Polyamine analog-producing yeast

Abstract

The present invention relates to the production of polyamine analogs in yeast cells capable of producing at least one polyamine. The yeast cells also contain a 4-coumarate:CoA ligase-encoding gene, at least one polyamine N-acyltransferase gene, and at least one polyamine synthase-encoding gene, but lack a polyamine oxidase-encoding gene or contain a disrupted polyamine oxidase-encoding gene. The yeast cells are capable of producing mono- and / or poly-substituted N-acylated polyamines.

Description

Technical Field

[0001] The present invention generally relates to genetically modified yeasts, and in particular, to yeasts capable of producing polyamine analogs.

Background Art

[0002] Polyamine analogs widely distributed in nature are used to address problems in the health and agricultural fields. For example, amide bond-containing polyamine analogs, such as N 1 -coumaroyl-spermine, N 1 -guanyl-1,7-diamino-heptane, and N 1 ,N 11 -diethyl-norspermine represent an important class of antiviral agents, antioxidants, antagonists, and chemotherapeutic agents that have the potential to be used to combat human diseases such as cancer and emerging viral threats such as COVID-19. Similarly, the widely distributed amide bond-containing hydroxycinnamic acid amides of diamines and polyamines, such as di-p-coumaroyl-caffeoyl-spermidine, can significantly reduce Blumeria graminis infection and demonstrate the potential to be used to combat fungal pathogens.

[0003] However, due to the complexity of their structures and their low abundance in nature, it is difficult to obtain polyamine analogs from either conventional synthetic chemistry or extraction from natural sources. Microbial-based production in rapidly growing, genetically tractable species has been promoted as an alternative to traditional supply chains for natural products and their derivatives. In particular, Saccharomyces cerevisiae has functioned as a cell factory for the production of many different fuels, chemicals, food ingredients, and pharmaceuticals, especially for the production of natural products. Indeed, Microbial Cell Factories (2016) 15:198 discloses the co-expression of BAHD acyltransferases and At4CL5 in Saccharomyces cerevisiae (S. cerevisiae), and the cloning of different BAHD acyltransferase coding sequences into vectors containing the Arabidopsis thaliana gene At4CL5 for the production of various hydroxycinnamate and benzoate conjugates.

[0004] Unfortunately, the elucidation of the chemical space of polyamine analogs for further pharmacological and pesticidal studies is hampered by several limitations: (i) the difficulty in accessing diverse polyamines, i.e., the precursors for the synthesis or biosynthesis of polyamine analogs from either conventional synthetic chemistry or extraction from natural resources, which limits the diversity of polyamine analogs; (ii) the lack of knowledge about biosynthetic enzymes, e.g., for the biosynthesis of polyamines and polyamine analogs; (iii) the lack of knowledge about the biochemical functions of polyamines, which limits the clinical use of polyamines.

[0005] As a result, there is a desire to develop new methods for the production of natural polyamines and their analogs, as well as new technologies and value chains to supply both natural and non-natural variants of these structures. SUMMARY OF THE INVENTION

Problems to be Solved by the Invention

[0006] A general object is to provide yeast cells capable of producing polyamine analogs.

Means for Solving the Problems

[0007] This object and other objects are satisfied by embodiments.

[0008] The present invention is defined in the independent claims. Further embodiments of the present invention are defined in the dependent claims.

[0009] The present invention relates to yeast cells capable of producing at least one polyamine analog. The yeast cells are capable of producing at least one polyamine. Further, the yeast cells contain a 4-coumaric acid:CoA ligase-encoding gene, at least one polyamine N-acyltransferase gene, and at least one polyamine synthase-encoding gene, but lack a polyamine oxidase-encoding gene or contain a disrupted polyamine oxidase-encoding gene.

[0010] The present invention also relates to a method for producing a polyamine analog. The method includes culturing yeast cells according to the present invention in a medium under culture conditions suitable for the production of polyamine analogs by the yeast cells. The method also includes collecting the polyamine analog from the medium and / or from the yeast cells.

[0011] The present invention provides an efficient means for producing various polyamine analogs including mono-substituted and / or multi-substituted N-acylated polyamines. Therefore, the present invention can be used as a cost-effective alternative to conventional synthetic chemistry for obtaining polyamine analogs or conventional techniques including extraction from natural sources.

[0012] The embodiments can be best understood by referring to the following description together with the accompanying drawings, along with its further objects and advantages.

Brief Description of the Drawings

[0013]

Figure 1a

Figure 1b

Figure 1c

Figure 1d

Figure 1e

Figure 1f

Figure 2a

Figure 2b

Figure 3a

Figure 3b

Figure 3c

Figure 3d

Figure 3e

Figure 3f

Figure 4a

Figure 4b

Figure 4c

Figure 5a

Figure 5b

Figure 6a

Figure 6b

Figure 6c

Figure 7a

Figure 7b

Figure 7c

Figure 7d

Figure 8a

Figure 8b

Figure 8c

Figure 8d

Figure 8e

Mode for Carrying Out the Invention

[0014] To enable efficient access to the diversity of polyamine analogs, the inventors modified yeast metabolism to overproduce complex polyamine categories such as spermidine, homospermidine, thermospermine, and spermine. The versatility of this yeast platform is demonstrated by the biosynthesis of diverse polyamine analogs via tailoring pathways. In particular, the inventors were able to enable yeast to produce >400 mg / l spermidine in deep-well scale fermentation by systematically refactoring yeast central carbon and nitrogen metabolism, the methionine salvage pathway, the adenine salvage pathway, the polyamine transport machinery, and the polyamine degradation pathway. Furthermore, by plugging into the tailoring pathway and creating a synthetic consortium, the inventors demonstrated de novo biosynthesis of polyamine analogs including trisubstituted N-acylated spermidine phenolamides in yeast.

[0015] Next, the present invention will be described below with reference to the accompanying drawings and examples showing embodiments of the present invention. This description is not intended to be a detailed catalog of all different ways in which the present invention can be practiced or all features that can be added to the present invention. For example, features shown with respect to one embodiment may be incorporated into another embodiment, and features shown with respect to a particular embodiment may be deleted from that embodiment. Thus, the present invention contemplates that, in some embodiments of the present invention, any feature or combination of features described herein may be excluded or omitted. Also, numerous modifications and additions to the various embodiments proposed herein will be apparent to those skilled in the art in light of the present disclosure without departing from the present invention. Therefore, the following description is intended to illustrate some specific embodiments of the present invention and is not intended to comprehensively identify all rearrangements, combinations, and variations thereof.

[0016] Unless otherwise defined herein, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art.

[0017] Generally, the nomenclature used in connection with the biochemical, enzymological, molecular and cell biological, microbiological, genetic, protein and nucleic acid chemical, and hybridization techniques described herein is well-known and generally used in the art.

[0018] Conventional methods and techniques referred to herein are described in more detail, for example, in Molecular Cloning, a laboratory manual [2nd edition] Sambrook et al. Cold Spring Harbor Laboratory, 1989, for example, in its Sections 1.21 "Extraction And Purification Of Plasmid DNA", 1.53 "Strategies For Cloning In Plasmid Vectors", 1.85 "Identification Of Bacterial Colonies That Contain Recombinant Plasmids", 6 "Gel Electrophoresis Of DNA", 14 "In vitro Amplification Of DNA By The Polymerase Chain Reaction", and 17 "Expression Of Cloned Genes In Escherichia coli".

[0019] The Enzyme Commission (EC) numbers (also referred to herein as "classes") referenced throughout this specification follow those of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (NC-IUBMB) in its resource "Enzyme Nomenclature" (1992, including Supplements 6 - 17) (e.g., "Enzyme nomenclature 1992: recommendations of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology on the nomenclature and classification of enzymes", Webb, E.C. (1992), San Diego (published by Academic Press for the International Union of Biochemistry and Molecular Biology) (ISBN 0-12-227164-5), available as such). This is a numerical classification scheme based on the chemical reactions catalyzed by each enzyme class.

[0020] Unless the context indicates otherwise, it is specifically intended that the various features of the invention described herein can be used in any combination. Furthermore, the invention also contemplates that, in some embodiments of the invention, any feature or combination of features described herein can be excluded or omitted. By way of example, if the specification states that a composition comprises components A, B, and C, it is specifically intended that any one of A, B, or C, or combinations thereof, can be omitted and waived, either singly or in any combination.

[0021] As used in the description of the present invention and the appended claims, the singular forms "a", "an", and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. Also, as used herein, "and / or" refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the absence of combinations when interpreted alternatively ("or").

[0022] Throughout the description and claims of this specification, the words "comprise", "contain", and variations of the words, such as "comprising" and "comprises", mean "include but are not limited to", and do not exclude other parts, additives, components, integers, or steps. Throughout the description and claims of this specification, the singular form includes the plural form unless the context requires otherwise. In particular, when an indefinite article is used, this specification should be understood as contemplating both plural and singular, unless the context requires otherwise.

[0023] As used herein, the transitional phrase "consisting essentially of" is to be interpreted to mean that the claim includes what is specified in the claim, namely a particular material or step, and those that do not materially affect the basic and novel characteristics of the claimed invention. Thus, the phrase "consisting essentially of" is not intended to be interpreted as equivalent to "comprising" when used in the claims of the present invention.

[0024] To facilitate understanding of the present invention, some terms are defined below.

[0025] As used herein, the term "polyamine" refers to an organic compound having two or more primary amino groups. Examples of polyamines include putrescine (Put), spermidine (Spd), spermine (Spm), thermospermine (Tspm), sym-homospermidine (Hspd), 1,2-diaminopropane, cadaverine, agmatine, sym-norspermidine, and norspermine.

[0026] As used herein, the terms "polyamine analog", "polyamine analog", or "polyamine conjugate" refer to an organic compound formed by reacting a polyamine with at least one molecule to form an amide bond between the polyamine and the at least one molecule. In certain embodiments, the at least one molecule is at least one carboxyl group-containing molecule, thereby enabling coupling of the carboxylic acid moiety with the amine group of the polyamine. Non-limiting but preferred examples of such carboxyl group-containing molecules include aromatic organic acids such as hydroxycinnamic acids (α-cyano-4-hydroxycinnamic acid, caffeic acid, chicoric acid, cinnamic acid, chlorogenic acid, diferulic acid, dihydrocaffeic acid, coumaric acid, coumarin, ferulic acid, and sinapic acid); hydroxycinnamoyltartaric acid (cafutaric acid, coutaric acid, and fertaric acid); phenolic acids (monohydroxybenzoic acids such as 3-hydroxybenzoic acid, 4-hydroxybenzoic acid, salicylic acid, and p-hydroxybenzoic acid glucoside); dihydroxybenzoic acids (2,3-dihydroxybenzoic acid, 2,4-dihydroxybenzoic acid, 2,6-dihydroxybenzoic acid, 3,5-dihydroxybenzoic acid, ethyl protocatechuate, gentisic acid, homogentisic acid, orsellinic acid, and protocatechuic acid); trihydroxybenzoic acids (bergenin, kebuleic acid, ethyl gallate, eudesmic acid, gallic acid, tannic acid, norbergenin, phloroglucinolcarboxylic acid, syringic acid, and theogallin); vanillin; and ellagic acid. Polyamine analogs formed by reacting a polyamine with an aromatic organic acid are typically referred to as polyamine alkaloids.As other examples of carboxyl group-containing molecules, fatty acids (including, but not limited to, saturated fatty acids such as caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acid, lignoceric acid, and cerotic acid; and unsaturated fatty acids such as myristoleic acid, palmitoleic acid, sapienic acid, oleic acid, elaidic acid, vaccenic acid, linoleic acid, linolenic acid, α-linolenic acid, arachidonic acid, eicosapentaenoic acid, erucic acid, and docosahexaenoic acid) are included. Polyamine analogs formed by reacting polyamines with fatty acids are typically referred to as polyamine-fatty acid conjugates.

