Polyamine conjugate-producing yeast

Abstract

The present invention relates to the production of polyamine conjugates in yeast cells capable of producing at least one polyamine. The yeast cells also contain a polyamine:glutathione ligase-encoding 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 a variety of polyamine-glutathione conjugates.

Description

[Technical Field]

[0001] The present invention relates generally to genetically modified yeast, and in particular to yeast capable of producing polyamine conjugates. [Background technology]

[0002] Small molecules with novel mechanisms of action are needed to help address today's most challenging biomedical and agricultural problems. However, over the past few decades, over 135 million small molecules generated by synthetic chemistry aimed at identifying lead compounds for pharmaceutical and agricultural development have been phased out despite passing initial screening. It has become clear that improving the complexity and diversity of small molecules is essential. In fact, diverse compounds—structurally complex natural products and their derivatives—are readily available in nature, and they have played an important role in the study and treatment of disease for millennia. In particular, polyamine conjugates, a diverse and quantitatively major group of secondary metabolites in plants, exhibit extensive structural diversity and complexity. This has led to multiple studies reporting on the beneficial properties of these compounds. For example, polyamine-glutathione conjugates, also known as trypanothiones, have been proposed as specific molecular probes in medicinal chemistry and for the development of glutathione reductase inhibitors.

[0003] Unfortunately, due to their complex structures and low abundance in nature, polyamine analogs are difficult to obtain either through traditional synthetic chemistry or extraction from natural sources. Microbial-based production in fast-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 served as a cellular factory for producing many different fuels, chemicals, food ingredients, and pharmaceuticals, particularly for the production of natural products.

[0004] Consequently, it is desirable to develop new methods for the discovery and production of polyamine conjugates. Summary of the Invention [Problem to be solved by the invention]

[0005] The general objective is to provide a yeast cell capable of producing a polyamine conjugate. [Means for solving the problem]

[0006] This and other objects are met by the embodiments.

[0007] The invention is defined in the independent claims. Further embodiments of the invention are defined in the dependent claims.

[0008] The present invention relates to yeast cells capable of producing glutathione-polyamine conjugates. The yeast cells are capable of producing at least one polyamine. The yeast cells also contain a gene encoding a polyamine:glutathione ligase and at least one gene encoding a polyamine synthase, but lack a gene encoding a polyamine oxidase or contain a disrupted gene encoding a polyamine oxidase.

[0009] The present invention also relates to a method for producing a glutathione-polyamine conjugate, the method comprising culturing a yeast cell according to the invention in a medium under culture conditions suitable for production of the glutathione-polyamine conjugate by the yeast cell, and collecting the glutathione-polyamine conjugate from the medium and / or from the yeast cell.

[0010] The present invention provides an efficient means for producing a variety of glutathione-polyamine conjugates, including mono- and / or poly-substituted polyamines, such as trypanothione. Thus, the present invention can be used as a cost-effective alternative to prior art methods involving traditional synthetic chemistry or extraction from natural sources to obtain polyamine conjugates.

[0011] The embodiments, together with further objects and advantages thereof, may best be understood by reference to the following description taken in conjunction with the accompanying drawings, in which: [Brief explanation of the drawings]

[0012] [Figure 1a] 1 shows the modification of yeast metabolism for the oversynthesis of spermidine and higher polyamines. [Figure 1b] 1 shows the modification of yeast metabolism for the oversynthesis of spermidine and higher polyamines. [Figure 1c] 1 shows the modification of yeast metabolism for the oversynthesis of spermidine and higher polyamines. [Figure 1d] 1 shows the modification of yeast metabolism for the oversynthesis of spermidine and higher polyamines. [Figure 1e] 1 shows the modification of yeast metabolism for the oversynthesis of spermidine and higher polyamines. [Figure 1f] 1 shows the modification of yeast metabolism for the oversynthesis of spermidine and higher polyamines. [Figure 2a] Figure 1 shows trypanothione-(SH)2 production in yeast. [Figure 2b] Figure 1 shows trypanothione-(SH)2 production in yeast. [Figure 3a] Figure 1 shows an engineered pathway for the biosynthesis of complex phenolamides and trypanothione-(SH)2 in yeast. [Figure 3b] Figure 1 shows an engineered pathway for the biosynthesis of complex phenolamides and trypanothione-(SH)2 in yeast. DETAILED DESCRIPTION OF THE INVENTION

