WO2023035396A1

WO2023035396A1 - Olivetol synthetase variant and engineered microorganism expressing same

Info

Publication number: WO2023035396A1
Application number: PCT/CN2021/129656
Authority: WO
Inventors: 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: 2021-09-10
Filing date: 2021-11-09
Publication date: 2023-03-16
Also published as: CN114196648A; CN114196649B; CN114196645A; CN114196647B; CN113502254A; CN114196649A; CN114196647A; CN114196648B; CN114196646B; CN113502254B; CN114196646A; CN114196645B

Abstract

Provided are an olivetol synthetase variant and an engineered Escherichia coli expressing same for the production of olivetol and olivetolic acid. The engineered Escherichia coli is modified to express an olivetol synthetase variant which comprises one or more of the following mutants compared with a wild-type olivetol synthetase variant: I303T, I52L, S56A, H262M, and K263R. The engineered Escherichia coli achieves improved yields of olivetol and olivetolic acid.

Description

Olivol synthase variants and engineered microorganisms expressing them

technical field

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).

Background technique

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. Because it is the common precursor of other cannabinoids, it is called the "mother of cannabinoids". As the cannabis plant grows, most of the CBG is converted into CBD and THC, and only a small amount of CBG remains in the plant, and the supply is severely limited, which limits the development of CBG applications.

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. As for the biosynthesis of olivetol and olivetolic acid, according to the knowledge of the inventors, the yield of biosynthetic olivetol and olivetolic acid is not enough to meet the requirements of industrialization with Escherichia coli as the chassis bacteria. Tan Z et al (Tan Z, Clomburg J M, Gonzalez R.Synthetic pathway for the production of olivetolic acid in Escherichia coli[J].ACS synthetic biology,2018,7(8):1886-1896.) reported that in 500mL biological The yield of olive oil in the reactor was only 80mg/L. In 2020, the patent No. WO2020176547A1 reported that by screening the olive alcohol synthase derived from Cymbidium hybrid cultivar, the test found that the yield of olive alcohol was 40.7mg/L.

There remains a need in the art for novel microorganisms that biosynthesize olivetol and olivetolic acid in high yields.

Contents of the invention

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. 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.

Therefore, in one aspect, 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.

In some embodiments of this aspect, the engineered E. coli is modified to express olivate cyclase (OAC).

In some embodiments of this aspect, the engineered E. coli has a genomic fabH gene deleted.

In some embodiments of this aspect, the engineered E. coli has a genomic fadE gene deleted.

In some embodiments of this aspect, both the genomic fabH and fadE genes of the engineered E. coli are deleted.

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

In some embodiments of this aspect, the engineered E. coli is modified from wild-type E. coli, BW25113 or BL21.

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

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

In some embodiments of this 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 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.

In some embodiments of the aspect, the method comprises modifying the rod to express olivate cyclase (OAC).

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

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

In some embodiments of this aspect, the method comprises deleting both the fabH and fadE genes of the E. coli genome.

In some embodiments of this aspect, the method comprises modifying the E. coli to overexpress long-chain acyl-CoA synthetase (fadD).

In some embodiments of the aspects, the E. coli is wild type E. coli, BW25113 or BL21.

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

Detailed ways

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 fully explained in the literature: "Molecular Cloning: A Laboratory Manual," Second Edition (Sambrook et al., 1989); "Oligonucleotide Synthesis" (ed. M.J. Gait, 1984); "Animal Cell Culture" (R.I. Freshney, 1987); "Methods in Enzymology" (Academic Press, Inc.); "Current Protocols in Molecular Biology" (F.M. Ausubel et al., 1987, and regularly updated); "PCR: The Polymerase Chain Reaction," ( Mullis et al., 1994); Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York, N.Y. 1994) and March's Advanced Organic Chemistry Reactions, Mechanisms and Structure 4th ed., John Wiley & Sons (New York, N.Y. 1994) York, N.Y. 1992), provides those skilled 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/an" and "the/the" are used herein to refer to one or more than one (ie at least one) of said articles The grammatical object of the word. For example, "an element" means one/an element or more than one/an element.

The use of an alternative (eg, "or") should be understood to mean either, both, or any combination of the alternatives.

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 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. In one embodiment, 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.