[0027] In the art of polyamine nomenclature, generally, the number of atoms within the alkaloid and the reordering of locants are important. Primarily two numbering systems are used in the literature. In this specification, the numbering system disclosed by Bentz et al. in 2015 is used. The system is briefly summarized by the following rules: I. The numbering of the polyamine skeleton includes the entire polyamine structure including the terminal N atom; II. The numbering starts with the N atom at the end of the shortest carbon chain (e.g., for spermidine having a primary amino group of an aminopropyl subunit); III. For symmetric skeletons, such as in the case of spermine, the numbering starts from the site of the molecule that gives the lowest locant for the substituent; and IV. For N-derivatized polyamines, the locant of the N-substituent is predetermined as N n and n coincides with the locant number of the substituted N atom.

[0028] Also, as used herein, the terms “nucleotide sequence,” “nucleic acid,” “nucleic acid molecule,” “oligonucleotide,” and “polynucleotide” refer to RNA or DNA (including cDNA, DNA fragments or portions, genomic DNA, synthetic DNA, plasmid DNA, mRNA, and antisense RNA), all of which can be single-stranded or double-stranded, linear or branched, or hybrids thereof. The nucleic acid molecules and / or nucleotide sequences provided herein are shown herein in the 5' to 3' direction from left to right, and are represented using the standard codes for representing nucleotide letters as set forth in the U.S. sequence rules, 37 CFR §§ 1.821-1.825, and the World Intellectual Property Organization (WIPO) Standard ST.25. When dsRNA is synthetically produced, less common bases such as inosine, 5-methylcytosine, 6-methyladenine, and hypoxanthine can also be used for antisense, dsRNA, and ribozyme pairing. For example, polynucleotides containing C-5 propyne analogs of uridine and cytidine have been shown to bind to RNA with high affinity and be potent antisense inhibitors of gene expression. Other modifications can also be made, such as to the phosphodiester backbone or to the 2'-hydroxy in the ribose sugar of RNA. As used herein, the term “recombinant” when used results in a construct having a structural coding or non-coding sequence distinguishable from the endogenous nucleic acid found in the natural system, meaning a product of various combinations of cloning, restriction, and / or ligation steps.

[0029] As used herein, the term "gene" refers to a nucleic acid molecule that can be used to generate mRNA, antisense RNA, miRNA, and anti-microRNA antisense oligodeoxynucleotides (AMO), etc. A gene may or may not be used to produce a functional protein or gene product. A gene can include both coding and non-coding regions, such as introns, regulatory elements, promoters, enhancers, termination sequences, and / or 5' and 3' untranslated regions. A gene may be "isolated", which means a nucleic acid substantially or essentially free of components normally found associated with a nucleic acid in its natural state. Such components include other cellular materials, media from recombinant production, and / or various chemicals used in the chemical synthesis of nucleic acids.

[0030] As defined herein, a "disrupted gene" includes any mutation or modification to a gene that results in a partially or completely non-functional gene and gene product. Such mutations or modifications include, but are not limited to, missense mutations, nonsense mutations, deletions, substitutions, insertions, and addition of targeting sequences. Further, disruption of a gene can also, or alternatively, be achieved by mutation or modification of regulatory elements that control transcription of the gene, such as mutations or modifications in promoters, terminators, and / or enhancer elements. In such cases, such mutations or modifications result in a partial or complete loss of transcription of the gene, i.e., a decrease or reduction in transcription compared to a native and unmodified regulatory element. As a result, only a small amount of gene product, if any, is available after transcription and translation. Further, disruption of a gene can also involve addition or removal of a localization signal from the gene, reducing the presence of the gene product within its natural intracellular compartment.

[0031] The purpose of gene disruption is to reduce the amount of available gene product (including completely preventing any production of the gene product), or to express a gene product that lacks enzymatic activity or has lower enzymatic activity compared to the native or wild-type gene product.

[0032] As used herein, the terms "deletion" or "knockout" refer to a gene that is inoperative or has been knocked out.

[0033] The term "attenuated activity", when referring to an enzyme, refers to a decrease in the activity of the enzyme within its native compartment compared to a control or wild-type state. Modifications that result in attenuated enzyme activity include, but are not limited to, missense mutations, nonsense mutations, deletions, substitutions, insertions, addition of a targeting sequence, or removal of a targeting sequence. A cell containing a modification that results in attenuated enzyme activity has lower enzyme activity compared to a cell that does not contain such a modification. Attenuated enzyme activity may be achieved by encoding a non-functional gene product, e.g., a polypeptide having essentially no activity, e.g., less than about 10% or even less than 5% of the activity compared to the activity of a wild-type polypeptide.

[0034] The codon-optimized version of a gene refers to a foreign gene that is introduced into a cell and the codons of the gene are optimized for a particular cell. Generally, not all tRNAs are expressed equally or at the same level throughout a species. Thus, codon optimization of a gene sequence involves changing the codons so that they match the most common tRNAs, i.e., changing codons recognized by less common tRNAs to synonymous codons recognized by more common tRNAs in a given cell. In this way, mRNA derived from the codon-optimized gene will be translated more efficiently. Codons and synonymous codons preferably encode the same amino acid.

[0035] As used herein, the terms "peptide," "polypeptide," and "protein" are used interchangeably to refer to polymers of amino acid residues. Also, the terms "peptide," "polypeptide," and "protein" include modifications such as, but not limited to, lipid attachment, glycosylation, glycosylation of L-glutamic acid residues, sulfation, hydroxylation, and γ-carboxylation, as well as ADP-ribosylation.

[0036] As used herein, the term "enzyme" is defined as a protein that catalyzes a chemical or biochemical reaction within a cell. Typically, according to the present invention, a nucleotide sequence encoding an enzyme is operably linked to a nucleotide sequence (promoter) that causes expression of the corresponding gene in the cell sufficient to confer upon the cell the ability to produce spermidine.

[0037] As used herein, the term "open reading frame (ORF)" refers to a region of RNA or DNA that encodes a polypeptide, peptide, or protein.

[0038] As used herein, the term "genome" encompasses both plasmids and chromosomes in a host cell. For example, a coding nucleic acid of the present disclosure introduced into a host cell can be part of the genome regardless of whether the nucleic acid is integrated into the chromosome or localized to a plasmid.

[0039] As used herein, the term "promoter" refers to a nucleic acid sequence having a function of controlling the transcription of one or more genes, which is located upstream with respect to the direction of transcription of the transcription start site of the gene. Suitable promoters in this context include both natural constitutive promoters and inducible promoters, as well as modified promoters, which are well known to those skilled in the art.

[0040] Examples of promoters suitable for use in yeast cells include, but are not limited to, the promoters of PDC, GPD1, TEF1, PGK1, and TDH. Other suitable promoters include the promoters of GAL1, GAL2, GAL10, GAL7, CUP1, HIS3, CYC1, ADH1, PGL, GAPDH, ADC1, URA3, TRP1, LEU2, TPI, AOX1, and ENO1.

[0041] As used herein, the term "terminator" refers to a "transcription termination signal" unless otherwise specified. A terminator is a sequence that impedes or stops the transcription of a polymerase.

[0042] As used herein, a "recombinant eukaryotic cell" according to the present disclosure is defined as a cell that contains an additional copy of an endogenous nucleic acid sequence, or is transformed with a polypeptide or nucleotide sequence that is not naturally present in a eukaryotic cell, or is genetically modified. A wild-type eukaryotic cell is defined as the parental cell of the recombinant eukaryotic cell used herein.

[0043] As used herein, the terms "increasing", "increased", "increasing", "enhancing", "enhanced", "enhancing", and "enhancement" (and their grammatical variations) indicate an increase 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%, 300%, 400%, 500%, or more, or any range therein, compared to a control.

[0044] As used herein, the terms "reduce", "reduced", "reduction", "decrease", "inhibit", and "lower", and similar terms, mean a decrease 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%, 300%, 400%, 500%, or more, or any range therein, as compared to a control.

[0045] The reduction of gene expression as used herein includes genetic modifications that reduce the transcription of a gene, reduce the translation of mRNA transcribed from the gene, and / or reduce the post-translational processing of the protein translated from the mRNA. Such genetic modifications include insertions, deletions, substitutions, or mutations made to the regulatory sequences of the gene, such as promoters and enhancers. For example, the transcription of a gene can be reduced by replacing the promoter of the gene with a less active or less inducible promoter. Also, knockout of the promoter results in a reduction of gene expression, typically to zero.

[0046] As used herein, the term "a part, portion" or "fragment" of a nucleotide sequence of the present invention means a nucleotide sequence that is shorter in length compared to a reference nucleic acid or nucleotide sequence and is identical or substantially identical, e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 98%, or 99% identical, to the reference nucleic acid or nucleotide sequence, consisting essentially of, and / or consisting of, a contiguous nucleotide sequence of the reference nucleic acid or nucleotide sequence. Such nucleic acid fragments or portions according to the present invention may, where appropriate, be included as components within larger polynucleotides.

[0047] Different nucleic acids or proteins having identity are referred to herein as "homologs". The term homolog includes homologous sequences from the same species and other species, as well as orthologous sequences from the same species and other species. "Identity" refers to the level of similarity, expressed as a percentage of sequence similarity or identity, of position identity between two or more nucleic acid sequences and / or amino acid sequences. Identity also refers to the concept of similar functional properties between different nucleic acids or proteins. Thus, the compositions and methods of the present invention further include homologs to the nucleotide sequences and polypeptide sequences of the present invention. As used herein, "ortholog" refers to homologous nucleotide sequences and / or amino acid sequences in different species that arose from a common ancestral gene during speciation. Homologs of the nucleotide sequences of the present invention have substantial sequence identity with the nucleotide sequences, for example, at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and / or 100%.

[0048] As used herein, the term "overexpress" or "overexpression" refers to a higher level of activity of a gene, such as transcription of the gene; a higher level of translation of mRNA into protein; and / or a higher level of production of a gene product, such as a polypeptide, in a cell in its native or control state that has not been transformed with a particular heterologous or recombinant polypeptide that is to be overexpressed. A typical example of an overexpressed gene is a gene that is under the transcriptional control of a different promoter as compared to the native promoter of the gene. Also, or alternatively, other changes to regulatory elements of the gene, such as enhancers, can be used to overexpress a particular gene. Further, modifications that affect, i.e., increase, the translation of mRNA transcribed from a gene can be used, alternatively or additionally, to achieve an overexpressed gene as used herein. Also, the term can refer to an increase in the copy number of a gene in a cell, and / or an increase in the amount of mRNA and / or gene product. Further, overexpression can be achieved by including genes from different species that encode the same or homologous gene products, such as enzymes. Overexpression can result in a level in a cell that is 25%, 50%, 75%, 100%, 200%, 300%, 400%, 500%, 750%, 1000%, 1500%, 2000%, or higher than that level, or any range therebetween, as compared to a control level.

[0049] As used herein, the terms "foreign" or "heterologous," when used with respect to a nucleic acid (RNA or DNA), protein, or gene, refer to a nucleic acid, protein, or gene that is not naturally present as part of the cell, organism, genome, or RNA or DNA sequence into which it is introduced (including multiple copies of a naturally occurring nucleotide sequence that are not naturally present). Such foreign genes can be genes from another species or strain, a modified, mutated, or evolved version of a gene that naturally occurs in the host cell, or a chimeric version of a gene that naturally occurs in the host cell or a fusion gene. In the former cases, the modification, mutation, or evolution results in a modified, mutated, or evolved gene having a different nucleotide sequence compared to the gene that naturally occurs in the host cell by causing a change in the nucleotide sequence of the gene. An evolved gene refers to a gene obtained by genetic modification, such as mutation, or exposure to evolutionary pressure that encodes an evolved gene and has a different nucleotide sequence compared to the wild-type or original gene. A chimeric gene is formed by a combination of portions of one or more coding sequences such that a new gene is generated. These modifications integrate the entire gene sequence into a single reading frame and, in many cases, are different from fusion genes that retain their original function.

[0050] "Endogenous," "native," or "wild-type" nucleic acid, nucleotide sequence, polypeptide, or amino acid sequence refers to a nucleic acid, nucleotide sequence, polypeptide, or amino acid sequence that is naturally occurring or endogenous. Thus, for example, "wild-type mRNA" is mRNA that is naturally present or endogenous within an organism.