[0013] To enable efficient access to a diversity of polyamine conjugates, we engineered 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 conjugates via tailored pathways. In particular, with the aid of computational simulations, we systematically refactored yeast's central carbon-nitrogen metabolism, methionine salvage pathway, adenine salvage pathway, polyamine transport machinery, and polyamine degradation pathway, enabling yeast to produce >400 mg / L spermidine in deep-well-scale fermentation. Furthermore, by plugging into the tailored pathways and creating a synthetic consortium, we demonstrated the de novo biosynthesis of polyamine conjugates, including trypanothione, in yeast.

[0014] The present invention will now be described below with reference to the accompanying drawings and examples, which illustrate embodiments of the invention. This description is not intended to be a detailed catalog of all the different ways in which the invention may be practiced or all the features that may be added to the invention. For example, features shown with respect to one embodiment may be incorporated into other embodiments, and features shown with respect to a particular embodiment may be omitted from that embodiment. Thus, the present invention contemplates that some embodiments of the invention may exclude or omit any feature or combination of features described herein. Also, numerous modifications and additions to the various embodiments proposed herein that do not depart from the invention will be apparent to those skilled in the art in light of this disclosure. Therefore, the following description is intended to describe some particular embodiments of the invention, but is not intended to exhaustively identify all rearrangements, combinations, and modifications thereof.

[0015] Unless otherwise defined herein, scientific and technical terms used herein have the meanings that are commonly understood by those of ordinary skill in the art.

[0016] Generally, the nomenclature used in connection with the techniques of biochemistry, enzymology, molecular and cell biology, microbiology, genetics, protein and nucleic acid chemistry, and hybridization described herein is that well known and commonly used in the art.

[0017] Conventional methods and techniques referred to herein are described, for example, in Molecular Cloning, a laboratory manual [Second Edition] Sambrook et al., Cold Spring Harbor Laboratory, 1989, e.g., in Section 1.21 "Extraction and Purification of Plasmid DNA," Section 1.53 "Strategies for Cloning in Plasmid Vectors," Section 1.85 "Identification of Bacterial Colonies That Contain Recombinant Plasmids," Section 6 "Gel Electrophoresis of DNA," Section 14 "In Vitro Amplification of DNA by the Polymerase Chain Reaction," and Section 17 "Expression of Cloned Genes in Escherichia coli." This is described in more detail in "The Role of Streptococcus aureus in Escherichia coli."

[0018] Enzyme Commission (EC) numbers (also referred to herein as "classes") referenced throughout this specification are taken from the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology in its resource "Enzyme Nomenclature" (1992, including supplements 6-17) (available, for example, as "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, EC (1992), San Diego (published by the International Union of Biochemistry and Molecular Biology Academic Press) (ISBN 0-12-227164-5)). The International Union for the Study of Molecular Biology (NC-IUBMB) is a numerical classification scheme based on the chemical reactions catalyzed by each enzyme class.

[0019] Unless the context indicates otherwise, it is specifically intended that the various features of the invention described herein can be used in any combination. Moreover, the present invention also contemplates that in some embodiments of the invention, any feature or combination of features described herein can be excluded or omitted. For illustrative purposes, if the specification states that a composition includes components A, B, and C, it is specifically intended that any of A, B, or C, or any combination thereof, alone or in any combination, can be omitted and waived.

[0020] As used in the description of this 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 dictates otherwise. Also, as used herein, "and / or" refers to and includes any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted as alternatives ("or").

[0021] Throughout the description and claims of this specification, the words "comprise" and "contain," as well as variations of words such as "comprising" and "comprises," mean "including but not limited to" and do not exclude other moieties, additives, ingredients, integers, or steps. Throughout the description and claims of this specification, the singular encompasses the plural unless the context requires otherwise. In particular, where the indefinite article is used, the specification should be understood as contemplating both the plural and the singular unless the context requires otherwise.

