CN114196648A

CN114196648A - Olive alcohol synthetase variant T and application thereof

Info

Publication number: CN114196648A
Application number: CN202111509635.0A
Authority: CN
Inventors: 杜德尧; 王高艳; 张倩; 宗朕; 李明月; 邱悦悦; 尹进; 张浩千
Original assignee: Shenzhen Blue Crystal Biotechnology Co ltd; Bluepha Co ltd
Current assignee: Shenzhen Blue Crystal Biotechnology Co ltd; Bluepha Co ltd
Priority date: 2021-09-10
Filing date: 2021-09-10
Publication date: 2022-03-18
Anticipated expiration: 2041-09-10
Also published as: CN114196645A; WO2023035396A1; CN114196647B; CN114196649B; CN114196646B; CN114196648B; CN114196649A; CN114196647A; CN113502254A; CN114196646A; CN114196645B; CN113502254B

Abstract

The present invention provides an olivine synthase variant and an engineered microorganism expressing the same for the production of olivine and olivine acid, more particularly an engineered escherichia coli, wherein the engineered escherichia coli is modified to express an olivine synthase variant whose mutation compared to its wild type is I303T. The engineered escherichia coli achieves improved yield of olivetol and olivetol acid.

Description

Olive alcohol synthetase variant T and application thereof

The present application is a divisional application of invention patent application No. 202111058663.5 filed on 09/10/2021 and entitled "variant of olive alcohol synthase and engineered microorganism expressing the same".

Technical Field

The present invention relates to the field of microorganisms and enzymes, in particular to olivetol synthase (OLS) variants and engineered microorganisms expressing the same for the production of Olivetol (OL) and Olivetolic Acid (OA).

Background

Cannabinoids (cannabinoids) refer to a large class of chemical molecules derived from the plant Cannabis sativa (Cannabis sativa), of which over 150 species. The currently used cannabinoids are mostly Cannabidiol (CBD), Tetrahydrocannabinol (THC) and Cannabigerol (CBG). CBD is one of the main chemical components in the plant cannabis, and is a non-addictive component of cannabinoids. THC is the major psychoactive substance in cannabis, can be addictive and is a substance that is strictly regulated in various countries throughout the world. CBG is generally classified as a micro-cannabinoid or a rare cannabinoid because of its low content in the plant cannabis sativa, and is known as the "parent" to cannabinoids because it is a common precursor to other cannabinoids. With the growth of cannabis, most of CBG is converted into CBD and THC, only a trace amount of CBG remains in the plant body, and the supply is severely limited, so that the development of CBG application is greatly limited.

Oligol and olivetol are type III polyketides derived from Cannabis (Cannabis) plants, have antibacterial, antitumor and anti-UV activities, and are key biosynthetic precursors of cannabinoids. There are few reports of natural extraction of olivetol and olivetol from plants. Moreover, plant cultivation is limited by factors such as the difficulty in obtaining high purity products compared to biosynthesis, strict plant control, limited productivity, the need for high plant cultivation and downstream extraction input, and low production stability. The chemical synthesis of the olive alcohol and the olive alcohol acid has high cost, and has the problems of environmental pollution, harsh conditions and the like. As far as the inventor knows, the biosynthesis of the olive alcohol and the olive alcohol acid by taking the Escherichia coli as the Chassis bacteria is not enough to meet the industrialization requirement. Tan Z et al (Tan Z, Clomburg J M, Gonzalez R. synthetic pathway for the production of an oleatic acid in Escherichia coli [ J ]. ACS synthetic biology,2018,7(8): 1886-. In 2020, patent No. WO2020176547A1 reported that by screening for an olive alcohol synthase derived from Cymbidium hybrid multivar, the yield of olive alcohol was found to be 40.7 mg/L.

There remains a need in the art for novel microorganisms for the biosynthesis of olive alcohol and olive alcohol acid in high yields.

Disclosure of Invention

The present invention meets the above-described need in the art by providing olive alcohol synthase (OLS) variants that biosynthesize olive alcohol and olive alcohol acid in high yields. The engineered microorganism according to the present invention can produce high concentrations of olivetol and olivetol under small-scale culture, and since the cell concentration of the fermentation broth under small-scale culture is much lower than that of the fermentation broth under industrial culture, it can be expected that the engineered microorganism of the present invention has strong industrial production potential of olivetol and olivetol, which is advantageous for industrial biosynthesis of cannabinoids.

Accordingly, in one aspect, the present invention provides an engineered escherichia coli, wherein the engineered escherichia coli is modified to express an olivine synthase (OLS) variant, wherein the OLS variant comprises one or more mutations selected from the group consisting of: I303T, I52L, S56A, H262M and K263R.

In some embodiments of the aspects, the engineered escherichia coli is modified to express an Olivine Acid Cyclase (OAC).

In some embodiments of the aspect, the engineered escherichia coli genomic fabH gene is deleted.

In some embodiments of the aspects, the engineered escherichia coli genomic fadE gene is deleted.

In some embodiments of the aspect, both the genomic fabH and fadE genes of the engineered e.

In some embodiments of the aspects, the engineered E.coli is modified to overexpress a long-chain acyl-CoA synthetase (fadD).

In some embodiments of the aspects, the engineered escherichia coli is modified from wild-type escherichia coli, BW25113, or BL 21.

In some embodiments of the aspect, the modification is by introduction of a plasmid.

In some embodiments of this aspect, the OLS and OAC are expressed by the same plasmid.