[0051] As used herein, the term "modified", when used with respect to an organism, refers to a host organism that has been modified to enable the production of at least one polyamine analog (compared to the same host organism that is not so modified, which is normal). In principle, such "modifications" according to the present disclosure can include any physiological, genetic, chemical, or other modifications that appropriately alter the production of polyamine analogs in the host organism (compared to the same organism that is not modified, which is normal). However, in most embodiments, the modifications will include genetic modifications. In certain embodiments, the modifications described herein include introducing a gene into the host cell. Genetic modifications that boost the activity of a polypeptide include, but are not limited to: introducing one or more copies of a gene encoding a polypeptide (distinguishable from any gene already present in the host cell that encodes a polypeptide having the same activity); altering a gene present in the cell (e.g., changing, adding additional sequences, substituting one or more nucleotides, deleting, or exchanging sequences for regulatory sequences, promoter sequences, or other sequences) so as to increase the transcription or translation of the gene; and altering the sequence of a gene encoding a polypeptide (e.g., non-coding or coding sequences) so as to boost the activity (e.g., by increasing enzyme activity, reducing feedback inhibition, targeting a specific intracellular location, boosting mRNA stability, boosting protein stability).Genetic modifications that reduce the activity of a polypeptide include, but are not limited to, deleting some or all of the gene encoding the polypeptide; inserting a nucleic acid sequence that disrupts the gene encoding the polypeptide; and altering a gene present in a cell to reduce the transcription or translation of the gene, or the stability of the mRNA or polypeptide encoded by the gene (e.g., by adding additional sequences to one or more nucleotides, changing the nucleotides, deleting sequences from the nucleotides, substituting the nucleotides, or exchanging the nucleotides, such as by substituting one or more nucleotides, promoter sequences, regulatory sequences, or other sequences). The term "overproduction" as used herein with respect to the production of a product in a host cell indicates that, due to the introduction of nucleic acid sequences encoding various polypeptides involved in the metabolic pathways of the host cell or as a result of other modifications, the host cell produces more product compared to an unmodified host cell or a wild-type cell.

[0052] As used herein, the term "vector" is defined as a linear or circular DNA molecule that contains a polynucleotide encoding a polypeptide of the invention and is operably linked to additional nucleotides that ensure its expression.

[0053] "Introducing" in the context of a yeast cell means contacting the nucleic acid molecule with the cell such that the nucleic acid molecule gains access to the interior of the cell. Thus, a polynucleotide and / or nucleic acid molecule can be introduced into a yeast cell in a single transformation event in a separate transformation event. Therefore, the term "transformation" as used herein refers to the introduction of a heterologous nucleic acid into a cell. Transformation of a yeast cell may be stable or transient.

[0054] "Transient transformation" in the context of a polynucleotide means that the polynucleotide is introduced into a cell and not integrated into the genome of the cell.

[0055] "Stably introducing" or "stably introduced" in the context of a polynucleotide introduced into a cell means that the introduced polynucleotide is stably integrated into the genome of the cell, such that the cell is stably transformed by the polynucleotide. As used herein, "stable transformation" or "stably transformed" means that a nucleic acid molecule is introduced into a cell and integrated into the genome of the cell. Thus, the integrated nucleic acid molecule can be inherited by its progeny, more specifically by the progeny of multiple successive generations. Also, as used herein, stable transformation can refer to a nucleic acid molecule maintained extrachromosomally, for example as a minichromosome.

[0056] Transient transformation can be detected, for example, by enzyme-linked immunosorbent assay (ELISA) or Western blot, which can detect the presence of a peptide or polypeptide encoded by one or more nucleic acid molecules introduced into an organism. Stable transformation of a cell can be detected, for example, by Southern blot hybridization assay of nucleic acid sequences in the genomic DNA of the cell that specifically hybridize to the nucleotide sequence of the nucleic acid molecule introduced into an organism (e.g., yeast). Stable transformation of a cell can be detected, for example, by Northern blot hybridization assay of nucleic acid sequences in the RNA of the cell that specifically hybridize to the nucleotide sequence of the nucleic acid molecule introduced into yeast or other organism. Also, stable transformation of a cell can be detected, for example, by polymerase chain reaction (PCR) or other amplification reactions well known in the art using specific primer sequences that hybridize to a target sequence of the nucleic acid molecule and result in amplification of the target sequence (which can be detected according to standard methods). Also, transformation can be detected by direct sequencing and / or hybridization protocols well known in the art.

[0057] In addition, embodiments of the present invention include variants of the polypeptides defined herein. As used herein, a "variant" means a polypeptide whose amino acid sequence is different from the nucleotide sequence from which it is derived in that one or more amino acids within the sequence have been replaced with other amino acids. For example, a variant of SEQ ID NO: 1 may have an amino acid sequence that is at least about 50% identical to SEQ ID NO: 1, such as at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or about 100% identical. Variants and / or fragments are functional variants / fragments in that the variant sequence has similar or identical functional enzyme activity characteristics as the enzyme having the non-variant amino acid sequence specified herein (and this is the meaning of the term "functional variant" used throughout this specification).

[0058] Accordingly, any "functional variant" or "functional fragment" of the presented amino acid sequences is any amino acid sequence that remains within the same enzyme category (i.e., having the same EC number) as the non-variant sequence. Methods for determining whether an enzyme falls into a particular category are well known to those skilled in the art, and a person skilled in the art can determine the enzyme category without using the technology of the present invention. Suitable methods can be obtained, for example, from the International Union of Biochemistry and Molecular Biology.

[0059] Amino acid substitutions can be considered "conservative" when the amino acid is replaced with a different amino acid whose properties are generally similar. A non-conservative substitution is when the amino acid is replaced with a different type of amino acid.

[0060] "Conservative substitution" means the substitution of an amino acid by another amino acid of the same class, where the classes are defined as follows: Examples of amino acid classes Non-polar: A, V, L, I, P, M, F, W Uncharged polar: G, S, T, C, Y, N, Q Acidic: D, E Basic: K, R, H.

[0061] As is well known to those skilled in the art, even if the primary structure of a polypeptide is changed by conservative substitution, the activity of the polypeptide may not be significantly changed. This is because the side chains of the amino acids inserted into the sequence may be able to form bonds and contacts similar to those of the substituted amino acids. This is the case even when the substitution is in a region important for determining the higher-order structure of the polypeptide.

[0062] In embodiments of the present invention, non-conservative substitutions are possible as long as they do not interfere with the enzymatic activity of the polypeptide, as defined elsewhere herein. The substituted version of the enzyme must retain the characteristic of remaining in the same enzyme class as the unsubstituted enzyme, as determined using the above NC-IUBMB nomenclature.

[0063] Broadly speaking, fewer non-conservative substitutions than conservative substitutions that do not alter the biological activity of the polypeptide would be possible. Determining the effect of any substitution (or indeed any amino acid deletion or insertion) is entirely within the routine capabilities of those skilled in the art to readily determine whether the mutant polypeptide retains enzymatic activity, in accordance with aspects of the present invention. For example, when determining whether a mutant of a polypeptide falls within the scope of the present invention (i.e., is a "functional mutant or fragment" as defined above), one skilled in the art will determine whether the mutant or fragment retains the substrate-converting enzyme activity defined with reference to the NC-IUBMB nomenclature referred to elsewhere herein. All such mutants are within the scope of the present invention.

[0064] Using the standard genetic code, additional nucleic acid sequences encoding polypeptides can be readily conceived and produced by those skilled in the art in addition to those disclosed herein. The nucleic acid sequence may be DNA or RNA, and in the case of a DNA molecule, it may contain, for example, cDNA or genomic DNA. The nucleic acid may be contained within an expression vector as described elsewhere herein.

[0065] Accordingly, embodiments of the present invention include variant nucleic acid sequences encoding polypeptides contemplated by embodiments of the present invention. The term "variant" with respect to a nucleic acid sequence means any substitution, mutation, modification, replacement, deletion, or addition of one or more nucleotides from, or to, a polynucleotide sequence, provided that the resulting polypeptide sequence encoded by the polynucleotide exhibits at least the same, or similar, enzymatic properties as the polypeptide encoded by the nucleotide sequence. The term includes allelic variants and also includes polynucleotides that hybridize substantially to the polynucleotide sequences of embodiments of the present invention ("probe sequences"). Such hybridization can occur under low stringency conditions, high stringency conditions, or between them. Generally, the term low stringency conditions can be defined as hybridization that occurs at a temperature about 40 - 48°C lower than the calculated or actual melting temperature (Tm) of the probe sequence (e.g., about ambient laboratory temperature to about 55°C) in a 0.330 - 0.825 M NaCl buffer solution during the washing step. On the other hand, high stringency conditions include washing in a 0.0165 - 0.0330 M N buffer solution at a temperature about 5 - 10°C lower than the calculated or actual Tm of the probe sequence (e.g., about 65°C). The buffer solution can be, for example, a saline-sodium citrate (SSC) buffer (0.15 M NaCl and 0.015 M trisodium citrate), with low stringency washing performed in 3×SSC buffer and high stringency washing performed in 0.1×SSC buffer. The procedures related to hybridization of nucleic acid sequences are described, for example, in Molecular Cloning, a laboratory manual [2nd edition] Sambrook et al. Cold Spring Harbor Laboratory, 1989, for example, in its Section 11 "Synthetic Oligonucleotide Probes".

[0066] Preferably, the nucleic acid sequence variant has at least about 80% identity, more preferably at least 85%, even more preferably 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more of the nucleotides common to the nucleic acid sequence of the embodiments of the present invention.

[0067] The variant nucleic acids of the present invention may be codon-optimized for expression in a particular host cell.

[0068] As used herein, "sequence identity" refers to sequence similarity between two nucleotide sequences, or between two peptide or protein sequences. Similarity is determined by sequence alignment for the purpose of determining the structural and / or functional relationship between sequences.

[0069] The sequence identity between amino acid sequences can be determined by comparing the sequence alignments using the Needleman-Wunsch Global Sequence Alignment Tool available from the National Center for Biotechnology Information (NCBI), Bethesda, Md., USA, for example, via http: / / blast.ncbi.nlm.nih.gov / Blast.cgi, using the default parameter settings (for protein alignment, Gap costs Existence: 11, Extension: 1). The sequence comparisons and percentage identities referred to herein were determined using this software. When determining the level of sequence identity, for example, when comparing to SEQ ID NO: 1, the comparison should preferably be made against the full length of SEQ ID NO: 1 in order to avoid short regions of high identity overlap producing a high overall identity score (i.e., using the global alignment method). For example, a short polypeptide fragment having, for example, five amino acids may have a 100% identical sequence to a five amino acid region within the entirety of SEQ ID NO: 1, but this does not give 100% amino acid identity unless the fragment forms part of a longer sequence having the same amino acids at other positions corresponding to positions within SEQ ID NO: 1. Molecules are identical at a position if the equivalent position in the sequences being compared is occupied by the same amino acid. The scoring of the alignment as a percentage identity is a function of the number of identical amino acids at positions shared by the sequences being compared. When comparing sequences, the optimal alignment may require that gaps be introduced into one or more of the sequences to account for possible insertions and deletions within the sequences. The sequence comparison method may use a gap penalty such that a sequence alignment with as few gaps as possible, which reflects a higher relatedness between the two sequences being compared for the same number of identical molecules within the sequences being compared, will achieve a higher score than one with many gaps.Calculation of the maximum percent identity involves generating an optimal alignment taking into account a gap penalty. As described above, the percent sequence identity may be determined using the Needleman-Wunsch global sequence alignment tool with default parameter settings. The Needleman-Wunsch algorithm was published in J. Mol. Biol. (1970) 48:443-453.

[0070] One aspect of the invention relates to yeast cells capable of producing at least one polyamine analog. The yeast cells are capable of producing at least one polyamine. The yeast cells contain at least one coenzyme A (CoA) ligase-encoding gene, preferably a 4-coumarate:CoA ligase-encoding gene, at least one polyamine N-acyltransferase gene, and at least one polyamine synthase-encoding gene, and lack a polyamine oxidase-encoding gene or contain a disrupted polyamine oxidase-encoding gene.