As used herein, 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. In one embodiment, the term "substantially the same" refers to a quantity, level, value, amount, frequency, A percentage, measure, size, amount, weight, or length range.

As used herein, 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.

Throughout this specification, unless the context requires otherwise, the terms "comprising", "including", "containing" and "having" are to be understood as implying the inclusion of stated steps or elements or groups of steps or elements, but not the exclusion of any other Steps or elements or groups of steps or elements. In certain embodiments, the terms "comprising", "including", "containing" and "having" are used synonymously.

"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.

Throughout this specification, reference to "one embodiment," "some embodiments," "a particular embodiment" and similar expressions means that a particular feature, structure, or characteristic described in connection with the embodiment is included in the present invention. In at least one embodiment of . 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 are disclosed with respect to a particular aspect of the invention (eg, the product of the invention), such disclosure is also considered to apply mutatis mutandis to any other aspect of the invention (eg, the method and use of the invention).

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. Without wishing to be bound by theory, it is believed that by deleting the fabH gene, intracellular utilization of increased levels of malonyl by olivol synthase (OLS) and/or olivate cyclase (OAC) in engineered microorganisms is facilitated. Coenzyme A (Malonyl-CoA) and hexanoyl-CoA (Hexanoyl-CoA) are substrates for the synthesis of olivetol and/or olivetolic acid. In this way, 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.

Therefore, in one aspect, 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.

In some embodiments, the microorganism engineered to biosynthesize olivetol and olivetolic acid is Escherichia coli (E. coli) or a relative thereof. In a specific embodiment, 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.

In some embodiments, the olivetol synthase used herein may be derived from Cannabis sativa. Olivetol synthase may be a variant optimized for expression in E. coli. In some embodiments, 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. In some embodiments, 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. In some embodiments, the olivetol synthase used in the present invention has the activity of catalyzing the synthesis of olivetol from malonyl-CoA and hexanoyl-CoA.

In some embodiments, the olivate cyclase used herein may be derived from Cannabis sativa. Olivate cyclase may be a variant optimized for expression in E. coli. In some embodiments, 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. In some embodiments, 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. In some embodiments, the olivate cyclase used in the invention has activity to carboxylate olivetol to olivate.

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

In some embodiments, fadD as used herein can be derived from E. coli. fadD may be a variant optimized for expression in E. coli. In some embodiments, 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. In some embodiments, 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. In some embodiments, fadD used in the present invention has the activity of catalyzing the production of hexanoyl-CoA from hexanoic acid by acylation.

In some embodiments, one or more enzymes used in the invention may be mutants or variants of the enzymes described herein. As used 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. In some embodiments, 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. For example, 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, His), lysine (K, Lys ) and arginine (R, Arg)); and hydrophobic pairs of 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, mutants or variants may also comprise non-conservative substitutions.

In some embodiments, 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.

As is well known to those skilled in the art, 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. 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 the codon bias for a particular host. For example, in some embodiments, 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.

A polynucleotide or polypeptide has a certain percentage of "sequence identity" or "identity" with another polynucleotide or polypeptide, meaning that when two sequences are aligned, that percentage of bases or amino acids are the same and at the same relative position. Determining the percent identity of two amino acid sequences or two nucleotide sequences may involve aligning and comparing the amino acid residues or nucleotides at corresponding positions in the two sequences. Two sequences are considered to be 100% identical if all positions in the two sequences are occupied by the same amino acid residues or nucleotides. 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 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. Preferably, said expression construct is a plasmid. Preferably, 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. Preferably, said expression construct is a plasmid. Preferably, the expression construct can be used to express fadD in E. coli, preferably E. coli BW25113. In some embodiments, 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. In some embodiments, 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. For example, 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. In some embodiments, the plasmid used herein is shown in SEQ ID NO:7. In some embodiments, the plasmid used herein is shown in SEQ ID NO: 10. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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) . In some embodiments, the genomic fadE gene in a microorganism described herein is deleted such that acyl-CoA dehydrogenase is not expressed in the microorganism. In some embodiments, 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). In some embodiments, 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. In some embodiments, 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. In some embodiments, 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.