[0022] As used herein, the transitional phrase "consisting essentially of" means that the claims should be construed to include the specific materials or steps recited in the claims and that do not materially affect the basic and novel characteristics of the claimed invention. Thus, the term "consisting essentially of" when used in the claims of the present invention is not intended to be construed as the equivalent of "comprising."

[0023] To facilitate the understanding of this invention, several terms are defined below.

[0024] The term "polyamine" as used herein 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.

[0025] As used herein, the term "polyamine conjugate" or "glutathione-polyamine conjugate" refers to a conjugate of at least one glutathione (GSH) molecule with a polyamine. The glutathione-polyamine conjugate preferably contains an amide bond formed between the carboxyl group of GSH and the amine group of the polyamine. A non-limiting, illustrative example of a glutathione-polyamine conjugate is trypanothione (N 1 ,N 10 -bis(glutathionyl)spermidine), N 1 -Glutathionylspermidine, N 10 -Glutathionylspermidine, N 1 ,N 10 -Bis(glutathionyl)spermine, N 1 ,N 5 ,N 10 -tri(glutathionyl)spermine, N 1 ,N 5 ,N 10 ,N 14 -tetra(glutathionyl)spermine. Thus, a glutathione-polyamine conjugate can be a conjugate of one polyamine, such as spermidine or spermine, with one, two, or more, for example, three or four, glutathione molecules.

[0026] 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), any of which may be single-stranded or double-stranded, linear or branched, or a hybrid thereof. The nucleic acid molecules and / or nucleotide sequences provided herein are presented left to right in the 5' to 3' direction and are represented using the standard code for representing nucleotide characters as set forth in the U.S. sequence rules, 37 CFR §§ 1.821-1.825, and World Intellectual Property Organization (WIPO) Standard ST.25. When dsRNA is produced synthetically, 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 RNA with high affinity and to be potent antisense inhibitors of gene expression. Other modifications, such as modifications to the phosphodiester backbone or the 2'-hydroxy in the ribose sugar group of RNA, can also be made. As used herein, the term "recombinant" means that a particular nucleic acid (DNA or RNA) is the product of various combinations of cloning, restriction, and / or ligation steps that result in a construct having structural coding or non-coding sequences that are distinguishable from the endogenous nucleic acid found in natural systems.

[0027] As used herein, the term "gene" refers to a nucleic acid molecule that can be used to produce mRNA, antisense RNA, miRNA, anti-microRNA antisense oligodeoxyribonucleotides (AMOs), and the like. A gene may or may not be used to produce a functional protein or gene product. A gene may 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 that is substantially or essentially free from components normally found associated with the nucleic acid in its natural state. Such components include other cellular material, culture media from recombinant production, and / or various chemicals used in the chemical synthesis of the nucleic acid.

[0028] 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 the addition of targeting sequences. Furthermore, gene disruption can also or alternatively be achieved by mutations or modifications of regulatory elements that control gene transcription, such as mutations or modifications in promoters, terminators, and / or enhancement elements. In such cases, such mutations or modifications result in partial or complete loss of gene transcription, i.e., reduced or depressed transcription compared to the native, unmodified regulatory elements. As a result, only small amounts, if any, of the gene product are available after transcription and translation. Furthermore, gene disruption can also involve the addition or removal of localization signals from the gene, reducing the presence of the gene product in its native cellular compartment.

[0029] The purpose of gene disruption is to reduce the available amount of a gene product (including completely preventing any production of the gene product), or to express a gene product that lacks or has lower enzymatic activity compared to the native or wild-type gene product.

[0030] As used herein, the term "deletion" or "knockout" refers to a gene that is inoperative or knocked out.

[0031] The term "attenuated activity" in relation to an enzyme refers to a reduction in the activity of the enzyme in its natural 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, additions of targeting sequences, or removals of targeting sequences. Cells containing modifications that result in attenuated enzyme activity have lower enzyme activity compared to cells that do not contain such modifications. Attenuated enzyme activity can be achieved by encoding a non-functional gene product, for example, a polypeptide that has essentially no activity, for example, less than about 10% or even less than 5% of the activity of the wild-type polypeptide.