[0071] The yeast cells of the invention contain a gene encoding 4-coumarate:CoA ligase (EC 6.2.1.12) capable of converting a carboxyl group-containing molecule to a CoA ester. The corresponding CoA ester then serves as a substrate for polyamine N-acyltransferase together with at least one polyamine produced by the yeast cells, whereby at least one polyamine analog is obtained (by acetylating at least one polyamine analog and forming an amide bond between at least one polyamine and the CoA ester).

[0072] In one embodiment, the yeast cells are modified for overexpression of 4-coumarate:CoA ligase.

[0073] In one embodiment, overexpression of 4-coumarate:CoA ligase is achieved by placing the 4-coumarate:CoA ligase coding gene under the transcriptional control of a highly active promoter within yeast cells. Examples of promoters suitable for use in yeast cells include, but are not limited to, the promoters of PDC, GPD, GPD1, TEF1, PGK1, TDH, and TDH3. Other suitable promoters include the promoters of GAL1, GAL2, GAL10, GAL7, CUP1, HIS3, CYC1, ADH1, PGL, GAPDH, ADC1, URA3, TRP1, LEU2, TPI, AOX1, and ENO1.

[0074] Yeast cells contain one or more, i.e., at least two copies, of the 4-coumarate:CoA ligase coding gene, thereby increasing the copy number of the mRNA for 4-coumarate:CoA ligase and thereby increasing the amount of 4-coumarate:CoA ligase produced by the yeast cells. In such cases, multiple copies of the 4-coumarate:CoA ligase coding gene may be under the transcriptional control of one promoter, or each 4-coumarate:CoA ligase coding gene may be under the transcriptional control of its own promoter. In the latter case, the same type of promoter may be used to control the transcription of each 4-coumarate:CoA ligase coding gene, or different types of promoters may be used.

[0075] In one embodiment, the 4-coumaric acid:CoA ligase (4CL) coding gene is selected from the group consisting of the Arabidopsis thaliana 4-coumaric acid:CoA ligase 1 (At4CL1), At4CL2, At4CL3, At4CL4, or At4CL5, and a nucleotide sequence encoding a 4-coumaric acid:CoA ligase having at least 80% sequence identity with any of At4CL1, At4CL2, At4CL3, At4CL4, or At4CL5. In one embodiment, the nucleotide sequence has at least 85%, or even 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with any of Arabidopsis thaliana 4CL1 (SEQ ID NO: 1), 4CL2, 4CL3, 4CL4, or 4CL5 and encodes a 4-coumaric acid:CoA ligase. In one embodiment, this 4-coumaric acid:CoA ligase having at least 80% sequence identity can catalyze the conversion of a carboxyl group-containing molecule to a CoA ester, preferably the conversion of 4-coumarate to 4-coumaroyl-CoA. The enzyme efficiency of the 4-coumaric acid:CoA ligase having at least 80% sequence identity can be lower than, substantially equal to, or higher than the corresponding enzyme efficiency of the relevant 4-coumaric acid:CoA ligase, preferably at least substantially equal to or higher enzyme efficiency.

[0076] In a particular embodiment, the 4-coumaric acid:CoA ligase coding gene is At4CL1. The amino acid sequence of At4CL1 is shown in SEQ ID NO: 1, and the nucleotide sequence of At4CL1 is shown in SEQ ID NO: 2.

[0077] In one embodiment, the yeast cell is modified for overexpression of at least one polyamine N-acetyltransferase.

[0078] In one embodiment, overexpression of at least one polyamine N - acyltransferase is achieved by placing at least one polyamine N - acyltransferase - encoding gene under the transcriptional control of a highly active promoter within a yeast cell. Promoters suitable for use in yeast cells include, but are not limited to, the promoters of PDC, GPD, GPD1, TEF1, PGK1, TDH, and TDH3. Other suitable promoters include the promoters of GAL1, GAL2, GAL10, GAL7, CUP1, HIS3, CYC1, ADH1, PGL, GAPDH, ADC1, URA3, TRP1, LEU2, TPI, AOX1, and ENO1.

[0079] The yeast cells contain one or more copies of the polyamine N - acyltransferase - encoding gene, thereby increasing the copy number of the mRNA for polyamine N - acyltransferase and thereby increasing the amount of polyamine N - acyltransferase produced by the yeast cells. In such cases, multiple copies of the polyamine N - acyltransferase - encoding gene may be under the transcriptional control of one promoter, or each polyamine N - acyltransferase - encoding gene may be under the transcriptional control of its own promoter. In the latter case, the same type of promoter may be used to control the transcription of each polyamine N - acyltransferase - encoding gene, or different types of promoters may be used.

[0080] In one embodiment, the yeast cells contain at least one polyamine N - acyltransferase - encoding gene selected from the group consisting of a spermidine hydroxycinnamoyltransferase (EC 2.3.1.34) - encoding gene, a spermidine coumaroyl - CoA acyltransferase (EC 2.3.1.249) - encoding gene, and a putrescine hydroxycinnamoyltransferase (EC 2.3.1.138) - encoding gene.

[0081] In certain embodiments, the spermidine hydroxycinnamoyl transferase (SHT) coding gene is selected from the group consisting of nucleotide sequences encoding spermidine hydroxycinnamoyl transferases having at least 80% sequence identity to Arabidopsis thaliana spermidine hydroxycinnamoyl transferase (AtSHT), Nicotiana attenuata DH29 (NaDH29), and spermidine hydroxycinnamoyl transferase AtSHT (SEQ ID NO: 3) or spermidine hydroxycinnamoyl transferase NaDH29 (SEQ ID NO: 5). In one embodiment, the nucleotide sequence encodes a spermidine hydroxycinnamoyl transferase having at least 85%, or even 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to either Arabidopsis thaliana SHT or Nicotiana attenuata DH29. In one embodiment, this spermidine hydroxycinnamoyl transferase having at least 80% sequence identity is capable of catalyzing the conversion of spermidine and CoA esters to polyamine analogs, preferably spermidine and coumaroyl-CoA, feruloyl-CoA, caffeoyl-CoA, cinnamoyl-CoA, or sinapoyl-CoA to N 1 -(coumaroyl, feruloyl, caffeoyl, cinnamoyl, or sinapoyl)spermidine, N 10 -(coumaroyl, feruloyl, caffeoyl, cinnamoyl, or sinapoyl)spermidine, N 1 ,N 10 -bis(coumaroyl, feruloyl, caffeoyl, cinnamoyl, or sinapoyl)spermidine, and / or N 1 ,N 5 ,N 10- It can catalyze the conversion to tri(coumaroyl, feruloyl, caffeoyl, sinamoyl, or sinapoyl) spermidine. The enzyme efficiency of spermidine hydroxycinnamoyl transferase having at least 80% sequence identity can be lower than, substantially equal to, or higher than the corresponding enzyme efficiency of the relevant spermidine hydroxycinnamoyl transferase, preferably at least substantially equal to or higher enzyme efficiency.

[0082] The SHT-encoding gene catalyzes the production of polyamine analogs in yeast cells with respect to polyamine alkaloids, particularly mono-substituted, di-substituted (also referred to as bis-substituted), and / or tri-substituted N-acylated polyamines, preferably spermidine. In certain embodiments, by expressing such an SHT-encoding gene together with a 4CL-encoding gene, yeast cells can produce symmetrically tri-substituted N-acylated polyamines, preferably spermidine in the case of AtSHT, and symmetrically mono-substituted N-acylated polyamines, preferably spermidine in the case of NaDH29.

[0083] The amino acid sequence of AtSHT is shown in SEQ ID NO: 3, and the nucleotide sequence of AtSHT is shown in SEQ ID NO: 4. The corresponding amino acid sequence of NaDH29 is shown in SEQ ID NO: 5, and the nucleotide sequence of NaDH29 is shown in SEQ ID NO: 6.

[0084] In one embodiment, the spermidine coumaroyl-CoA acyltransferase (SCT) coding gene is selected from the group consisting of the Arabidopsis thaliana spermidine coumaroyl-CoA acyltransferase (AtSCT), and nucleotide sequences encoding spermidine coumaroyl-CoA acyltransferases having at least 80% sequence identity with AtSCT. In one embodiment, the nucleotide sequence has at least 85%, or even 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with Arabidopsis thaliana SCT (SEQ ID NO: 7) and encodes a spermidine coumaroyl-CoA acyltransferase. In one embodiment, this spermidine coumaroyl-CoA acyltransferase having at least 80% sequence identity can catalyze the conversion of spermidine and CoA esters to polyamine analogs, preferably, the conversion of spermidine and coumaroyl-CoA, feruloyl-CoA, caffeoyl-CoA, cinnamoyl-CoA, or sinapoyl-CoA to 1 ,N 10 -bis(coumaroyl, feruloyl, caffeoyl, cinnamoyl, or sinapoyl)spermidine. The enzyme efficiency of the spermidine coumaroyl-CoA acyltransferase having at least 80% sequence identity may be lower than, substantially equal to, or higher than the corresponding enzyme efficiency of AtSCT, preferably at least substantially equal to or higher enzyme efficiency.

[0085] The SCT-encoding gene catalyzes the production of polyamine analogs in yeast cells with respect to polyamine alkaloids, particularly disubstituted N-acylated polyamines, preferably spermidine. In certain embodiments, by expressing such an SCT-encoding gene together with a 4CL-encoding gene, yeast cells can produce symmetric disubstituted N-acylated polyamines, preferably spermidine, in the case of AtSCT.

[0086] The amino acid sequence of AtSCT is shown in SEQ ID NO: 7, and the nucleotide sequence of AtSCT is shown in SEQ ID NO: 8.

[0087] In one embodiment, the putrescine hydroxycinnamoyl transferase-encoding gene is selected from the group consisting of Nicotiana attenuata AT1 (NaAT1), and nucleotide sequences encoding putrescine hydroxycinnamoyl transferases having at least 80% sequence identity to putrescine hydroxycinnamoyl transferase NaAT1. In one embodiment, the nucleotide sequence encodes a putrescine hydroxycinnamoyl transferase having at least 85%, or even 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to Nicotiana attenuata AT1 (SEQ ID NO: 9). In one embodiment, this putrescine hydroxycinnamoyl transferase having at least 80% sequence identity can catalyze the conversion of putrescine and CoA esters to polyamine analogs, preferably the conversion of putrescine and coumaroyl-CoA, feruloyl-CoA, caffeoyl-CoA, cinnamoyl-CoA, or sinapoyl-CoA to N 1 -(coumaroyl, feruloyl, caffeoyl, cinnamoyl, or sinapoyl)putrescine, N 6 -bis(coumaroyl, feruloyl, caffeoyl, cinnamoyl, or sinapoyl)putrescine, and / or N 1 ,N 6- It can catalyze the conversion to bis(coumaroyl, feruloyl, caffeoyl, cinnamoyl, or sinapoyl) putrescine. The enzyme efficiency of putrescine hydroxycinnamoyl transferase having at least 80% sequence identity can be lower than, substantially equal to, or higher than the corresponding enzyme efficiency of NaAT1, preferably at least substantially equal to or higher enzyme efficiency.

[0088] The putrescine hydroxycinnamoyl transferase coding gene catalyzes the production of polyamine analogs in yeast cells with respect to polyamine alkaloids, particularly disubstituted N-acylated polyamines, preferably putrescine. In certain embodiments, by expressing such a putrescine hydroxycinnamoyl transferase coding gene together with a 4CL coding gene, yeast cells can produce symmetric disubstituted N-acylated polyamines, preferably putrescine, in the case of NaAT1.

[0089] The amino acid sequence of NaAT1 is shown in SEQ ID NO: 9, and the nucleotide sequence of NaAT1 is shown in SEQ ID NO: 10.