In some embodiments of the aspect, the fadD is overexpressed by a plasmid.

In another aspect, the present invention provides a method for preparing engineered escherichia coli, comprising modifying escherichia coli to express an olivol synthase (OLS) variant, wherein said OLS variant comprises one or more mutations compared to its wild type selected from the group consisting of: I303T, I52L, S56A, H262M and K263R.

In some embodiments of the aspect, the method comprises modifying the large intestine rod to express an Olivine Acid Cyclase (OAC).

In some embodiments of the aspect, the method comprises deleting the genomic fabH gene of the e.

In some embodiments of the aspect, the method comprises deleting the genomic fadE gene of the E.coli.

In some embodiments of the aspect, the method comprises deleting both of the genomic fabH and fadE genes of the e.

In some embodiments of the aspect, the method comprises modifying the escherichia coli to overexpress a long-chain acyl-coa synthetase (fadD).

In some embodiments of the aspect, the e.coli is wild-type e.coli, BW25113, or BL 21.

In some embodiments of the aspect, the fadD is overexpressed by a plasmid.

Detailed Description

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and synthetic biology, and the like, which are within the skill of the art. Such techniques are explained fully in the literature: "Molecular Cloning: A Laboratory Manual," second edition (Sambrook et al, 1989); "Oligonucleotide Synthesis" (edited by m.j. gate, 1984); "Animal Cell Culture" (ed. r.i. freshney, 1987); "Methods in Enzymology" (Academic Press, Inc.); "Current Protocols in Molecular Biology" (ed. F.M. Ausubel et al, 1987, and periodic updates); "PCR: The Polymerase Chain Reaction," (Mullis et al eds., 1994); singleton et al, Dictionary of Microbiology and Molecular Biology, second edition, J.Wiley & Sons (New York, N.Y.1994) and March's Advanced Organic Chemistry Reactions, fourth edition Mechanisms and Structure, John Wiley & Sons (New York, N.Y.1992), provide one of skill in the art with a general guide to many of the terms used in this application.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. For the purposes of the present invention, the following terms are defined below.

The articles "a" and "the" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. For example, "an element" means one/one element or more than one/one element.

The use of an alternative (e.g., "or") should be understood to mean either, both, or any combination thereof.

The term "and/or" should be understood to mean either or both of the alternatives.

As used herein, the term "about" or "approximately" refers to a quantity, level, value, quantity, frequency, percentage, dimension, size, amount, weight, or length that varies by up to 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% as compared to a reference quantity, level, value, quantity, frequency, percentage, dimension, size, amount, weight, or length. In one embodiment, the term "about" or "approximately" refers to a quantity, level, value, quantity, frequency, percentage, dimension, size, amount, weight, or length that surrounds a reference quantity, level, value, quantity, frequency, percentage, dimension, size, amount, weight, or length by 15%, ± 10%, ± 9%, ± 8%, ± 7%, ± 6%, ± 5%, ± 4%, ± 3%, ± 2%, or ± 1%.

As used herein, the term "substantially" refers to an amount, level, value, amount, frequency, percentage, dimension, size, amount, weight, or length that is about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more as compared to a reference amount, level, value, amount, frequency, percentage, dimension, size, amount, weight, or length. In one embodiment, the term "substantially the same" refers to a range of numbers, levels, values, amounts, frequencies, percentages, dimensions, sizes, amounts, weights, or lengths that are about the same as a reference number, level, value, amount, weight, or length.

As used herein, the term "substantially free," when used to describe a composition, e.g., a population of cells or a culture medium, refers to a composition that is free of a specified substance, e.g., 95% free, 96% free, 97% free, 98% free, 99% free of the specified substance, or is undetectable as measured by conventional means. Similar meanings may apply to the term "absent" when referring to the absence of a particular substance or component of a composition.

Throughout this specification, unless the context requires otherwise, the terms "comprise", "comprising" and "have" are to be construed as implying that the recited step or element or group of steps or elements is included, but not excluding any other step or element or group of steps or elements. In certain embodiments, the terms "comprising," "including," "containing," and "having" are used synonymously.

"consisting of … …" is intended to include, but is not limited to, anything following the phrase "consisting of … …". Thus, the phrase "consisting of … …" is intended to indicate that the listed elements are required or mandatory, and that no other element may be present.

"consisting essentially of … …" is intended to include any elements listed after the phrase "consisting essentially of … …" and is limited to other elements that do not interfere with or contribute to the activities or actions specified in the disclosure of the listed elements. Thus, the phrase "consisting essentially of … …" is intended to indicate that the listed elements are required or mandatory, but that no other elements are optional and may or may not be present depending on whether they affect the activity or action of the listed elements.

Reference throughout this specification to "one embodiment," "some embodiments," "a particular embodiment," or similar language means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, the appearances of the foregoing phrases in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

Where features relating to a particular aspect of the invention, for example the product of the invention, are disclosed, such disclosure is also to be considered as applicable to any other aspect of the invention, for example the method and use of the invention, mutatis mutandis.

The present invention is based, at least in part, on the discovery that: host microorganisms expressing an olivine synthase (OLS) and/or an Olivine Acid Cyclase (OAC) can produce high concentrations of olivine and/or olivine acid by deleting the genomic fabH coding sequence in the host microorganism (e.g., escherichia coli). Without wishing to be bound by theory, it is believed that the deletion of the fabH gene promotes synthesis of olivine and/or olivine acid by the olive alcohol synthase (OLS) and/or Olive Acid Cyclase (OAC) in the engineered microorganism using intracellular increased levels of Malonyl-CoA (Malonyl-CoA) and Hexanoyl-CoA (Hexanoyl-CoA) as substrates. In this way, the engineered microorganism can be used to produce olivetol or olivetol on demand, thereby surpassing the cumbersome and costly prior art that still relies on complex synthetic chemistry.