[0032] A codon-optimized version of a gene refers to a foreign gene that is introduced into a cell and has its codons optimized for a specific cell. Generally, not all tRNAs are expressed equally or at the same level across all species. Therefore, codon optimization of a gene sequence involves changing codons to 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 a codon-optimized gene will be translated more efficiently. Codons and synonymous codons preferably code for the same amino acid.

[0033] As used herein, the terms "peptide," "polypeptide," and "protein" are used interchangeably to refer to a polymer of amino acid residues. The terms "peptide," "polypeptide," and "protein" also include modifications, including, but not limited to, lipid conjugation, glycosylation, glycosylation of L-glutamic acid residues, sulfation, hydroxylation, and gamma-carboxylation, and ADP-ribosylation.

[0034] 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 the enzyme is operably linked to a nucleotide sequence (promoter) that causes expression of the corresponding gene in the cell sufficient to confer on the cell the ability to produce spermidine.

[0035] As used herein, the term "open reading frame (ORF)" refers to a region of RNA or DNA that encodes a polypeptide, peptide, or protein.

[0036] As used herein, the term "genome" encompasses both plasmids and chromosomes in a host cell. For example, an encoding nucleic acid of the present disclosure that is introduced into a host cell can be part of the genome, whether the nucleic acid is integrated into a chromosome or located on a plasmid.

[0037] As used herein, the term "promoter" refers to a nucleic acid sequence that functions to control the transcription of one or more genes, and is located upstream of the transcription start site of the gene in the direction of transcription. Suitable promoters in this context include both natural constitutive promoters and inducible promoters, as well as modified promoters, and are well known to those skilled in the art.

[0038] Suitable promoters for use in yeast cells include, but are not limited to, the promoters PDC, GPD1, TEF1, PGK1, and TDH. Other suitable promoters include the promoters GAL1, GAL2, GAL10, GAL7, CUP1, HIS3, CYC1, ADH1, PGL, GAPDH, ADC1, URA3, TRP1, LEU2, TPI, AOX1, and ENO1.

[0039] As used herein, unless otherwise specified, the term "terminator" refers to a "transcription termination signal." A terminator is a sequence that prevents or stops transcription of a polymerase.

[0040] As used herein, a "recombinant eukaryotic cell" according to this disclosure is defined as a cell that contains additional copies of an endogenous nucleic acid sequence or that has been transformed or genetically modified with a polypeptide or nucleotide sequence that does not naturally occur in a eukaryotic cell. A wild-type eukaryotic cell is defined as the parent cell of a recombinant eukaryotic cell as used herein.

[0041] As used herein, the terms "enhance," "enhanced," "enhancing," "enhance," "enhancing," and "enhancement" (and grammatical variations thereof) refer to 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, as compared to a control.

[0042] As used herein, the terms "reduce," "reduced," "reduction," "reduce," "inhibit," and "reduce," and similar terms, refer to a reduction 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.

[0043] As used herein, the reduction of gene expression 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 proteins translated from mRNA. Such genetic modifications include insertions, deletions, substitutions, or mutations made to the regulatory sequences of a gene, such as promoters and enhancers. For example, the promoter of a gene can be replaced with a less active or less inducible promoter, thereby reducing the transcription of the gene. Also, knocking out a promoter results in the reduction, typically to zero, of the expression of a gene.

[0044] As used herein, the term "portion, portion" or "fragment" of a nucleotide sequence of the invention is understood to mean a nucleotide sequence that is shorter in length compared to a reference nucleic acid or nucleotide sequence and that comprises, consists essentially of, and / or consists of a nucleotide sequence of contiguous nucleotides that is identical or nearly 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. Such nucleic acid fragments or portions according to the invention may, where appropriate, be comprised componently within a larger polynucleotide.