[0090] In one embodiment, the yeast cell contains multiple types of polyamine N - acyltransferase - encoding genes. Therefore, the embodiments include yeast cells containing a 4CL - encoding gene such as At4CL1, an SHT - encoding gene such as AtSHT and / or NaDH29, and an SCT - encoding gene such as AtSCT; yeast cells containing a 4CL - encoding gene such as At4CL1, an SHT - encoding gene such as AtSHT and / or NaDH29, and a putrescine hydroxycinnamoyltransferase - encoding gene such as NaAT1; yeast cells containing a 4CL - encoding gene such as At4CL1, an SCT - encoding gene such as AtSCT, and a putrescine hydroxycinnamoyltransferase - encoding gene such as NaAT1; and yeast cells containing a 4CL - encoding gene such as At4CL1, an SHT - encoding gene such as AtSHT and / or NaDH29, an SCT - encoding gene such as AtSCT, and a putrescine hydroxycinnamoyltransferase - encoding gene such as NaAT1. For example, yeast cells containing a 4CL - encoding gene such as At4CL1, an SCT - encoding gene such as AtSCT, and an SHT - encoding gene such as AtSHT can produce an asymmetrically trisubstituted N - acylated polyamine, preferably spermidine.

[0091] In one embodiment, a carboxyl group-containing molecule such as an aromatic organic acid, a fatty acid, a halogenated aromatic organic acid, a halogenated fatty acid, or a combination thereof is added to the medium in which yeast cells are cultured. Therefore, in this embodiment, the yeast cells are supplied with a carboxyl group-containing molecule that is converted into a CoA ester by 4-coumaric acid:CoA ligase expressed by the yeast cells. Then, the yeast cells may be supplied with a single carboxyl group-containing molecule or a mixture of different carboxyl group-containing molecules. For example, yeast cells containing a 4CL coding gene such as At4CL1 and an SHT coding gene such as AtSHT can produce an asymmetrically trisubstituted N-acylated polyamine such as spermidine when supplied with a mixture of aromatic organic acids. Correspondingly, yeast cells containing a 4CL coding gene such as At4CL1 and an SCT coding gene such as AtSCT can produce an asymmetrically disubstituted N-acylated polyamine such as spermidine when supplied with a mixture of aromatic organic acids. Yeast cells containing a 4CL coding gene such as At4CL1 and an SHT coding gene such as NaDH29 can produce a symmetrically monosubstituted N-acylated polyamine such as spermidine when supplied with a mixture of aromatic organic acids.

[0092] Instead of supplying carboxyl group-containing molecules such as aromatic organic acids and / or fatty acids and / or their halogenated versions to yeast cells, or as a supplement to the supply, the yeast cells can be modified to produce or overproduce carboxyl group-containing molecules. Therefore, in certain embodiments, the yeast cells can produce at least one organic acid selected from the group consisting of aromatic organic acids, halogenated aromatic organic acids, fatty acids, halogenated fatty acids, and combinations thereof. Yeast cells modified for the production of such aromatic organic acids and / or fatty acids are disclosed in Yu et al. 2018; Zhou et al. 2016; Liu et al. 2019; and Rodriguez et al. 2015. The teachings regarding the ability of these yeast cells to produce at least one organic acid selected from the group consisting of aromatic organic acids, halogenated aromatic organic acids, fatty acids, halogenated fatty acids, and combinations thereof are hereby incorporated by reference into this specification.

[0093] However, overproduction of such carboxyl group-containing molecules in the yeast cells of the present invention may cause a flux imbalance between polyamine metabolism and the metabolism of carboxyl group-containing molecules, for example, with respect to aromatic amino acid (AAA) metabolism. Another source of carboxyl group-containing molecules is to co-culture the yeast cells of the present invention with microbial cells, preferably yeast cells, that can produce and secrete carboxyl group-containing molecules, instead of or as a supplement to supplying carboxyl group-containing molecules to the yeast cells by adding them to the medium. Examples of such microbial cells that can be co-cultured with the yeast cells of the present invention, although non-limiting and exemplary, are disclosed in Yu et al. 2018; Zhou et al. 2016; Liu et al. 2019; and Rodriguez et al. 2015.

[0094] As the halogenated aromatic organic acids used in this specification, halogen-substituted aromatic organic acids are exemplified, and as the halogenated fatty acids, halogen-substituted fatty acids are exemplified. Examples of such halogen-substituted aromatic organic acids and halogen-substituted fatty acids include fluorine-substituted, chlorine-substituted, bromine-substituted, and iodine-substituted aromatic organic acids and fatty acids, preferably fluorine-substituted aromatic organic acids and fatty acids.

[0095] In one embodiment, at least one polyamine is selected from the group consisting of spermine, thermospermine, sym-homospermidine, 1,3-diaminopropane, putrescine, cadaverine, agmatine, spermidine, sym-norspermidine, norspermine, and combinations thereof.

[0096] The yeast cells of the present invention lack a polyamine oxidase (EC1.5.3.17) coding gene or contain a disrupted polyamine oxidase coding gene. Further, the yeast cells contain at least one polyamine synthase coding gene.

[0097] At least one polyamine synthase expressed by the yeast cells catalyzes the production of at least one polyamine in the yeast cells. Polyamine oxidase is an enzyme that catalyzes the conversion of spermine back to spermidine. Therefore, the yeast cells lack any polyamine oxidase coding gene or contain a disrupted polyamine oxidase coding gene. This means that the yeast cells preferably lack any polyamine oxidase, or if such polyamine oxidase is expressed in the yeast cells, the polyamine oxidase is preferably enzymatically inactive or at least has a much lower enzyme efficiency compared to the natural polyamine oxidase.

[0098] In one embodiment, the yeast cells are modified for overexpression of at least one polyamine synthase.

[0099] In one embodiment, overexpression of at least one polyamine synthase is achieved by placing at least one polyamine synthase-encoding gene under the transcriptional control of a highly active promoter in yeast cells. Promoters suitable for use in yeast cells include, but are not limited to, the promoters of PDC, GPD, GPD1, TEF1, PGK1, TDH, and TDH3. Other suitable promoters include the promoters of GAL1, GAL2, GAL10, GAL7, CUP1, HIS3, CYC1, ADH1, PGL, GAPDH, ADC1, URA3, TRP1, LEU2, TPI, AOX1, and ENO1.

[0100] The yeast cells contain one or more copies of the polyamine synthase-encoding gene, thereby increasing the copy number of the mRNA for polyamine synthase and thereby increasing the amount of polyamine synthase produced by the yeast cells. In such cases, multiple copies of the polyamine synthase-encoding gene may be under the transcriptional control of one promoter, or each polyamine synthase-encoding gene may be under the transcriptional control of its own promoter. In the latter case, the same type of promoter may be used to control the transcription of each polyamine synthase-encoding gene, or different types of promoters may be used.

[0101] In one embodiment, the polyamine synthase-encoding gene is selected from the group consisting of the spermidine synthase (EC 2.5.1.22)-encoding gene, the thermospermidine synthase (EC 2.5.1.79)-encoding gene, and the homospermidine synthase (EC 2.5.1.44 or EC 2.5.1.45)-encoding gene.

[0102] In one embodiment, the spermidine synthase coding gene is selected from the group consisting of nucleotide sequences encoding Saccharomyces cerevisiae spermidine synthase, preferably ScSPE4, Arabidopsis thaliana spermidine synthase (AtSPMS), and a spermidine synthase having at least 80% sequence identity with spermidine synthase ScSPE4 or spermidine synthase AtSPMS. In one embodiment, the nucleotide sequence encodes a spermidine synthase having at least 85%, or even 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with ScSPE4 or AtSPMS. In one embodiment, this spermidine synthase having at least 80% sequence identity can catalyze the conversion of spermidine to spermine. The enzyme efficiency of the spermidine synthase having at least 80% sequence identity may be lower than, substantially equal to, or higher than the corresponding enzyme efficiency of ScSPE4 or AtSPES, preferably at least substantially equal to or higher enzyme efficiency.

[0103] The amino acid sequence of ScSPE4 is shown in SEQ ID NO: 11, and the nucleotide sequence of ScSPE4 is shown in SEQ ID NO: 12. The corresponding amino acid sequence of AtSPMS is shown in SEQ ID NO: 13, and the nucleotide sequence of AtSPMS is shown in SEQ ID NO: 14.

[0104] In one embodiment, the thermospermine synthase coding gene is selected from the group consisting of a nucleotide sequence encoding Arabidopsis thaliana thermospermine synthase, preferably AtACL5, and a thermospermine synthase having at least 80% sequence identity with thermospermine synthase AtACL5. In one embodiment, the nucleotide sequence encodes a thermospermine synthase having at least 85%, or even 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with AtACL5. In one embodiment, this thermospermine synthase having at least 80% sequence identity can catalyze the conversion of spermidine to thermospermine. The enzyme efficiency of the thermospermine synthase having at least 80% sequence identity may be lower than, substantially equal to, or higher than the corresponding enzyme efficiency of AtACL5, preferably at least substantially equal to or higher enzyme efficiency.

[0105] The amino acid sequence of AtACL5 is shown in SEQ ID NO: 15, and the nucleotide sequence of AtACL5 is shown in SEQ ID NO: 16.

[0106] In one embodiment, the homospermidine synthase (HSS) coding gene is selected from the group consisting of the nucleotide sequences encoding Senecio vernalis homospermidine synthase (SvHSS), Blastochloris viridis homospermidine synthase (BvHSS), and a homospermidine synthase having at least 80% sequence identity with homospermidine synthase SvHSS or homospermidine synthase BvHSS. In one embodiment, the nucleotide sequence encodes a homospermidine synthase having at least 85%, or even 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SvHSS or BvHSS. In one embodiment, this homospermidine synthase having at least 80% sequence identity can catalyze the conversion of putrescine to sym-homospermidine, or the conversion of putrescine or spermidine to sym-homospermidine. The enzyme efficiency of the homospermidine synthase having at least 80% sequence identity may be lower than, substantially equal to, or higher than the corresponding enzyme efficiency of SvHSS or BvHSS, preferably at least substantially equal to or higher enzyme efficiency.

[0107] The amino acid sequence of SvHSS is shown in SEQ ID NO: 17, and the nucleotide sequence of SvHSS is shown in SEQ ID NO: 18. The corresponding amino acid sequence of BvHSS is shown in SEQ ID NO: 19, and the nucleotide sequence of BvHSS is shown in SEQ ID NO: 20.

[0108] In one embodiment, the yeast cell is selected from the group consisting of the genera Saccharomyces, Kluyveromyces, Zygosaccharomyces, Candida, Hanseniaspora, Pichia, Hansenula, Schizosaccharomyces, Trigonopsis, Brettanomyces, Debaromyces, Nadsonia, Lipomyces, Cryptococcus, Aureobasidium, Trichosporon, Rhodotorula, Yarrowia, Rhodosporidium, Phaffia, Schwanniomyces, Aspergillus, and Ashbya. In certain embodiments, the yeast cell is selected from the group consisting of Saccharomyces cerevisiae, Saccharomyces boulardii, Zygosaccharomyces bailii, Kluyveromyces lactis, Rhodosporidium toruloides, Yarrowia lipolytica, Schizosaccharomyces pombe, Pichia pastoris, Hansenula anomala, Candida sphaerica, or Schizosaccharomyces malidevorans.Saccharomyces cerevisiae is a preferred yeast species.

[0109] In one embodiment, the yeast cells are Saccharomyces cerevisiae cells and the polyamine oxidase is FMS1. Therefore, in one embodiment, the Saccharomyces cerevisiae (S. cerevisiae) cells lack FMS1 or contain disrupted FMS1.

[0110] Another aspect of the present invention relates to yeast cells capable of producing at least one polyamine analog. The yeast cells are capable of producing at least one polyamine. The yeast cells contain a 4-coumaric acid:CoA ligase coding gene and at least one polyamine N-acyltransferase gene.

[0111] Also, various embodiments of the above-described yeast cells can be utilized in this aspect of the present invention.

