellules modifiées de production améliorée de cannabinoïdes
WO2020214951A1 (fr) * 2019-04-19 2020-10-22 Genomatica, Inc. Variants d'olivétol synthase et procédés de production d'acide olivétolique et de ses composés analogues
WO2021108617A1 (fr) * 2019-11-27 2021-06-03 Genomatica, Inc. Cellules génétiquement modifiées pour la production de cannabinoïdes et d'autres produits dérivés de malonyl-coa
CN113366009A (zh) * 2018-11-27 2021-09-07 科纳科学有限责任公司 用于生物合成大麻素的双向多酶支架
CN113502254A (zh) * 2021-09-10 2021-10-15 北京蓝晶微生物科技有限公司 橄榄醇合成酶变体和表达其的工程化微生物

Family Cites Families (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA3130705A1 (fr) * 2010-04-15 2011-10-20 National Research Council Of Canada Genes et proteines servant a la synthese de polycetides aromatiques
CA2851316C (fr) * 2011-07-13 2021-10-19 National Research Council Of Canada Genes et proteines pour la synthese d'alcanoyl-coa
GB201715919D0 (en) * 2017-09-29 2017-11-15 Gw Res Ltd use of cannabinoids in the treatment of epilepsy
CN110791466B (zh) * 2018-08-01 2021-04-30 中国科学院青岛生物能源与过程研究所 一种合成丁三醇油酸酯的重组菌及其构建方法和应用
CN113227353A (zh) * 2018-11-14 2021-08-06 马努斯生物合成股份有限公司 用于产生大麻素的微生物细胞及方法
EP3931330A4 (fr) * 2019-02-25 2023-03-15 Ginkgo Bioworks, Inc. Biosynthèse de cannabinoïdes et de précurseurs cannabinoïdes
US20220315969A1 (en) * 2019-06-06 2022-10-06 Genomatica Inc. Olivetolic acid cyclase variants and methods for their use
WO2021042057A1 (fr) * 2019-08-30 2021-03-04 Lygos, Inc. Systèmes et procédés de préparation de cannabinoïdes et de dérivés
CN114729386A (zh) * 2019-10-01 2022-07-08 杭州恩和生物科技有限公司 用于大麻素合成的酶及其制备和使用方法
WO2021081647A1 (fr) * 2019-10-29 2021-05-06 Algae-C Inc. Micro-organisme génétiquement modifié pour la production de cannabinoïdes
CN113355300B (zh) * 2020-08-05 2022-04-01 深圳蓝晶生物科技有限公司 芳香族异戊烯基转移酶突变体、用于其表达的重组菌的构建方法及由其构建的重组菌

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20190352679A1 (en) * 2016-03-16 2019-11-21 William Marsh Rice University Microbial synthesis of isoprenoid precursors, isoprenoids and derivatives including prenylated aromatic compounds
CN110914416A (zh) * 2017-04-27 2020-03-24 加州大学董事会 产生大麻素和大麻素衍生物的微生物和方法
CN113366009A (zh) * 2018-11-27 2021-09-07 科纳科学有限责任公司 用于生物合成大麻素的双向多酶支架
WO2020160289A1 (fr) * 2019-01-30 2020-08-06 Genomatica, Inc. Cellules modifiées de production améliorée de cannabinoïdes
WO2020214951A1 (fr) * 2019-04-19 2020-10-22 Genomatica, Inc. Variants d'olivétol synthase et procédés de production d'acide olivétolique et de ses composés analogues
WO2021108617A1 (fr) * 2019-11-27 2021-06-03 Genomatica, Inc. Cellules génétiquement modifiées pour la production de cannabinoïdes et d'autres produits dérivés de malonyl-coa
CN113502254A (zh) * 2021-09-10 2021-10-15 北京蓝晶微生物科技有限公司 橄榄醇合成酶变体和表达其的工程化微生物