Accordingly, in one aspect, the present invention provides an engineered microorganism, wherein the engineered microorganism is modified to express an olivine synthase (OLS), wherein the engineered microorganism has a deletion of the genomic fabH gene.

In some embodiments, the microorganism engineered to biosynthesize olivetol and olivetol is escherichia coli (e. In a specific embodiment, the microorganism engineered to biosynthesize olivetol and olivetol is Escherichia coli BW25113(ATCC No.; available from American Type Culture Collection). The BW25113 strain is derived from E.coli K-12W1485, is a derivative strain of K-12W1485, is similar to MG1655, and is an engineered Escherichia coli strain which is slightly modified and is closer to a wild type. Improved in the production of malonyl-CoA and hexanoyl-CoA compared to WT.

In some embodiments, the olivetol synthase used herein may be derived from Cannabis sativa (Cannabis sativa). The olivetol synthase can be a variant optimized for expression in e. In some embodiments, the olivine synthase may comprise, consist essentially of, or consist of: an amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO. 12 or an amino acid sequence encoded by a nucleotide sequence having 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% identity to SEQ ID NO. 12 or a range of any two of the foregoing. In some embodiments, the olivine synthase may comprise, consist essentially of, or consist of: 13 or an amino acid sequence which has 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 a range of any two of the aforementioned values as compared to SEQ ID No. 13.

Exemplary olive alcohol synthetases for use herein may include full-length olive alcohol synthetases, fragments of olive alcohol synthetases, variants of olive alcohol synthetases, truncated olive alcohol synthetases, or fusion enzymes having at least one activity of an olive alcohol synthetase. In some embodiments, the olive alcohol synthase used in the present invention has an activity of catalyzing the synthesis of olive alcohol from malonyl-coa and hexanoyl-coa.

In some embodiments, the olivine cyclase used herein may be derived from Cannabis sativa (Cannabis sativa). The olivine acid cyclase may be a variant optimized for expression in E.coli. In some embodiments, the olivine acid cyclase may comprise, consist essentially of, or consist of: an amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO. 14 or an amino acid sequence encoded by a nucleotide sequence having 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% identity to SEQ ID NO. 14 or a range of any two of the foregoing. In some embodiments, the olivine acid cyclase may comprise, consist essentially of, or consist of: 15 or an amino acid sequence which has 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 a range of any two of the aforementioned values as compared to SEQ ID No. 15.

Exemplary olive-alcohol-acid cyclases for use herein may include full-length olive-alcohol-acid cyclase, fragments of olive-alcohol-acid cyclase, variants of olive-alcohol-acid cyclase, truncated olive-alcohol-acid cyclase, or a fusion enzyme having at least one activity of olive-alcohol-acid cyclase. In some embodiments, the olivine acid cyclase used in the present invention has activity to carboxylate olivine to olivine acid.

In some embodiments of the invention, the long-chain acyl-CoA synthetase (fadD), acyl-CoA dehydrogenase (fadE), and β -ketoacyl-acyl carrier protein synthase (fabH) are components inherent in E.coli.

In some embodiments, fadD as used herein may be derived from e. fadD can be a variant optimized for expression in e. In some embodiments, the fadD may comprise, consist essentially of, or consist of: an amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO. 8 or an amino acid sequence encoded by a nucleotide sequence having 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% identity to SEQ ID NO. 8 or a range consisting of any two of the foregoing. In some embodiments, the fadD may comprise, consist essentially of, or consist of: 9 or an amino acid sequence which has 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 a range of any two of the aforementioned values as compared to SEQ ID No. 9.

Exemplary fadD as used herein may include full-length fadD, fragments of fadD, variants of fadD, truncated fadD, or a fusion enzyme having at least one activity of fadD. In some embodiments, fadD used in the present invention has activity to catalyze the production of hexanoyl-coa from hexanoic acid by acylation.

In some embodiments, the one or more enzymes used in the present invention may be a mutant (mutant) or variant (variant) of an enzyme described herein. As used herein, "mutant" and "variant" refer to molecules that retain the same or substantially the same biological activity as the original sequence. The mutants or variants may be from the same or different species, or may be synthetic sequences based on natural or existing molecules. In some embodiments, the terms "mutant" and "variant" refer to polypeptides having an amino acid sequence that differs from a corresponding wild-type polypeptide by at least one amino acid. For example, mutants and variants may comprise conservative amino acid substitutions: i.e. replacing the original corresponding amino acid with an amino acid having similar properties. Conservative substitutions may be polar para-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)); nonpolar versus nonpolar amino acids (alanine (a, Ala), valine (V, Val), tryptophan (W, Trp), leucine (L, Leu), proline (P, Pro), methionine (m, Met), phenylalanine (F, Phe)); acidic versus acidic amino acids (aspartic acid (D, Asp), glutamic acid (E, Glu)); basic pair basic amino acids (arginine (R, Arg), histidine (H, His), lysine (K, Lys)); charged amino acids (aspartic acid (D, Asp), glutamic acid (E, Glu), histidine (H, His), lysine (K, Lys) and arginine (R, Arg)); and hydrophobic versus hydrophobic amino acids (alanine (a, Ala), leucine (ULeu), isoleucine (I, Ile), valine (V, Val), proline (P, Pro), phenylalanine (F, Phe), tryptophan (W, Trp), and methionine (M, Met)). In some other embodiments, the mutant or variant may also comprise non-conservative substitutions.