[0045] Different nucleic acids or proteins that share homology are referred to herein as "homologues." The term homologue includes homologous sequences from the same species and other species, as well as orthologous sequences from the same species and other species. "Homology" refers to the level of positional identity, i.e., sequence similarity or similarity in terms of percentage identity, between two or more nucleic acid and / or amino acid sequences. Homology 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 homologues to the nucleotide and polypeptide sequences of the present invention. As used herein, "ortholog" refers to homologous nucleotide and / or amino acid sequences in different species that arose from a common ancestral gene during speciation. Homologues of the nucleotide sequences of the present invention have substantial, e.g., 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% sequence identity with said nucleotide sequence.

[0046] As used herein, the term "overexpress" or "overexpression" refers to a higher level of gene activity, e.g., transcription of a gene; a higher level of translation of mRNA into protein; and / or a higher level of production of a gene product, e.g., a polypeptide, than exists in a cell in its native or control state, e.g., not transformed with a particular heterologous or recombinant polypeptide to be overexpressed. A typical example of an overexpressed gene is a gene under the transcriptional control of a different promoter compared to the gene's native promoter. Alternatively or alternatively, other changes to the gene's regulatory elements, e.g., enhancers, can be used to overexpress a particular gene. Furthermore, as used herein, overexpression can be achieved alternatively or additionally by using modifications that affect, i.e., increase, the translation of mRNA transcribed from the gene. The term can also 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. Furthermore, overexpression can be achieved by including genes from different species that encode the same or homologous gene products, e.g., enzymes. Overexpression can result in 25%, 50%, 75%, 100%, 200%, 300%, 400%, 500%, 750%, 1000%, 1500%, 2000% or higher levels in a cell compared to control levels, or any range therein.

[0047] As used herein, the terms "foreign" or "heterologous," when used with reference to a nucleic acid (RNA or DNA), protein, or gene, refer to a nucleic acid, protein, or gene (including multiple non-naturally occurring copies of a naturally occurring nucleotide sequence) that does not naturally occur as part of the cell, organism, genome, or RNA or DNA sequence into which it is introduced. Such a foreign gene can be a gene from another species or strain, a modified, mutated, or evolved version of a gene naturally occurring in the host cell, or a chimeric version of a gene naturally occurring in the host cell or a fusion gene. In these former cases, the modification, mutation, or evolution causes a change in the nucleotide sequence of the gene, resulting in a modified, mutated, or evolved gene with a different nucleotide sequence compared to the gene naturally occurring in the host cell. An evolved gene refers to a gene obtained by genetic modification, e.g., mutation, or exposure to evolutionary pressure, that encodes the evolved gene and leads to a new gene with a different nucleotide sequence compared to the wild-type or original gene. A chimeric gene is formed by the combination of portions of one or more coding sequences to generate a new gene. These modifications differ from fusion genes, which integrate entire gene sequences into a single reading frame and often retain their original function.

[0048] An "endogenous," "natural," or "wild-type" nucleic acid, nucleotide sequence, polypeptide, or amino acid sequence refers to a naturally occurring or endogenous nucleic acid, nucleotide sequence, polypeptide, or amino acid sequence. Thus, for example, a "wild-type mRNA" is an mRNA that occurs naturally in or is endogenous to an organism.

[0049] As used herein, the term "modified," when used with reference to an organism, refers to a host organism that has been modified to produce polyamine conjugates (compared to an otherwise identical host organism that has not been so modified). In principle, such "modification" in accordance with the present disclosure can include any physiological, genetic, chemical, or other modification that suitably alters the production of polyamine conjugates in the host organism (compared to an otherwise identical organism that has not been modified). However, in most embodiments, the modification will include a genetic modification. In certain embodiments, the modification described herein involves 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 the polypeptide (distinguishable from any gene already present in the host cell that encodes a polypeptide with the same activity); altering a gene present in the cell to increase transcription or translation of the gene (e.g., by altering, adding additional sequences, substituting one or more nucleotides, deleting sequences, or replacing regulatory sequences, promoter sequences, or other sequences); and altering the sequence (e.g., non-coding sequence or coding sequence) of a gene encoding a polypeptide to boost activity (e.g., by increasing enzymatic activity, reducing feedback inhibition, targeting a specific subcellular location, boosting mRNA stability, boosting protein stability).Genetic modifications that reduce the activity of a polypeptide include, but are not limited to, deleting part or all of a gene encoding the polypeptide; inserting a nucleic acid sequence that disrupts a gene encoding a 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, altering, deleting sequences from, substituting, or replacing one or more nucleotides, e.g., by substituting one or more nucleotides, promoter sequences, regulatory sequences, or other sequences). The term "overproduction," as used herein in reference to the production of a product in a host cell, indicates that the host cell produces more product than an unmodified host cell or a wild-type cell, as a result of the introduction of nucleic acid sequences encoding various polypeptides involved in a metabolic pathway of the host cell, or as a result of other modifications.