Deletion of genomic sequences of interest in microorganisms can be performed by methods known in the art. For example, the genome sequence can be deleted by designing an artificial sequence for λ-Red homologous recombination with respect to the genome sequence, and integrating the artificial sequence into a target position in the genome by using λ-Red homologous recombination. For specific experimental schemes, please refer to the examples described herein. In some embodiments, the artificial sequence used to delete the fabH gene is shown in SEQ ID NO:1. In some embodiments, 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. In some embodiments, the artificial sequence used to delete the fadE gene is as shown in SEQ ID NO:4. In some embodiments, 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.

In addition to deleting the genomic sequence of interest in the microorganism, 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 The deletion of all or part of the promoter, the insertion of one or more nucleotides in the middle of the genomic sequence encoding the protein of interest or its regulatory elements such as a promoter, such as a stop codon, or one or more nucleotides that affect its transcription Methods of acid mutating the genomic sequence to the extent that the genomic sequence cannot be properly transcribed, introducing agents such as siRNA or dsRNAi agents that interfere with or silence the mRNA encoding the protein of interest, or inhibiting or stopping delivery of the protein of interest, e.g., to extracellular systems ( methods for the function of eg chaperones, signal sequences, transporters).

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.). In some embodiments, the medium can be supplemented with fermentable sugars (eg, hexoses, eg, glucose, xylose, etc.). In some embodiments, a suitable medium comprises an inducing agent. In certain such embodiments, 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.

Materials and methods suitable for the maintenance and growth of the microorganisms of the invention are described herein, for example in the Examples section. Other materials and methods suitable for the maintenance and growth of microorganisms such as E. coli are well known in the art. Exemplary techniques can be found in WO2009/076676; US12/335,071 (US2009/0203102); WO2010/003007; US2010/0048964; WO2009/132220; of methods for general bacteriology[J].1981.; Crueger W, Crueger A, Brock T D, et al.Biotechnology: a textbook of industrial microbiology[J].1990., which describes the standard culture conditions and fermentation that can be used Mode, such as batch, fed-batch or continuous fermentation, the entire contents of which are incorporated herein by reference in their entirety.

For small-scale production, 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. For large-scale production, 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. In some embodiments, 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. In some embodiments, 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, 600, 650, 700, 750, 800, 850, 900, 950, 1000mg/L or higher or the output of any two of the foregoing values.

The present invention also provides some following preferred embodiments:

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.

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).

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.

Item 12. The method according to any one of the preceding items, comprising modifying the coliform rod to express olivate cyclase (OAC).

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.

Example

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

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

The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.

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.

50×M: 1.25mol/L Na ₂ HPO ₄ , 1.25mol/L KH ₂ PO ₄ , 2.5mol/L NH ₄ Cl, 0.25mol/L Na ₂ SO ₄ , autoclaved for 30 minutes.

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

1M MgSO ₄ : Weigh 24.6g MgSO ₄ ·7H ₂ O and add H ₂ O to dissolve, make the volume to 100mL, and then 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 μL 1M MgSO ₄ , 100 μL 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.

Design and synthesize the H1-kana-H2 described in SEQ ID NO:1, according to the literature (Datsenko K A, Wanner B L. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products[J].Proceedings of the National Academy of Sciences, 2000,97(12): 6640-6645.) The method provided is to integrate SEQ ID NO: 1 into the fabH gene position of the genome of BW25113 by λ-Red homologous recombination so that the fabH gene is deleted, and the steps are as follows :

1. Prepare the competent state of BW25113;

2. Introduce plasmid pKD46;

3. Inoculate BW25113 (pKD46) into 3 mL of LB containing ampicillin, the concentration of ampicillin is 100 μg/L, and culture overnight in a shaker at 30°C;

4. Take 100 μL of overnight cultured BW25113 (pKD46) cell suspension and add it to 10 mL of SOB medium, then add 100 μL of 1M arabinose and 10 μL of 100 mg/L ampicillin. Cultivate on a shaker at 30°C until OD600=0.4-0.6;

Under the condition of 5.4°C, collect the bacteria by centrifugation, and resuspend the bacteria with 10 mL pre-cooled ultrapure water, wash the bacteria twice in the same way, and finally resuspend the bacteria with 50 μL of 10% glycerol solution;