[0112] A further aspect of the present invention relates to a method for producing a polyamine analog. The method includes culturing yeast cells according to the present invention in a medium under culture conditions suitable for the production of the polyamine analog by the yeast cells. The method also includes collecting the polyamine analog from the medium and / or from the yeast cells.

[0113] In one embodiment, culturing the yeast cells includes culturing the yeast cells in a medium containing at least one organic acid selected from the group consisting of aromatic organic acids, fatty acids, and combinations thereof. Therefore, in this embodiment, carboxyl group-containing molecules in the form of at least one organic acid are included in the medium. Therefore, at least one organic acid is supplied to the yeast cells.

[0114] In certain embodiments, the method comprises adding at least one organic acid to the medium. Thus, at least one organic acid becomes available to the yeast cells in the medium by adding at least one organic acid to the medium. In this particular embodiment, a single organic acid is added to the medium, or multiple different organic acids are added to the medium.

[0115] In another particular embodiment, culturing the yeast cells comprises co-culturing the yeast cells in the medium with a microorganism, preferably a yeast cell, that is capable of producing at least one organic acid and releasing at least one organic acid into the medium.

[0116] The two particular embodiments described can be combined. That is, at least one organic acid is added to the medium, in which the yeast cells of the present invention are co-cultured with at least one microorganism, preferably a yeast cell, capable of producing at least one organic acid. The at least one organic acid added to the medium may be the same as or different from the at least one organic acid produced by the at least one microorganism.

[0117] The medium in this aspect of the present invention can be any medium in which yeast cells can be cultured to produce polyamine analogs. The culturing can be in the form of, for example, batch, fed-batch, or perfusion culture, or fermentation, bioreactor fermentation, etc.

[0118] Example Example 1: Improvement of Spermidine Production by Systematic Rewiring of Natural Metabolism in Yeast In Example 1, the inventors systematically refactored the metabolism in a yeast strain, including central carbon-nitrogen metabolism, the methionine salvage pathway, the adenine salvage pathway, the polyamine transport mechanism, and the polyamine consumption / degradation pathway. In addition, the inventors also introduced additional potential positive gene targets. This yeast strain was constructed by a new modular genetic design. Specifically, the de novo Spd biosynthetic pathway is divided into multiple genetic modules containing coding sequences for a number of biosynthetic enzymes to redirect a larger carbon flux from the sugar carbon source to Spd.

[0119] The precursor overproduction module (I) designed to increase the accumulation of L-ornithine (Orn) contains the overexpression of eight proteins: NADP (+) -dependent glutamate dehydrogenase (GDH1) [SEQ ID NO: 21] from Saccharomyces cerevisiae, mitochondrial aspartate-glutamate carrier protein (AGC1) [SEQ ID NO: 22] from S. cerevisiae, mitochondrial L-ornithine carrier protein (ORT1) [SEQ ID NO: 23] from S. cerevisiae, glutamate N-acetyltransferase (EcargA) [SEQ ID NO: 24] from Escherichia coli, acetylglutamate kinase (EcargB) [SEQ ID NO: 25] from E. coli, N-acetyl-gamma-glutamyl-phosphate reductase (CgargC) [SEQ ID NO: 26] from Corynebacterium glutamicum, acetylornithine aminotransferase (CgargD) [SEQ ID NO: 27] from C. glutamicum, and ornithine acetyltransferase (CgargJ) [SEQ ID NO: 28] from C. glutamicum. Furthermore, the attenuation or removal of two proteins: the native promoter P ARG3 was replaced with a weaker promoter P KEX2Attenuation of yeast native ornithine carbamoyltransferase (ARG3) [SEQ ID NO: 29] by exchange, and removal of the activity of L-ornithine transaminase (CAR2) [SEQ ID NO: 30] by knockout of CAR2 were also included in this module (I).

[0120] The putrescine (Put) module (II) designed to overproduce Put from L-ornithine included two genetic modifications; overexpression of ornithine decarboxylase (SPE1) [SEQ ID NO: 31] from Saccharomyces cerevisiae and deletion of the native ornithine decarboxylase antizyme (OAZ1) [SEQ ID NO: 32].

[0121] The spermidine biosynthesis module (III) was designed for overproduction of spermidine (Spd) from putrescine (characterized by overexpression of two proteins from Saccharomyces cerevisiae: adenosylmethionine decarboxylase (AdoMetDC; SPE2) [SEQ ID NO: 33] and spermidine synthase (SpdSyn; SPE3) [SEQ ID NO: 34]). Also included in this module were deletions of two native proteins to avoid consumption or degradation of spermidine: deletion of SPE4 [SEQ ID NO: 12] encoding spermine synthase and deletion of FMS1 [SEQ ID NO: 35] encoding non-specific polyamine oxidase.

[0122] The S-adenosyl-L-methionine (AdoMet) module (IV) was designed to enhance the accessibility of the cofactor AdoMet. This modification included overexpression of a number of proteins: 5'-methylthioadenosine phosphorylase (MEU1) [SEQ ID NO: 36] from Saccharomyces cerevisiae, branched-chain amino acid aminotransferase (BAT2) [SEQ ID NO: 37] from Saccharomyces cerevisiae, adenine phosphoribosyltransferase (APT1) [SEQ ID NO: 38] from Saccharomyces cerevisiae, ribose-phosphate pyrophosphokinase (PRS5) [SEQ ID NO: 39] from Saccharomyces cerevisiae, and S-adenosylmethionine synthetase (LiMAT) [SEQ ID NO: 40] from Leishmania infantum. Also included in this module was the deletion of adenine deaminase activity (AAH1) [SEQ ID NO: 41].

[0123] The polyamine efflux module (V) was designed to reduce cytotoxicity to cells or inhibition of polyamine biosynthesis. This module included overexpression of the yeast native polyamine transporter encoded by TPO5 [SEQ ID NO: 42].

[0124] Finally, and more importantly, an additional spermidine biosynthesis module (VI) was designed for the overproduction of spermidine from putrescine and AdoMet. This module included overexpression of the AdoMetDC-SpdSyn fusion protein encoded by SPE2-SPE3 [SEQ ID NO: 43].

[0125] Overexpression of the gene in Example 1 was obtained by chromosomal integration via the CRISPR / cas9 system or traditional genetic marker-based methods into regions predicted by the inventors to have no growth defects and no active expression as integration loci. The implementation of CRISPR / cas9-based genome editing followed the protocol developed by Mans et al. 2015. In particular, the Saccharomyces cerevisiae strain CEN.PK113-11C with plasmid pL-CAS9-HIS having the HIS3 marker, which enables constitutive expression of Cas9, was the starting strain for all gene modifications. To enable efficient genome editing at the selected loci, multiple guide RNA (gRNA) plasmids were constructed. Genetic modules containing various combinations of genetic parts, namely promoters, terminators, ORFs, and homologous arms, were constructed as integration cassettes according to the overlap extension PCR (OE-PCR) procedure. The following gene and promoter combinations were used in Example 1: TPI1p-ORT1-pYX212t; tHXT7p-AGC1-CYC1t; TEF1p-GDH1-DIT1t; PGK1p-SPE3-pYX212t; TEF1p-SPE1-PRM9t; TDH3p-SPE2-DIT1t; TDH3p-CgargJ-TDH2t; PGK1p--EcargB-ADH1t; TEF1p-CgargC-FBA1t; tHXT7p-CgargD-TPI1t; TPI1p-EcargA-CYC1t; TPI1p-MEU1p-FBA1t; PGK1p-BAT2-CYC1t; TDH3p-APT1-DIT1t; TEF1p-PRS5-PRM9t; TEF1p-LiMAT-PRM9; TDH3p-TPO5-CYC1t; TEF1p-SPE2-SPE3-PRM9t.

[0126] All natural genetic parts, namely, natural promoters, terminators, ORFs, and homologous arms, were PCR amplified using CEN.PK113-11C genomic DNA as a template. For optimized heterologous genes, synthetic fragments or plasmids (obtained from GenScript) were used for PCR amplification. High-fidelity Phusion DNA polymerase was utilized throughout the molecular cloning procedure. Cassettes or plasmids were introduced into yeast by the standard LiAc / SS DNA / PEG transformation method. Strains containing URA3-based plasmids or cassettes were selected on synthetic complete medium without uracil (SC-URA) consisting of 6.7 g / l yeast nitrogen base (YNB) without amino acids, 0.77 g / l complete supplement mixture without uracil (CSM-URA), 20 g / l glucose, and 20 g / l agar. The URA3 marker was removed and selected against 5-fluoroorotic acid (5’-FOA) plates. Additionally, a CRISPR / cas9-based system was also used to perform deletions of AAH1, SPE4, and FMS1. Other gene knockout experiments were conducted by conventional methods. All primers used in this specification are listed in Table 1, all plasmids are listed in Table 2, and all strains are listed in Table 3.

Table 1-1

Table 1-2

Table 1-3

Table 1-4

Table 1-5

Table 1-6

Table 1-7

Table 1-8

Table 1-9

Table 1-10

Table 1-11

Table 1-12

Table 1-13

Table 1-14

Table 1-15

Table 1-16

Table 2

Table 3-1

Table 3-2

Table 3-3

[0127] The resulting strain JQSPD_AA was evaluated using an assay that combined deep well scale fermentation with high performance liquid chromatography (HPLC). In particular, 24 deep well batch fermentations of the resulting JQSPD_AA strain for polyamine production were carried out in minimal medium developed by Verduyn et al 1992. Cultures from 24-hour precultures were used at an initial OD of 0.2 600It was inoculated into 2 ml of minimal medium in 24 deep-well plates and cultured at 300 rpm and 30 °C for 120 hours. The pH of the minimal medium containing 7.5 g / l (NH4)2SO4, 14.4 g / l KH2PO4, 0.5 g / l MgSO4·7H2O, 20 g / l glucose, 2 ml / l trace metals, and 1 ml / l vitamin solution (supplemented with 40 mg / l uracil and 40 mg / l histidine as needed) was adjusted to 4.5. Samples were prepared by taking 0.1 ml of the liquid culture and subjected to hot water (HW) extraction. In this method, the inventors used minimal medium for the fermentation in deep-well plates as the situation of the extract. Tubes containing 0.9 ml of the fermentation medium were preheated in a water bath at 100 °C for 10 minutes. Then, the hot fermentation medium was quickly poured onto 0.1 ml of the liquid culture; the mixture was immediately vortexed and the sample was placed in the water bath. After 30 minutes, each tube was placed on ice for 5 minutes. After centrifugation, the supernatant was used directly for derivatization. For derivatization, 0.125 ml of saturated NaHCO3 solution and 0.25 ml of dansyl chloride solution (5 mg / ml in acetone) were added to 0.25 ml of the sample. Then, the reaction mixture was incubated at 40 °C in the dark for 1 hour with occasional shaking. The reaction was stopped by adding 0.275 ml of methanol. The sample was filtered through a 25 mm syringe filter (0.45 μm nylon) for HPLC detection. The following chromatographic conditions were used: C18 (100 mm × inner diameter 4.6 mm, 2.6 μm, Phenomenex Kinetex), excitation wavelength 340 nm, emission wavelength 515 nm, sample injection 1.5 μl, column temperature 40 °C, detector sensitivity 7, acquisition started at 4.0 minutes. The mobile phase was water and methanol at a flow rate of 1 ml / min. The elution program was as follows: 0 - 5 minutes 50% - 65% methanol, 5 - 7.5 minutes 65% - 75% methanol, 7.5 - 9.5 minutes 75% - 87.5% methanol, 9.5 - 10.5 minutes 87.5% - 100% methanol, 10.5 - 11.5 minutes 100% methanol, 11.5 - 13.5 minutes 100% - 50% methanol, 13.5 - 16 minutes 50% methanol.

[0128] Strain JQSPD_AA yielded an Spd titer at a concentration of >400 mg / l, and the Spd titer increased significantly compared to strains having only partially the modifications used herein (see the examples in WO 2016 / 144247 pamphlet and WO 2019 / 013696 pamphlet).