In some embodiments, a mutant or variant polypeptide can have substitutions, additions, insertions, or deletions of amino acids in a range of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more or any two of the foregoing. A mutant or variant may have an activity of at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% or a range of any two of the foregoing values as compared to an unaltered enzyme. Enzyme activity can be determined by conventional techniques known in the art, such as colorimetric enzymatic assays.

As is well known to those skilled in the art, expression of a heterologous nucleic acid in a host can be improved (i.e., codon optimized) by replacing one or more encoding nucleotides (i.e., codons) in a nucleotide sequence encoding a polypeptide, such as an enzyme, with another codon for better expression in the host. One reason for this effect is because different organisms show a preference for different codons. In some embodiments, a nucleotide sequence encoding a polypeptide, such as an enzyme, disclosed herein is modified or optimized such that the resulting nucleotide sequence reflects codon bias for a particular host. For example, in some embodiments, the nucleotide sequence encoding a polypeptide, such as an enzyme, is modified or optimized for e. See, e.g., Gouy M, gateway C. Codon usage in bacteria: correlation with gene expression [ J ]. Nucleic acids research,1982,10(22): 7055-7074; Eyre-Walker A. Synonymus code bis is related to gene length in Escherichia coli: selection for translational access [ J ]. Molecular biology and evolution,1996,13(6):864 872.; nakamura Y, Gojobori T, Ikemura T. Codon use configured from interactive DNA sequences databases: status for the year 2000[ J ]. Nucleic acids research,2000,28(1): 292-.

A polynucleotide or polypeptide has a certain "sequence identity" or percentage of "identity" to another polynucleotide or polypeptide, meaning that when two sequences are aligned, the percentage of bases or amino acids are the same and in the same relative position. Determining the percent identity of two amino acid sequences or two nucleotide sequences can include aligning and comparing the amino acid residues or nucleotides at corresponding positions in the two sequences. A sequence is considered 100% identical if all positions in both sequences are occupied by the same amino acid residue or nucleotide. Sequence identity can be determined in a number of different ways, for example, sequences can be aligned using various methods and computer programs (e.g., BLAST, T-COFFEE, MUSCLE, MAFFT, etc.).

Some embodiments of the invention relate to expression constructs, e.g. vectors, such as plasmids, preferably expression constructs comprising one or more nucleotide sequences encoding OLS and/or OAC. The nucleotide sequence encoding OLS or OAC is as described above. Preferably, the expression construct is a plasmid. Preferably, the expression construct may be used to express OLS and/or OAC, more preferably to co-express both OLS and OAC, in escherichia coli, preferably in escherichia coli BW 25113.

Some embodiments of the invention relate to expression constructs, e.g., vectors, such as plasmids, comprising a nucleotide sequence encoding fadD. The nucleotide sequence encoding fadD is as described above. Preferably, the expression construct is a plasmid. Preferably, the expression construct can be used for the expression of fadD in e.coli, preferably e.coli BW 25113. In some embodiments, the host microorganism expresses fadD itself, and thus an expression construct encoding fadD is introduced into the host microorganism to overexpress fadD.

Engineering a microorganism may comprise expressing an enzyme of interest in the microorganism. In some embodiments, the expression constructs described herein are introduced into a host microorganism by transformation to express an enzyme of interest. The transformation can be carried out by methods well known in the art. For example, plasmids described herein comprising a nucleotide sequence encoding fadD 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 the particular host.

Expression of the enzyme of interest in the host microorganism to achieve the desired aim may be achieved as described above by converting the expression construct encoding the enzyme into the host microorganism, by integrating the expression construct encoding the enzyme into the genomic sequence of the host microorganism in a variety of ways, or by enhancing transcription and/or expression of enzyme encoding genes, such as fadD encoding genes, native to the host microorganism in a variety of ways, for example by using stronger regulatory elements such as promoters. Such means are generally well known to those skilled in the art. In some embodiments, the plasmids used herein are set forth in SEQ ID NO 7. In some embodiments, the plasmids used herein are set forth in SEQ ID NO 10. In some embodiments, the plasmids used herein are set forth in SEQ ID NO 11.

Engineering a microorganism may comprise intervening in the function of the protein of interest in said microorganism, e.g. reducing or eliminating the expression of said protein, which may be achieved, for example, by deleting genomic sequences of interest in said microorganism. In some embodiments, the genomic fabH gene in a microorganism described herein is deleted such that 3-oxoacyl- [ acyl carrier protein ] synthetase 3(3-oxoacyl- [ acyl carrier protein ] synthase 3) is not expressed in the microorganism. In some embodiments, the amino acid sequence of 3-oxoacyl- [ acyl carrier protein ] synthetase 3 is shown as NCBI ACCESSION NO: NP-415609 (https:// www.ncbi.nlm.nih.gov/protein/16129054). In some embodiments, a genomic fadE gene in a microorganism described herein is deleted such that an acyl-CoA dehydrogenase (acyl-CoA dehydrogenase) is not expressed in the microorganism. In some embodiments, the amino acid sequence of the acyl-CoA dehydrogenase is shown as NCBI ACCESSION NO: NP-414756 (https:// www.ncbi.nlm.nih.gov/protein/90111100). In some embodiments, both genomic fabH and fadE genes in a microorganism described herein are deleted such that both 3-oxoacyl- [ acyl carrier protein ] synthetase 3 and acyl-CoA dehydrogenase are not expressed in the microorganism. In some embodiments, the fabH gene sequence that is deleted is shown in SEQ ID NO. 3, or is a variant sequence of SEQ ID NO. 3 that is deleted such that a protein having the same or similar function as 3-oxoacyl- [ acyl carrier protein ] synthetase 3 is not expressed. In some embodiments, the deleted fadE gene sequence is as shown in SEQ ID NO 6, or a variant sequence of SEQ ID NO 6 that is deleted such that a protein having the same or similar function as the acyl-CoA dehydrogenase is not expressed.