[0050] The term "vector" as used herein is defined as a linear or circular DNA molecule that contains a polynucleotide encoding a polypeptide of the present invention and is operably linked to additional nucleotides that ensure its expression.

[0051] "Introducing" in the context of yeast cells means contacting a nucleic acid molecule with a cell so that the nucleic acid molecule has access to the interior of the cell. Thus, polynucleotides and / or nucleic acid molecules can be introduced into yeast cells in a single transformation event in separate transformation events. Thus, the term "transformation" as used herein refers to the introduction of heterologous nucleic acid into a cell. Transformation of yeast cells can be stable or transient.

[0052] "Transient transformation" in the context of a polynucleotide means that the polynucleotide is introduced into a cell but does not integrate into the genome of the cell.

[0053] "Stable introduction" 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, thereby stably transforming the cell with 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 its progeny for multiple successive generations. As used herein, stable transformation can also refer to a nucleic acid molecule that is maintained extrachromosomally, for example, as a minichromosome.

[0054] Transient transformation can be detected, for example, by enzyme-linked immunosorbent assay (ELISA) or Western blot, which can detect the presence of peptides or polypeptides encoded by one or more nucleic acid molecules introduced into an organism. Stable transformation of cells can be detected, for example, by Southern blot hybridization assays of the cell's genomic DNA with a nucleic acid sequence that specifically hybridizes with the nucleotide sequence of the nucleic acid molecule introduced into the organism (e.g., yeast). Stable transformation of cells can be detected, for example, by Northern blot hybridization assays of the cell's RNA with a nucleic acid sequence that specifically hybridizes with the nucleotide sequence of the nucleic acid molecule introduced into yeast or other organisms. Stable transformation of cells can also be detected by polymerase chain reaction (PCR) or other amplification reactions well known in the art, for example, using specific primer sequences that hybridize with target sequences in the nucleic acid molecule, resulting in amplification of the target sequence, which can be detected according to standard methods. Transformation can also be detected by direct sequencing and / or hybridization protocols well known in the art.

[0055] Embodiments of the present invention also encompass variants of the polypeptides defined herein. As used herein, "variant" refers to a polypeptide whose amino acid sequence differs from the nucleic acid sequence from which it is derived in that one or more amino acids in 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, e.g., at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or about 100% identical. The variants and / or fragments are functional variants / fragments (and this is the meaning of the term "functional variant" as used throughout this specification) in that the variant sequence has similar or identical functional enzymatic activity characteristics as the enzyme having the non-variant amino acid sequence specified herein.

[0056] Thus, a "functional variant" or "functional fragment" of any of the presented amino acid sequences is any amino acid sequence that remains within the same enzyme category (i.e., has 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 of skill in the art, and one of skill in the art can determine the enzyme category without using the techniques of the present invention. Suitable methods can be obtained, for example, from the International Union of Biochemistry and Molecular Biology.

[0057] Amino acid substitutions may be considered "conservative" when an amino acid is replaced with a different amino acid having broadly similar properties. Non-conservative substitutions are when an amino acid is replaced with an amino acid of a different type.

[0058] "Conservative substitution" means the replacement of an amino acid with another amino acid of the same class, where the class is defined as follows: Examples of Class Amino Acids Non-polar: A, V, L, I, P, M, F, W Uncharged polarity: G, S, T, C, Y, N, Q Acidic: D, E Basic: K, R, H.

[0059] As is well known to those skilled in the art, conservative substitutions may alter the primary structure of a polypeptide without significantly altering the activity of the polypeptide, because the side chain of the amino acid inserted into the sequence may be able to form similar bonds and contacts with the side chain of the substituted amino acid, even if the substitution is in a region important for determining the higher-order structure of the polypeptide.