[0129] Example 2: Higher-order polyamine production in yeast Life has evolved diverse pathways for synthesizing structural variants of polyamines. Indeed, Put and Spd are typically found in most cells as common polyamines, while rare polyamines such as sym-homospermidine (Hspd), thermospermine (Tspm), spermine (Spm), branched-chain polyamines, and long-chain polyamines (LCPA) have also been identified in nature. Example 2 investigated the biosynthesis of sym-homospermidine (Hspd), thermospermine (Tspm), and spermine (Spm) by designing genetic module (VII) and introducing it into the Spd platform strain JQSPD_AA of Example 1.

[0130] The inventors first set out to heterologously synthesize triamine Hspd, which is present in both plants and bacteria. In plants, Hspd is the first pathway-specific intermediate in the biosynthesis of pyrrolizidine alkaloids, which are formed by homospermidine synthase (plant HSS; EC 2.5.1.45). This enzyme is more specific than bacterial homospermidine synthase (bacterial HSS; EC 2.5.1.44), because the latter cannot use Put as a donor of the aminobutyl group. To investigate the potential of both plant and bacterial HSS for the microbial production of Hspd, genetic submodules (VII-a) and (VII-b), designed for the biosynthesis of Hspd in yeast, encoded the expression of SvHSS from Senecio vernalis and BvHSS13 from Blastochloris viridis, respectively. The submodules were introduced as high-copy plasmids SvHSS_p426GPD and BvHSS_p426GPD ordered from GenScript, retaining the yeast-codon-optimized SvHSS gene [SEQ ID NO: 18] and BvHSS gene [SEQ ID NO: 20] in the Spd platform strain JQSPD_AA, respectively. The transformation experiments followed the same procedure as in Example 1. The resulting strains JQSPD_AA (SvHSS_p426GPD) and JQSPD_AA (BvHSS_p426GPD) were assayed for Hspd production by the same procedure as described in Example 1.

[0131] The inventors found that overexpression of both HSS enabled the biosynthesis of Hspd. In particular, SvHSS enabled an Hspd titer of 40.9 mg / , while BvHSS enabled an Hspd titer of 31.1 mg / (see FIGS. 1a and 1d).

[0132] Subsequently, the inventors also utilized the Spd platform (Example 1) for the production of tetraamines Spm and Tspm by introducing submodules (VII-c), (VII-d), and (VII-e). Spm is the most common tetraamine found throughout metazoans, flowering plants, and yeast. A specific aminopropyltransferase, namely spermine synthase (SpmSyn; EC 2.5.1.22), is responsible for Spm biosynthesis. The inventors first investigated the yeast native SpmSyn Spe4p for Spm overproduction.

[0133] When the codon-optimized yeast SPE4 [SEQ ID NO: 12] was overexpressed as a high-copy plasmid SPE4_p426GPD (submodule (VII-c)) in JQSPD_AA, 53.1 mg / l of Spm was obtained (see Figures 1c and 1f). The inventors also tested the SpmSyn from Arabidopsis thaliana by overexpressing AtSPMS [SEQ ID NO: 14] as a high-copy plasmid AtSPMS_p426GPD (submodule (VII-d)) in the JQSPD_AA strain. This resulted in the production of Spm (41.8 mg / l; see Figures 1c and 1f). The plant ACL5 aminopropyltransferase (TspmSyn; EC 2.5.1.79) from Arabidopsis thaliana (A. thaliana) was shown to synthesize the Spm isomer Tspm. The inventors also overexpressed AtACL5 [SEQ ID NO: 16] as a high-copy plasmid AtACL5_p426GPD (submodule (VII-e)) in the JQSPD_AA strain. This strategy enabled the production of 43.8 mg / l of Tspm (see Figures 1b and 1e). All plasmids with yeast codon-optimized genes were purchased from GenScript. In Example 2, the same transformation and product assays as used in Example 1 were used. All plasmids are listed in Table 2 and all strains are listed in Table 3.

[0134] Figure 5a shows a modified pathway for the biosynthesis of spermidine and higher order polyamines in yeast.

[0135] Example 3: Biosynthesis of cocamine in yeast Next, the inventors set out to synthesize cocamine (a series of plant polyamine analogs composed of a polymethylene polyamine backbone, such as Put, Spd, and Spm), and at least one dihydrocaffeic acid fragment. Due to its versatile bioactivities, such as antihypertensive, antitrypanosomal, anti-lipid peroxidation, as well as lipoxygenase, disinfectant, and neuroprotective effects, cocamine has attracted attention in recent years as a functional food and drug candidate. Cocamine was first discovered in Cortex Lycii and later in other plants of the Solanaceae family, such as tomato, potato, and tobacco. The coupling of the dihydrocaffeoyl moiety and the amine moiety is a committed step and can be considered the actual entry point into cocamine biosynthesis. However, the enzymes mediating this reaction in these plants have been scarcely described so far. Nevertheless, a panel of N-hydroxysinamiloyltransferases belonging to the BAHD acyltransferase superfamily has been demonstrated to catalyze the N-acylation of polyamines by acylating the amine (-NH2) group with coenzyme A-activated hydroxycinnamic acid. Its specificity / indiscrimination towards acyl acceptors and acyl donors varies depending on the plant source.

[0136] Example 3 investigated the biosynthesis of cocoamine by designing three genetic submodules that express multiple N - hydroxycinnamoyltransferases and introducing them into the Spd platform strain JQSPD_AA of Example 1. Since N - hydroxycinnamoyltransferase only accepts coenzyme A - activated hydroxycinnamic acid, the inventors also co - expressed promiscuous 4 - coumarate:CoA ligase (EC6.2.1.12) in the module. Sub - module (VIII - b) encoded the co - expression of two proteins: Arabidopsis thaliana promiscuous 4 - coumarate:CoA ligase 1 (At4CL1) [SEQ ID NO:2] (which has been shown to most efficiently convert caffeic acid to its CoA ester compared to other members of the Arabidopsis thaliana 4CL family); and spermidine dicaumoyltransferase (AtSCT; EC2.3.1.249) [SEQ ID NO:8] from Arabidopsis thaliana. This module, constructed as the high - copy plasmid pLAt4CL - AtACT, was ordered from GenScript and had expression cassettes for yeast - codon - optimized At4CL1 and AtSCT. All plasmids are listed in Table 2 and all strains are listed in Table 3.

[0137] Sub - module (VIII - c) encoded the co - expression of two proteins: Arabidopsis thaliana promiscuous 4 - coumarate:CoA ligase 1 (At4CL1), and spermidine hydroxycinnamoyltransferase (AtSHT; EC2.3.1M34) [SEQ ID NO:4] from Arabidopsis thaliana. This module, constructed as the high - copy plasmid pLAt4CL1 - AtSHT, was ordered from GenScript and had expression cassettes for yeast - codon - optimized At4CL1 and AtSHT.

[0138] Sub-module (VIII-d) encoded the co-expression of two proteins: Arabidopsis thaliana promiscuous 4-coumaric acid:CoA ligase 1 (At4CL1), and spermidine hydroxycinnamoyl transferase (NaDH29; EC 2.3.1.34) from Nicotiana attenuata [SEQ ID NO:6]. This module, constructed as the high-copy plasmid pLAt4CL1-NaDH29, was ordered from GenScript and had expression cassettes for yeast-codon optimized At4CL1 and NaDH29.

[0139] Finally, sub-module (VIII-e) encoded the co-expression of two proteins - Arabidopsis genus promiscuous 4-coumaric acid:CoA ligase 1 (At4CL1) and N. attenuata putrescine hydroxycinnamoyl transferase (NaAT1; EC 2.3.1.138) [SEQ ID NO:10]. This module, constructed as the high-copy plasmid pLAt4CL1-NaAT1, was ordered from GenScript and had expression cassettes for yeast-codon optimized At4CL1 and NaAT1.

[0140] These plasmids were transformed into Spd platform strain JQSPD_AA by the same procedure as described in Example 2 to obtain strains JQSPD_AA(pLAt4CL-AtSCT), JQSPD_AA(pLAt4CL1-AtAHT), JQSPD_AA(pLAt4CL1-NaDH29), and JQSPD_AA(pLAt4CL1-NaAT1), respectively. These strains were assayed by supplying 2 mM dihydrocaffeic acid (3,4-dihydroxyhydrocinnamic acid) for 120 hours, and the growth medium was analyzed for polyamine analog production according to the following procedure. Detection of polyamine analogs was performed by liquid chromatography-mass spectrometry (LC-MS) measurements using a Dionex UltiMate 3000 UHPLC (Fisher Scientific, San Jose, CA) coupled to an Orbitrap Fusion mass spectrometer (Thermo Fisher Scientific, San Jose, CA). The system used an Agilent Zorbax Eclipse Plus C18 2.1×100 mm, 1.8 μm column maintained at 35°C. The flow rate was 0.350 mL / min using 0.1% formic acid in acetonitrile (A) and 0.1% formic acid (B) as the mobile phase. The gradient started at 5% B for 1 minute and then continued with a linear gradient to 95% B until 5 minutes. This solvent composition was held for 1.5 minutes. Then, it was changed to 5% B and held until 8 minutes. The sample (5 μl) was passed through the MS equipped with a heated electrospray ionization source (HESI) in positive ion mode or negative ion mode. The sheath gas was set to 50 (a.u.), the auxiliary gas to 10 (a.u.), and the sweep gas to 1 (a.u.). The cone temperature and probe temperature were 325°C and 380°C, respectively, and the spray voltage was 3500 V. The scan range was from 80 to 500 Da, and the time between scans was 50 ms.

[0141] The inventors were delighted to find that these efforts had led to the biosynthesis of cocamine. In particular, in the NaDH29 strain, 310.2128 [M+H] +A large single LC-MS peak with an m / z value corresponding to [NaDH29] was detected (see Fig. 2a). This indicates that NaDH29 enables the biosynthesis of N 1 - or N 10 -dihydrocaffeoyl spermidine, and that At4CL1 can also accept dihydrocaffeic acid as a substrate. The inventors also observed a single LC-MS peak with an m / z value corresponding to 310.2128 [M+H] + in the AtSCT overexpressing line. Furthermore, when dihydrocaffeic acid was supplied to the line JQSPD_AA (pLAt4CL1-NaAT1), a large single LC-MS peak with an m / z value corresponding to 417.2010 [M+H] + was detected (see Fig. 2b). This indicates that NaAT1 enabled the biosynthesis of N 1 ,N 6 -bis(dihydrocaffeoyl) putrescine.