Deletion of genomic sequences of interest in a microorganism can be performed by methods known in the art. The genomic sequence may be deleted, for example, by designing an artificial sequence for lambda-Red homologous recombination against the genomic sequence, which is integrated into the genome at the target location using lambda-Red homologous recombination. Specific experimental protocols can be found in the examples described herein. In some embodiments, the artificial sequence for deletion of the fabH gene is shown in SEQ ID NO 1. In some embodiments, the portion of the genomic sequence of E.coli in which the fabH gene is deleted, such as BW25113, that is upstream and downstream of the original fabH gene site is SEQ ID NO 2. In some embodiments, the artificial sequence for deletion of the fadE gene is set forth in SEQ ID NO 4. In some embodiments, the portion of the genomic sequence of E.coli in which the fadE gene is deleted, e.g., BW25113, that is upstream and downstream of the original fadE gene site is SEQ ID NO 5.

In addition to deleting the genomic sequence of interest in the microorganism, the function of the protein of interest can be interfered with by other methods known in the art, including, but not limited to, interfering with transcription of the genomic sequence encoding the protein of interest, interfering with expression of mRNA encoding the protein of interest, interfering with delivery of the protein of interest, e.g., to the outside of the cell; more specifically, including, but not limited to, methods of deleting all or part of the genomic sequence encoding the protein of interest or its regulatory elements such as a promoter, inserting one or more nucleotides, e.g., a stop codon, that affects its transcription or mutating one or more nucleotides thereof to such an extent that the genomic sequence cannot be normally transcribed, in the middle of the genomic sequence encoding the protein of interest or its regulatory elements such as a promoter, introducing an agent that interferes with or silences mRNA encoding the protein of interest, e.g., an siRNA or dsRNAi agent, or a method of inhibiting or stopping the function of delivering the protein of interest, e.g., to an extracellular system (e.g., chaperones, signal sequences, transporters).

Suitable media for culturing the host may include standard media (e.g., Luria-Bertani broth, optionally supplemented with one or more other agents, such as an inducer; standard yeast media; and the like). In some embodiments, the medium can be supplemented with fermentable sugars (e.g., hexoses, such as glucose, xylose, and the like). In some embodiments, a suitable medium comprises an inducer. In certain such embodiments, the inducing agent comprises rhamnose.

The carbon source in a suitable medium for host culture may vary from simple sugars such as glucose to more complex hydrolysates of other biomass such as yeast extract. The addition of salts typically provides the necessary elements, such as magnesium, nitrogen, phosphorus, and sulfur, to allow the cell to synthesize polypeptides and nucleic acids. Suitable media may also be supplemented with selective agents, such as antibiotics, to select for maintenance of certain plasmids and the like. For example, if the microorganism is resistant to an antibiotic, such as ampicillin, tetracycline or kanamycin, the antibiotic can be added to the culture medium to prevent growth of cells that lack resistance. Suitable media may be supplemented with other compounds as necessary to select for desired physiological or biochemical properties, such as particular amino acids, and the like.

Materials and methods suitable for the maintenance and growth of microorganisms of the present invention are described herein, for example, in the examples section. Other materials and methods suitable for the maintenance and growth of microorganisms (e.g., E.coli) are well known in the art. Exemplary techniques may be found in WO 2009/076676; US12/335,071(US 2009/0203102); WO 2010/003007; US 2010/0048964; WO 2009/132220; US 2010/0003716; gerhardt P, Murray R G E, Costalow R N, et al, Manual of methods for genetic biology [ J ]. 1981; crueger W, Crueger A, Brock T D, et al.Biotechnology: a textbook of industrial microbiology [ J ] 1990, in which standard culture conditions and fermentation modes such as batch, fed-batch or continuous fermentation are described, the entire contents of which are incorporated herein by reference.

For small scale production, the engineered microorganism may be grown, fermented, and induced to express a desired nucleotide sequence, such as a nucleotide sequence encoding an OLS, OAC, and/or fadD, and/or to synthesize a desired fermentation product, such as olivetol and/or olivolic acid, in bulk, for example, on a scale of about 100mL, 500mL, 1L, 5L, or 10L. For large scale production, the engineered microorganism may be grown, fermented, and induced to express a desired nucleotide sequence, such as a nucleotide sequence encoding an OLS, OAC, and/or fadD, and/or to synthesize a desired fermentation product, such as olivetol and/or olivolic acid, in bulk on a scale of about 10L, 100L, 1000L, 10,000L, 100,000L, or greater.

Analysis of the fermentation product may be performed by separating the fermentation product of interest by chromatography, preferably HPLC, to determine the concentration at one or more times during the cultivation. The microbial culture and fermentation products can also be detected photometrically (absorbance, fluorescence).