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
DATABASE PROTEIN ANONYMOUS : "RecName: Full=3,5,7-trioxododecanoyl-CoA synthase; AltName: Full=Olivetol synthase; AltName: Full=Polyketide synthase-1; AltName: Full=Tetraketide synthase", XP093043954, retrieved from NCBI *
FLORES-SANCHEZ ISVETT J., LINTHORST HUUB J.M., VERPOORTE ROBERT: "In silicio expression analysis of PKS genes isolated from Cannabis sativa L.", GENETICS AND MOLECULAR BIOLOGY, SOCIEDADE BRASILEIRA DE GENETICA, RIBEIRAO PRETO, BR, vol. 33, no. 4, 31 December 2010 (2010-12-31), BR , pages 703 - 713, XP093043951, ISSN: 1415-4757, DOI: 10.1590/S1415-47572010005000088 *
TAURA, F. ; TANAKA, S. ; TAGUCHI, C. ; FUKAMIZU, T. ; TANAKA, H. ; SHOYAMA, Y. ; MORIMOTO, S.: "Characterization of olivetol synthase, a polyketide synthase putatively involved in cannabinoid biosynthetic pathway", FEBS LETTERS, ELSEVIER, AMSTERDAM., NL, vol. 583, no. 12, 18 June 2009 (2009-06-18), NL , pages 2061 - 2066, XP026185379, ISSN: 0014-5793, DOI: 10.1016/j.febslet.2009.05.024 *

Also Published As

Publication number Publication date
CN114196645A (zh) 2022-03-18
CN114196647B (zh) 2022-08-12
CN114196649B (zh) 2022-08-16
CN114196646B (zh) 2022-07-26
CN114196648B (zh) 2022-09-13
CN114196649A (zh) 2022-03-18
CN114196647A (zh) 2022-03-18
CN113502254A (zh) 2021-10-15
CN114196646A (zh) 2022-03-18
CN114196645B (zh) 2022-08-09
CN113502254B (zh) 2022-01-07
CN114196648A (zh) 2022-03-18

Similar Documents

Publication Publication Date Title
JP6254728B2 (ja) イソプレンシンターゼおよびそれをコードするポリヌクレオチド、ならびにイソプレンモノマーの製造方法
KR102390716B1 (ko) 코리스메이트-유도된 산물의 생산을 위한 유전적으로 조작된 미생물들
US20220002773A1 (en) Production of 3-fucosyllactose and lactose converting alpha-1,3-fucosyltransferase enzymes
WO2023035527A1 (fr) Micro-organisme génétiquement modifié pour la production d'olivetol et d'acide olivetolique
WO2023035396A1 (fr) Variant d'olivétol synthétase et micro-organisme génétiquement modifié exprimant celui-ci
US11459592B2 (en) Processes for the production of cannabinoids from a carbon source precursor
CN111979163B (zh) 一种重组罗氏真氧菌及其制备方法和应用
JP7317255B1 (ja) Ｐｈａ生産量の高い遺伝子組換え細菌及びその構築方法
CA3153641A1 (fr) Production basee sur des cellules de nonulosonates
US20220049235A1 (en) Engineering Bacteria for Ferulic Acid Production, Preparation Method and Use Thereof
CN117083382A (zh) 细菌中非天然单不饱和脂肪酸的产生
CN113355300B (zh) 芳香族异戊烯基转移酶突变体、用于其表达的重组菌的构建方法及由其构建的重组菌
CN111662892A (zh) β-酮己二酸代谢相关三个基因的结构优化与应用
CN114174506B (zh) 芳樟醇合酶
KR20210047992A (ko) 알파-휴물렌 생산용 형질전환 메탄자화균 및 이의 용도
CN114292825B (zh) 一种托品酮的合成方法
WO2023138679A1 (fr) Procédé de régulation et de contrôle d'un composé flavonoïde synthétique hétérologue et son utilisation
CN117402846B (zh) L-丙氨酸脱氢酶突变体及其制备方法和应用
JP6635535B1 (ja) Ｅｆｐタンパク質を発現する大腸菌およびそれを用いたフラボノイド化合物製造方法
US20210355434A1 (en) Methods of Producing Cannabinoids
CN112522231A (zh) 一种酰基转移酶及其编码基因和应用
KR20210046912A (ko) α-bisabolene 생산용 형질전환 메탄자화균 및 이의 용도
WO2017132924A1 (fr) Enzyme de biosynthèse d'isoprène et d'isopentényle et son mutant
CN111321098A (zh) 一种具有桧烯高耐受能力的大肠杆菌基因工程菌及其构建方法与应用

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 21956568

Country of ref document: EP

Kind code of ref document: A1