[0142] Example 4: Biosynthesis of complex phenolamides in yeast The successful demonstration of the biosynthesis of cocoamine in the polyamine platform gave the inventors the confidence to further utilize this platform for the biosynthesis of more diverse and complex phenolic amides, which constitute a quantitatively major group of nitrogen-containing secondary metabolites resulting from the conjugation of the phenolic moiety with polyamines. Thus, in Example 4, the production of complex phenolic amides became possible by overexpressing a specific polyamine N-hydrocinnamoyltransferase. Following the same strategy demonstrated in Example 3, the inventors also assayed these strains, namely, JQSPD_AA(pLAt4CL-AtACT), JQSPD_AA(pLAt4CL1-AtAHT), JQSPD_AA(pLAt4CL1-NaDH29), and JQSPD_AA(pLAt4CL1-NaAT1), by supplying 2 mM p-coumaric acid, 2 mM caffeic acid, or 2 mM ferulic acid for 120 hours and analyzed the medium for the production of polyamine analogs. The fermentation, sample preparation, or LC-MS confirmation procedures were the same as those in Example 3. Indeed, supplying hydroxycinnamic acids, namely p-coumaric acid, caffeic acid, or ferulic acid, to the JQSPD_AA strains co-expressing At4CL1 with AtSCT, AtSHT, or NaDH29 resulted in phenolic amide biosynthesis. In particular, when p-coumaric acid was supplied, a large single LC-MS peak with an m / z value corresponding to 584.2748[M+H] + was detected in the AtSHT strain (see Figure 3a). This indicates that AtSHT enabled the biosynthesis of N 1 ,N 5 ,N 10 -tri(coumaroyl)spermidine. Similarly, when caffeic acid was supplied, a large single LC-MS peak with an m / z value corresponding to 632.2599[M+H] + was detected in the AtSHT strain (see Figure 3b). This indicates that AtSHT enabled the biosynthesis of N 1 ,N 5 ,N 10- It has been shown to enable the biosynthesis of tri(caffeoyl) spermidine. Furthermore, when p-coumaric acid was supplied, a large single LC-MS peak with an m / z value corresponding to 438.2383 [M+H] + was detected in the AtSCT strain (see Figure 3c). This indicates that AtSCT enables the biosynthesis of N 1 ,N 10 -bis(coumaroyl) spermidine. Similarly, when caffeic acid was supplied, a large single LC-MS peak with an m / z value corresponding to 470.2282 [M+H] + was detected in the AtSCT strain (see Figure 3d). This indicates that AtSCT enables the biosynthesis of N 1 ,N 10 -bis(caffeoyl) spermidine. In addition, when ferulic acid was supplied, a large single LC-MS peak with an m / z value corresponding to 498.2599 [M+H] + was detected in the AtSCT strain (see Figure 3e). This indicates that AtSCT enables the biosynthesis of N 1 ,N 10 -bis(feruloyl) spermidine. Correspondingly, when p-coumaric acid, caffeic acid, or ferulic acid was supplied to the NaDH29 strain, the biosynthesis of N 1 - or N 10 -coumaroyl spermidine, N 1 - or N 10 -caffeoyl spermidine, or N 1 - or N 10 -feruloyl spermidine was successfully achieved. Therefore, by selecting various N-hydroxysinnamoyltransferases with different regioselectivities, the inventors achieved the regioselective biosynthesis of mono-substituted, di-substituted, and tri-substituted spermidine phenolic amides. Similarly, when p-coumaric acid, caffeic acid, or ferulic acid was supplied to the NaAT1 strain, N 1 -coumaroyl putrescine, N 1 ,N 6 -bis(caffeoyl) putrescine, N 1- Caffeoylputrescine, and N 1 - The biosynthesis of feruloylputrescine was successfully achieved (see Figure 3f). All plasmids are listed in Table 2, and all strains are listed in Table 3.

[0143] Example 5: Biosynthesis of complex phenolamides in yeast co - cultures In Example 4, the inventors demonstrated that by supplying multiple aromatic organic acids, such as p - coumaric acid, caffeic acid, or ferulic acid, to their polyamine platform strains, the biosynthesis of various polyamine - derived phenolamides was possible. However, these aromatic organic acids used in titration experiments are generally expensive to obtain, and to some extent, the economic feasibility of this titration - based process for phenolamide production is sacrificed. In contrast, the inventors believe that the de novo production of these phenolamides without supplying any aromatic organic acids can be an economically viable bioprocess. Indeed, recent advances in metabolic engineering and synthetic biology of microorganisms, such as yeast, have already provided many platform strains for the production of these aromatic organic acids, such as p - coumaric acid. To demonstrate the concept of de novo production of polyamine - derived phenolamides by the inventors' polyamine platform, the inventors introduced an additional genetic module sub - module (VIII - f), a p - coumaric acid - overproducing yeast strain, into their system. The inventors demonstrated this by designing a synthetic consortium containing a polyamine - producing strain and a p - coumaric acid - overproducing strain. In particular, the inventors co - cultured the JQSPD_AA strain that co - overexpresses At4CL1 and one of AtSHT, AtSCT, NaDH29, and NaAT1 with the p - coumaric acid - overproducing strain QL58 (Liu et al., 2019), and a series of polyamine - p - coumaric acid conjugates, namely, N 1 ,N 5 ,N 10 - Tri(coumaroyl) spermidine, N 1 ,N 10 - Bis(coumaroyl) spermidine, N 1 - Or N10 -Coumaroyl spermidine and N 1 -Denovo biosynthesis of coumaroyl putrescine occurred (see FIGS. 4a-4c). It must be emphasized that all positive genetic targets in the p-coumaric acid overproducing strain can be introduced into our polyamine platform strains, such as JQSPD_AA and its derivatives, and the submodule (VIII-f) used herein can also be alternatively introduced.

[0144] FIG. 5b shows a modified pathway for the biosynthesis of complex phenolic amides in yeast.

[0145] Example 6: Biosynthesis of halogenated phenolic amides in yeast co-cultures In Example 5, the inventors demonstrated that de novo production of naturally occurring polyamine-derived phenolic amides, i.e., production using simple sugars as the sole carbon source, can be achieved by designing a synthetic consortium containing a polyamine-producing strain and a p-coumaric acid overproducing strain. However, the inventors also noticed that, in addition to their natural counterparts, non-natural polyamine-hydroxycinnamic acid conjugates are being actively investigated for their potentially improved pharmaceutical properties (Mounce et al., 2017; Antoniou et al., 2016). One of the major pharmacophores of interest in this search is the halogenated derivatives, such as fluorine substituents. This is because organofluorine is known to affect the absorption, distribution, metabolism, excretion, and toxicity (ADMET) properties of lead compounds (Muller et al., 2007). The inventors set out to establish a biosynthetic approach for the production of this class of fluorine-substituted polyamine-hydroxycinnamic acid conjugates, assuming that the observed promiscuity of the 4CLs-NAT system for hydroxycinnamic acids, i.e., 4-coumaric acid:CoA ligase + N-acyltransferase, would be converted to fluorine-substituted precursors. To access fluorine-substituted hydroxycinnamic acids, the inventors used a strain (QL58) that overproduces aromatic chemicals (Liu et al., 2019) and supplied this strain with a fluorine-substituted aromatic amino acid (3-fluoro-L-phenylalanine). From this, 3-fluoro-cinnamic acid ([M-H] - =165.0358), 3-fluoro-p-coumaric acid ([M-H] - =181.0305), and fluorine-substituted hydrogenated p-coumaric acid ([M-H] -Peaks corresponding to the predicted m / z values (m / z = 183.0463) were detected (see FIGS. 6a to 6c). This suggests that the heterologous pathway mobilized here for the biosynthesis of aromatic compounds from aromatic amino acids is promising. Subsequently, when 3-fluoro-L-phenylalanine was supplemented to the co-culture system of Example 5 containing both the polyamine overproducing strains (At4CL1 and the JQSPD_AA strain co-overexpressing one of AtSHT, AtSCT, NaDH29, and NaAT1) and the aromatic overproducing strain QL58, a series of mono- and di-non-natural fluorine-substituted putrescine-hydroxycinnamic acid conjugates (see FIGS. 7a to 7d), as well as a list of mono-substituted, di-substituted, and tri-substituted non-natural fluorine-substituted spermidine-hydroxycinnamic acid conjugates (see FIGS. 8a to 8e) were obtained.

[0146] The above embodiments should be understood as some examples of the present invention. Those skilled in the art will understand that various modifications, combinations, and changes can be made to the embodiments without departing from the scope of the present invention. In particular, the solutions of different parts in different embodiments can be combined in other configurations if technically possible. However, the scope of the present invention is defined by the appended claims.

[0147] References JPEG0007695715000021.jpg53165 JPEG0007695715000022.jpg213165 JPEG0007695715000023.jpg154165

Sequence Listing Free-Text

[0148] Sequence Listing 44 - 266 <223>Primer

Claims

1. A yeast cell that produces at least one polyamine alkaloid, producing at least one polyamine; containing and expressing a 4-coumaric acid:CoA ligase-encoding gene; containing and expressing at least one polyamine N-acyltransferase gene; containing and expressing at least one polyamine synthase-encoding gene; and lacking a polyamine oxidase-encoding gene or containing a disrupted polyamine oxidase-encoding gene, wherein the 4-coumaric acid:CoA ligase-encoding gene is selected from the group consisting of a nucleotide sequence encoding Arabidopsis thaliana At4CL1, a nucleotide sequence encoding Arabidopsis thaliana At4CL2, a nucleotide sequence encoding Arabidopsis thaliana At4CL3, a nucleotide sequence encoding Arabidopsis thaliana At4CL4, a nucleotide sequence encoding Arabidopsis thaliana At4CL5, and a nucleotide sequence encoding a 4-coumaric acid:CoA ligase having at least 90% sequence identity with 4-coumaric acid:CoA ligase At4CL1 shown in SEQ ID NO: 2, The at least one polyamine N - acyltransferase gene is selected from the group consisting of a nucleotide sequence encoding Arabidopsis thaliana AtSHT, a nucleotide sequence encoding Nicotiana attenuata NaDH29, a nucleotide sequence encoding spermidine hydroxycinnamoyltransferase having at least 90% sequence identity with spermidine hydroxycinnamoyltransferase AtSHT shown in SEQ ID NO: 3 or spermidine hydroxycinnamoyltransferase NaDH29 shown in SEQ ID NO: 5, a nucleotide sequence encoding Arabidopsis thaliana AtSCT, a nucleotide sequence encoding spermidine coumaroyl - CoA acyltransferase having at least 90% sequence identity with spermidine coumaroyl - CoA acyltransferase AtSCT shown in SEQ ID NO: 8, a nucleotide sequence encoding Nicotiana attenuata NaAT1, and a nucleotide sequence encoding putrescine hydroxycinnamoyltransferase having at least 90% sequence identity with putrescine hydroxycinnamoyltransferase NaAT1 shown in SEQ ID NO:

9. The yeast cell.

2. The yeast cell according to claim 1, wherein the 4 - coumaric acid: CoA ligase - encoding gene is Arabidopsis thaliana At4CL1.

3. The yeast cell according to claim 1 or 2, which produces at least one organic acid selected from the group consisting of aromatic organic acids, fatty acids, halogenated aromatic organic acids, halogenated fatty acids, and combinations thereof.

4. The yeast cell according to any one of claims 1 to 3, wherein the at least one polyamine is selected from the group consisting of spermine, thermospermine, sym-homospermidine, 1,3-diaminopropane, putrescine, cadaverine, agmatine, spermidine, sym-norspermidine, norspermine, and combinations thereof.

5. The yeast cell according to any one of claims 1 to 4, wherein the polyamine synthase-encoding gene is selected from the group consisting of a spermine synthase-encoding gene, a thermospermine synthase-encoding gene, a homospermidine synthase-encoding gene, an ornithine decarboxylase-encoding gene, and a spermidine synthase-encoding gene.

6. The yeast cell according to claim 5, wherein the spermine synthase-encoding gene is selected from the group consisting of a nucleotide sequence encoding Saccharomyces cerevisiae SPE4, a nucleotide sequence encoding Arabidopsis thaliana AtSPMS, and a nucleotide sequence encoding a spermine synthase having at least 90% sequence identity with the spermine synthase SPE4 shown in SEQ ID NO: 12 or the spermine synthase AtSPMS shown in SEQ ID NO:

14.

7. The yeast cell according to claim 5 or 6, wherein the thermospermine synthase-encoding gene is selected from the group consisting of a nucleotide sequence encoding Arabidopsis thaliana AtACL5 and a nucleotide sequence encoding a thermospermine synthase having at least 90% sequence identity with the thermospermine synthase AtACL5 shown in SEQ ID NO:

16.

8. The yeast cell according to any one of claims 5 to 7, wherein the homospermidine synthase-encoding gene is selected from the group consisting of a nucleotide sequence encoding SvHSS of Senecio vernalis, a nucleotide sequence encoding BvHSS of Blastochloris viridis, and a nucleotide sequence encoding a homospermidine synthase having at least 90% sequence identity with the homospermidine synthase SvHSS shown in SEQ ID NO: 18 or the homospermidine synthase BvHSS shown in SEQ ID NO:

20.

9. The yeast cell according to any one of claims 1 to 8, wherein the yeast cell is a Saccharomyces cerevisiae cell and the polyamine oxidase is FMS1.

10. A method for producing a polyamine alkaloid, the method comprising: culturing the yeast cell according to any one of claims 1 to 9 in a medium under culture conditions suitable for the production of the polyamine alkaloid by the yeast cell; collecting the polyamine alkaloid from the medium and / or from the yeast cell and the above method.

Images