The engineered microorganisms described herein achieve improved production of olivetol and/or olivetol acid. In some embodiments, the engineered microorganisms described herein achieve a higher yield in terms of olivine alcohol and/or olivine acid production than a suitable un-engineered or partially engineered microorganism control of 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, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000-fold or more or a range of any two of the foregoing values. In some embodiments, the engineered microorganism described herein achieves 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, 8.6, 8, 8.0, 6.2, 6.4, 6.6, 6, 6.8, 7.8, 7.0, 7.2, 7.8, 8, 8.0, 8, 6, 8, 9.0, 9.50, 9.8, 30, 25, 20, 19, 25, 40, 25, 40, 25, 40, 25, 40, 25, 40, 25, 40, 19, 25, 19, 25, 40, 25, 40, 25, 40, 19, 40, 25, 40, 25, 40, 19, 25, 40, 25, 40, 25, 40, 25, 19, 40, 19, 40, 23, 19, 40, 25, 19, 25, 40, 19, 40, 19, 40, 19, 40, 19, 40, 19, 40, 23, 40, 25, 40, 19, 25, 40, 19, 25, 40, 25, 40, 19, 40, 25, 40, 200. 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000mg/L or higher or a range of any two of the foregoing values.

The invention also provides some preferred embodiments as follows:

item 1. an engineered escherichia coli, wherein said engineered escherichia coli is modified to express an olivine synthase (OLS) variant, wherein said OLS variant comprises one or more mutations compared to its wild type selected from the group consisting of: I303T, I52L, S56A, H262M and K263R.

Item 2. the engineered escherichia coli according to any one of the preceding items, wherein the engineered escherichia coli is modified to express an Olivine Acid Cyclase (OAC).

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 genomic fadE gene of the engineered escherichia coli is deleted.

Item 5. the engineered escherichia coli according to any one of the preceding items, wherein both of the genomic fabH and fadE genes of the engineered escherichia coli 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 a long-chain esteracyl-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 BL 21.

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 olivol synthase (OLS) variant, wherein said OLS variant comprises one or more mutations compared to its wild type selected from the group consisting of: I303T, I52L, S56A, H262M and K263R.

Item 12. the method of any one of the preceding items, comprising modifying the large intestine rod to express Olivine Acid Cyclase (OAC).

Item 13. the method of any one of the preceding items, comprising deleting the genomic fabH gene of the e.

Item 14. the method of any one of the preceding items, comprising deleting the genomic fadE gene of e.

Item 15. the method of any one of the preceding items, comprising deleting both the genomic fabH and fadE genes of the e.

Item 16. the method of any one of the preceding items, comprising modifying the escherichia coli to overexpress a long-chain esteracyl-coa synthetase (fadD).

Item 17. the method of any one of the preceding items, wherein the e.coli is wild-type e.coli, BW25113, or BL 21.

Item 18. the method of any one of the preceding items, wherein the modification is performed by introducing a plasmid.

Item 19. the method of any one of the preceding items, wherein the OLS and OAC are expressed by the same plasmid.

Item 20. the method of any one of the preceding items, wherein the fadD is overexpressed by a plasmid.

Examples

Hereinafter, the present invention will be described in detail by examples. However, the examples provided herein are for illustrative purposes only and are not intended to limit the present invention.

The experimental procedures used in the following examples are all conventional unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The enzymatic reagents used were purchased from ThermoFisher and New England Biolabs (NEB), the small molecule standards used were purchased from Sigma, the kits used for plasmid extraction were purchased from Tiangen Biotechnology, Inc. (Beijing), and the kits for DNA fragment recovery were purchased from Omega, USA, and the corresponding procedures were performed strictly according to the product instructions. All media were prepared with deionized water unless otherwise specified, yeast extract and peptone were purchased from OXID, UK, and other reagents were purchased from Chemicals, national institutes. The service of gene synthesis is provided by the Huada institute of genetics.

Coli culture media that can be used herein:

LB culture medium: 5g/L yeast extract, 10g/L peptone, 10g/L NaCl. Adjusting pH to 7.0-7.2, and autoclaving for 30 min.

SOB medium: 5g/L yeast extract, 20g/L peptone, 0.5g/L NaCl, 2.5mL of 1M KCl. Adjusting pH to 7.0-7.2, and sterilizing with high pressure steam.

ZY medium: 10g/L of peptone and 5g/L of yeast extract, and after dissolving in distilled water, adjusting the pH to 7.0. Autoclaving for 30 minutes.

50×M：1.25mol/L Na₂HPO₄，1.25mol/L KH₂PO₄，2.5mol/L NH₄Cl，0.25mol/L Na₂SO₄And (5) sterilizing by high-pressure steam for 30 minutes.

50X 5052: 25% glycerol, 2.5% glucose, autoclaved for 30 minutes.

1M MgSO₄: 24.6g MgSO were weighed out₄·7H₂O plus H₂Dissolving O, diluting to 100mL, and then sterilizing for 30 minutes by high-pressure steam.

1000 times trace elements: 50mmol/L FeCl₃，20mmol/L CaCl₂，10mmol/L MnCl₂，10mmol/L ZnSO₄，CoCl₂，NiCl₂，Na₂MO₄，Na₂SeO₃，H₃BO₃2mmol/L each.

ZYM medium: 2mL of 50X 5052, 2mL of 50X M, 200. mu.L of 1M MgSO 2 was added to the ZY medium₄100 μ L1000 Xtrace elements.