6. Add 50ng of the nucleotide sequence fragment of SEQ ID NO:1, mix well and add the mixture into the electric shock cup;

7. Put the electric shock cup into the electric shock instrument and give an electric shock. The electric shock conditions are 200Ω, 25μF, and 2.5KV;

8. Add 1 mL of pre-cooled SOB medium, transfer the mixture into a sterile EP tube, and incubate on a shaker at 30°C for 1 hour;

9. Evenly spread the bacterial solution on the LB plate medium containing ampicillin and kanamycin, and culture at 30°C for 16-20 hours;

10. Verify the outgrowth of transformants on the plate.

After integrating SEQ ID NO: 1, 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:

1. Prepare the competent state of the bacterial strain integrated with SEQ ID NO:1;

2. Transform pCP20 into a competent state, and the culture temperature is 30 degrees;

3. Pick a single clone into 3mL SOB medium and culture at 30 degrees overnight;

4. Take 100μL and transfer to 10mL SOB, culture on a 30-degree shaker for 3-4 hours, OD600=0.4;

Collect the bacteria by centrifugation at 5.4°C, and wash the bacteria twice with sterile water in an ice bath;

6. Resuspend in 50μL-100μL sterile water for electroporation transformation;

7. Add 100ng of pCP20, transform by electric shock (electric shock instrument setting: 1.8KV, 5.5ms), recover at 30 degrees for 1 hour;

8. Evenly spread on LB plates containing ampicillin, and culture at 30 degrees for 2-3 days;

9. Pick a single clone, streak it on an LB plate without antibiotics, and culture it at 42 degrees overnight;

10. Pick 20-30 clones to the kanamycin-resistant plate and the non-resistant plate respectively, and the clones that cannot grow on the kanamycin-resistant plate but grow on the non-resistant plate at the same time are the target clones, and carry out PCR verification.

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

In the engineered BW25113 obtained in Example 1, 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 .

Design and synthesize the H3-kana-H4 described in SEQ ID NO: 4, as described in Example 1, integrate SEQ ID NO: 4 into the fadE gene position of the genome of BW25113 that lacks the fabH gene so that the fadE gene is deleted and integrated SEQ ID NO: 4 The KanR resistance gene was deleted after ID NO:4.

Embodiment 3: import fadD expression plasmid

In the engineered BW25113 obtained in Example 2, the long-chain acyl-CoA synthetase (fadD) was overexpressed to convert hexanoic acid (hexanoic acid) into hexanoyl-CoA, thereby increasing the accumulation of intracellular hexanoyl-CoA to further increase Synthesis of the target product OA.

Design and synthesize the 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

Design and synthesize 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, which are respectively transformed into the engineered BW25113 obtained in Example 3 , to obtain engineered BW25113 for the synthesis of OL or OA, called CZ-OL or CZ-OA, respectively.

Example 5: Introduction of OLS mutations

Design and synthesize OLS variant & OAC expression plasmid p15A-Prha-OLS variant-OAC with different OLS mutations, and transform it into the engineered BW25113 obtained in Example 3 as described in Example 4 to obtain a series of synthetic OA-containing OLS variants Body engineering BW25113.

Embodiment 6: the performance test that contains OLS variant engineered bacterial strain

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

Fermentation and sample preparation:

1. Inoculate the recombinant bacteria into 3mL LB liquid medium, cultivate overnight at 37 degrees, 220 rpm, about 14 hours, and the final OD600 value reaches 2-3;

2. Add ZYM medium to 24 deep-well plate, add 2mL to each well;

3. Transfer the bacterial solution in step 1 to the ZYM medium in step 2, and the OD600 after transfer is 0.01;

4. When the OD600 of the bacterial solution grows to 0.2, add an inducer (addition amount: 0.2% rhamnose) and precursor hexanoic acid (1 mM), a total of 24 hours from inoculation to the end of fermentation;

5. Take 1mL of fermentation broth, add 3mL of ethyl acetate, shake and mix for 10min, then centrifuge to collect the upper organic phase;

6. Repeat operation 5, and centrifuge twice to obtain organic phases, combine them and transfer about 6mL to a 10mL test tube.

7. Evaporate all the organic phase in the test tube to dryness with a vacuum concentrator, and add 1 mL of methanol to resuspend all the samples in the test tube.