Example 1: deletion of fabH Gene

The fabH gene in the genome of the Escherichia coli BW25113 is deleted to reduce the intracellular Malonyl-CoA flow to the branch metabolism, thereby improving the accumulation of intracellular Malonyl-CoA to further increase the synthesis of the target product OA.

Synthesis of H1-kana-H2 as described in SEQ ID NO:1, integration of SEQ ID NO:1 into the fabH gene position of the genome of BW25113 by lambda-Red homologous recombination to delete the fabH gene according to the method provided in the literature (Datsenko K A, Wanner B L. one-step inactivation of chromogenes in Escherichia coli K-12using PCR products [ J ]. Proceedings of the National Academy of Sciences,2000,97(12):6640-6645.) as follows:

1. preparation of BW25113 competence;

2. introducing a plasmid pKD 46;

3. BW25113(pKD46) was inoculated into 3mL of LB containing ampicillin at a concentration of 100. mu.g/L and subjected to shaking overnight at 30 ℃;

4. 100. mu.L of an overnight cultured BW25113(pKD46) cell suspension was added to 10mL of the OB medium, followed by 100. mu.L of arabinose at a concentration of 1M and 10. mu.L of ampicillin at a concentration of 100 mg/L. Shaking culturing at 30 deg.C until OD600 is 0.4-0.6;

centrifuging at 5.4 deg.C to collect thallus, re-suspending thallus with 10mL precooled ultrapure water, washing thallus twice in the same way, and finally re-suspending thallus with 50 μ L10% glycerol solution;

6. adding 50ng of the nucleotide sequence fragment of SEQ ID NO. 1, uniformly mixing, and adding the mixed solution into an electric shock cup;

7. putting the electric shock cup into an electric shock instrument for one-time electric shock, wherein the electric shock conditions are 200 omega, 25 muF and 2.5 KV;

8. adding 1mL of precooled SOB culture medium, transferring the mixed solution into a sterile EP tube, and carrying out shake culture at 30 ℃ for 1 hour;

9. uniformly coating the bacterial liquid on an LB plate culture medium containing ampicillin and kanamycin, and carrying out static culture at 30 ℃ for 16-20 hours;

10. transformants grown on the plates were verified.

After integration of SEQ ID NO:1, the KanR resistance gene was deleted according to the method provided in the literature (Datsenko K A, Wanner B L., supra) as follows:

1. preparing the competence of the strain integrating SEQ ID NO. 1;

2. transforming pCP20 into competence, and culturing at 30 ℃;

3. selecting a single clone to 3mL of SOB culture medium, and culturing at 30 ℃ overnight;

4. transferring 100 mu L of the suspension to 10mL of SOB, and culturing the suspension for 3-4 hours at 30 ℃ by using a shaking table, wherein OD600 is 0.4;

centrifugally collecting thalli under the condition of 5.4 ℃, and washing the thalli twice by using ice-bath sterile water;

6. resuspending in 50-100. mu.L sterile water for electroporation;

7. adding 100ng of pCP20, performing electric shock transformation (setting of an electric shock instrument: 1.8KV, 5.5ms), and resuscitating at 30 ℃ for 1 hour;

8. uniformly coating on an LB plate containing ampicillin, and culturing at 30 ℃ for 2-3 days;

9. selecting a single clone, streaking the single clone on an LB (Langmuir-Blodgett) plate without antibiotics, and culturing the single clone at 42 ℃ overnight;

10. 20-30 clones were picked up on a kanamycin-resistant plate and a non-resistant plate, respectively, and clones which could not grow on the kanamycin plate but simultaneously grown on the non-resistant plate were targeted for PCR verification.

The partial sequence of the genomic sequence of the engineered BW25113 lacking fabH gene, which is up-and-down stream of the original fabH gene site, is SEQ ID NO 2.

Example 2: deletion of fadE Gene

The engineered BW25113 obtained in example 1 further has the fadE gene deleted in its genome 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.

Synthesis of H3-kana-H4 as described in SEQ ID NO:4, integration of SEQ ID NO:4 into the fadE gene position of the genome of BW25113 lacking the fabH gene as described in example 1 to delete the fadE gene and the KanR resistance gene after integration of SEQ ID NO:4 was designed.

Example 3: introduction of fadD expression plasmid

Long-chain acyl-coa synthetase (fade) was overexpressed in engineered BW25113 obtained in example 2 to convert hexanoic acid (hexanoic acid) to hexanoyl-coa, thereby increasing intracellular hexanoyl-coa accumulation to further increase the synthesis of the target product OA.

The fadD (long-chain acyl-coenzyme A synthetase) expression plasmid pL-Prha-fadD shown in SEQ ID NO. 7 was designed and synthesized, and transformed into BW25113 lacking both of the fabH and fadE genes to obtain an engineered E.coli strain in which fadD is overexpressed and both of the fabH and fadE genes are deleted.

Example 4: introduction into OLS expression plasmid or OLS&OAC expression plasmid

The OLS expression plasmid p15A-Prha-OLS shown in SEQ ID NO 10 and the OLS & OAC expression plasmid p15A-Prha-OLS-OAC shown in SEQ ID NO 11 were designed and transformed into engineered BW25113 obtained in example 3, respectively, to obtain engineered BW25113 synthesizing OL or OA, referred to as CZ-OL or CZ-OA, respectively.

Example 5: introduction of OLS mutations

The OLS variant & OAC expression plasmid p 15A-pra-OLS variant-OAC with different OLS mutations was designed to be synthesized and transformed into the engineered BW25113 obtained in example 3 as described in example 4 to obtain a series of OLS variant-containing engineered BW25113 of synthetic OA.

Example 6: performance testing of engineered strains containing OLS variants

The engineered strains obtained in the examples were tested in triplicate according to the procedure shown below.

Fermentation and sample preparation:

1. the recombinant strain is inoculated into 3mL of LB liquid culture medium, and is cultured at 37 ℃ at 220 rpm overnight for about 14 hours, and the final OD600 value reaches 2-3;

2. adding ZYM culture medium into a 24-deep-well plate, and adding 2mL of ZYM culture medium into each well;

3. transferring the bacterial liquid in the step 1 to the ZYM culture medium in the step 2, wherein OD600 is 0.01 after transferring;

4. when the bacterial liquid OD600 grows to 0.2, adding an inducer (the addition amount is 0.2 percent of rhamnose) and a precursor caproic acid (1mM), and totally 24 hours from inoculation to fermentation end;

5. adding 3mL of ethyl acetate into 1mL of fermentation liquor, shaking and uniformly mixing for 10min, and centrifuging to collect an upper organic phase;

6. repeating the operation 5, centrifuging twice to obtain organic phases, combining the organic phases together, transferring about 6mL of the organic phases into a 10mL test tube;

7. evaporating all organic phases in the test tube to dryness by using a vacuum concentrator, and adding 1mL of methanol to resuspend all samples in the test tube;

8. the sample from step 7 was filtered through a 0.22 μ M filter and then transferred to an HPLC sample bottle.

TABLE 1 cell concentration in fermentation broths at fermentation stop

The fermentation broth extract was analyzed for the amount of OL produced by the HPLC assay method described above.

The OA yield in the fermentation broth extract was analyzed by the HPLC assay method described above.

The OA yield in the fermentation broth extract was calculated from the peak area. The results showed that, in the above engineered strain, the yield of OA was 260.49mg/L for OLS (I52L); for OLS (S56A), the yield of OA was 320.31 mg/L; for OLS (H262M), the yield of OA was 311.60 mg/L; for OLS (K263R), the OA yield was 300.89mg/L, which was much higher than that achieved in the prior art (Tan Z, Clomburg J M, Gonzalez R. synthetic pathway for the production of aliphatic acid in Escherichia coli [ J ]. ACS synthetic biology,2018,7(8): 1886. 1896.), with a yield of 80 mg/L. In addition, the CZ-OL was tested to have an OL yield of 116.02mg/L, which is higher than that reported in the prior art patent No. WO2020176547A1, which found an olive alcohol synthase derived from Cymbium hybride multivar to have an olive alcohol yield of 40.7 mg/L.

TABLE 2 OA yields corresponding to the various OLS variants

Example 7: performance testing of OLS variant-containing strains

To further test whether the effect of the OLS variants on OA production was limited to that of a particular engineered strain, the effect of the OLS variants was tested in two non-engineered escherichia coli strains BW25113 and BL 21.

The p15A-Prha-OLS (WT) -OAC plasmid and the four p15A-Prha-OLS (mutant) -OAC plasmids were transformed into these two E.coli strains, respectively, to obtain strains BW (WT), BW (I52L), BW (S56A), BW (H262M), BW (K263R), BL21(WT), BL21(I52L), BL21(S56A), BL21(H262M), BL21 (K263R). The strain was tested for OA production as described in example 6.

TABLE 3 cell concentration in fermentation broths at fermentation stop

TABLE 4 OA yields corresponding to the various OLS variants

Note: "-" indicates that OA was not detected

It can be seen that the above-described OLS variants achieve improved OA production in both wild-type and engineered strains. It was concluded that the OA production-improving effect of the OLS variant is not limited to a specific strain.

In addition, the corresponding version of the strain used herein for OA production without OAC expression (i.e., producing OL instead of OA) also obtained beneficial OL yields relative to the corresponding version of the OA production control strain herein without OAC expression (i.e., producing OL instead of OA).

While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention.

Claims

1. An olivetol synthase variant, characterized in that the mutation of the olivetol synthase variant compared to the wild type olivetol synthase represented by SEQ ID NO 13 is I303T.

2. A gene encoding the olivine synthase variant according to claim 1.

3. A recombinant expression vector comprising the gene of claim 2.

4. An engineered E.coli modified to express the olivine synthase variant of claim 1, wherein the engineered E.coli has a deletion of the genomic fabH gene according to SEQ ID NO. 3 and/or the genomic fadE gene according to SEQ ID NO. 6.

5. The engineered E.coli of claim 4, wherein said engineered E.coli is modified to express an olivine acid cyclase.

6. The engineered E.coli of claim 5, wherein the engineered E.coli has been modified to overexpress a long-chain acyl-CoA synthetase.

7. The engineered E.coli of any one of claims 4 to 6, wherein the modification is by introduction of a plasmid.

8. Engineered escherichia coli according to claim 7, characterized in that the olivil synthase and the olivil acid cyclase are expressed by the same plasmid.

9. The engineered E.coli of any one of claims 4 to 8, wherein the engineered E.coli is modified from wild-type E.coli, BW25113 or BL 21.

10. Use of the variant of olive alcohol synthase according to claim 1 or the gene according to claim 2 or the recombinant expression vector according to claim 3 or the engineered escherichia coli according to any one of claims 4 to 9 for the production of olive alcohol and/or olive alcohol acid.