8. Filter the sample from step 7 with a 0.22 μM filter and transfer to an HPLC vial.

Table 1. Cell concentration in the broth when the fermentation was stopped

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]. ACS synthetic biology, 2018, 7 (8): 1886-1896.) The realized yield, the yield is 80mg/L. In addition, 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.

Table 2. OA yields corresponding to various OLS variants

Embodiment 7: the performance test that contains OLS variant bacterial strain

To further test whether the effect of OLS variants on OA production was limited to the effects of specific engineered strains, the effects of OLS variants were tested in two unengineered E. coli strains, BW25113 and BL21.

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.

Table 3. Cell concentration in the fermentation broth when the fermentation was stopped

Table 4. OA yields corresponding to various OLS variants

Note: "-" means no OA was detected

It can be seen that the above OLS variants have achieved an increase in OA production in wild-type or engineered strains. It was concluded that the OA production enhancement effect brought about by the OLS variant was not limited to a specific strain.

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

While preferred embodiments of the present invention have been shown and described herein, it should 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 of ordinary skill 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

An engineered escherichia coli, wherein said engineered escherichia coli is modified to express olive alcohol synthase variant, it is characterized in that said olive alcohol synthase variant comprises compared with its wild type shown in SEQ ID NO:13 One or more selected from the following mutations: I303T, I52L, S56A, H262M and K263R.
The engineered escherichia coli according to claim 1, characterized in that the engineered escherichia coli is modified to express olivate cyclase.
The engineered escherichia coli according to claim 1, characterized in that the genome fabH gene shown in SEQ ID NO: 3 of said engineered escherichia coli is deleted.
According to the engineering escherichia coli described in any one of claim 1-3, it is characterized in that the genome fadE gene shown in the SEQ ID NO:6 of described engineering escherichia coli is deleted.
According to the engineered escherichia coli any one in the claim 1-3, it is characterized in that both of genome fabH and fadE gene shown in SEQ ID NO:3 and 6 of said engineered escherichia coli are deleted.
The engineered Escherichia coli according to any one of claims 1-3, characterized in that the engineered Escherichia coli is modified to overexpress long-chain acyl-CoA synthetase.
The engineered Escherichia coli according to any one of claims 1-3, characterized in that the engineered Escherichia coli is modified from wild-type Escherichia coli, BW25113 or BL21.
The engineered Escherichia coli according to any one of claims 1-3, characterized in that said modification is performed by introducing a plasmid.
The engineered Escherichia coli according to any one of claims 1-3, characterized in that the olivetol synthase and olivetol acid cyclase are expressed by the same plasmid.
The engineered Escherichia coli according to any one of claims 1-3, characterized in that the long-chain acyl-CoA synthetase is overexpressed through a plasmid.
A method for preparing engineered escherichia coli, comprising modifying escherichia coli to express olive alcohol synthase variant, characterized in that the olive alcohol synthase variant is compared with its wild type shown in SEQ ID NO:13 Comprising one or more of the following mutations: I303T, I52L, S56A, H262M and K263R.
The method according to claim 11, characterized in that said method comprises modifying said E. coli rod to express olivate cyclase.
The method according to claim 11, characterized in that the method comprises deletion of the fabH gene of the genome shown in the SEQ ID NO:3 of the escherichia coli.
The method according to any one of claims 11-13, characterized in that the method comprises deletion of the genome fadE gene shown in SEQ ID NO: 6 of the escherichia coli.
Method according to any one of claims 11-13, characterized in that said method comprises the deletion of both the fabH and fadE genes of the genome of said E. coli.
The method according to any one of claims 11-13, characterized in that said method comprises modifying said Escherichia coli to overexpress long-chain acyl-CoA synthetase.
The method according to any one of claims 11-13, characterized in that the Escherichia coli is wild type Escherichia coli, BW25113 or BL21.
Method according to any one of claims 11-13, characterized in that said modification is carried out by introducing a plasmid.
The method according to any one of claims 11-13, characterized in that said olivetol synthase and olivetate cyclase are expressed by the same plasmid.
The method according to any one of claims 11-13, characterized in that the long-chain acyl-CoA synthetase is overexpressed by a plasmid.