[0060] In embodiments of the 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 characteristics such that it remains in the same enzyme class as the unsubstituted enzyme, as determined using the NC-IUBMB nomenclature system described above.

[0061] Generally speaking, fewer non-conservative than conservative substitutions will be possible without altering the biological activity of a polypeptide. Determining the effect of any substitution (and indeed of any amino acid deletion or insertion) is entirely within the routine ability of one of ordinary skill in the art, who can readily determine whether a variant polypeptide retains enzymatic activity in accordance with aspects of the present invention. For example, when determining whether a variant of a polypeptide falls within the scope of the present invention (i.e., is a "functional variant or fragment" as defined above), one of ordinary skill in the art will determine whether the variant or fragment retains substrate-converting enzyme activity as defined with reference to the NC-IUBMB nomenclature referred to elsewhere herein. All such variants are within the scope of the present invention.

[0062] Using the standard genetic code, other nucleic acid sequences encoding polypeptides can be easily conceived and produced by those skilled in the art in addition to those disclosed herein.The nucleic acid sequence can be DNA or RNA, and if it is a DNA molecule, it can include, for example, cDNA or genomic DNA.The nucleic acid can be contained in an expression vector, as described elsewhere herein.

[0063] Thus, embodiments of the present invention encompass variant nucleic acid sequences that encode polypeptides contemplated by embodiments of the present invention. The term "variant" with respect to a nucleic acid sequence refers to any substitution, mutation, modification, substitution, 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 nucleic acid sequence. The term includes allelic variants and also includes polynucleotides ("probe sequences") that substantially hybridize to the polynucleotide sequences of embodiments of the present invention. Such hybridization may occur under low stringency conditions, high stringency conditions, or in between. Generally, the term low stringency conditions can be defined as hybridization in which washing steps occur in a 0.330-0.825 M NaCl buffer solution 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). High stringency conditions, on the other hand, involve 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, saline-sodium citrate (SSC) buffer (0.15 M NaCl and 0.015 M trisodium citrate), with low stringency washes being performed in 3x SSC buffer and high stringency washes being performed in 0.1x SSC buffer. The steps involved in hybridization of nucleic acid sequences can be described in, for example, Molecular Cloning, a laboratory manual. manual) [2nd ed.] Sambrook et al., Cold Spring Harbor Laboratory, 1989, for example, in Section 11 "Synthetic Oligonucleotide Probes."

[0064] Preferably, nucleic acid sequence variants share about 80% or more, more preferably at least 85%, and even more preferably 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity of nucleotides in common with the nucleic acid sequences of the present embodiments.

[0065] The variant nucleic acids of the invention may be codon optimized for expression in a particular host cell.

[0066] As used herein, "sequence identity" refers to the sequence similarity between two nucleotide sequences or between two peptide or protein sequences. Similarity is determined by sequence alignment to determine structural and / or functional relationships between the sequences.

[0067] Sequence identity between amino acid sequences can be determined by comparing 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 default parameter settings (for protein alignment, gap costs Existence: 11, Extension: 1). The sequence comparisons and identity percentages referred to herein were determined using this software. When comparing the level of sequence identity with, for example, SEQ ID NO: 1, the comparison should preferably be made over the entire length of SEQ ID NO: 1 (i.e., using the global alignment method) to avoid short regions of high identity overlap from causing a high overall assessment of identity. For example, a short polypeptide fragment, e.g., five amino acids, may have a 100% identical sequence over a five-amino acid region of SEQ ID NO: 1, but this does not confer 100% amino acid identity unless the fragment forms part of a longer sequence that also has identical amino acids at other positions corresponding to positions in SEQ ID NO: 1. If an equal position in the compared sequences is occupied by the same amino acid, the molecules are identical at that position. The scoring of the alignment as a percentage of identity is a function of the number of identical amino acids at positions shared by the compared sequences. When comparing sequences, optimal alignment may require the introduction of gaps into one or more of the sequences to account for possible insertions and deletions within the sequences. Sequence comparison methods may use gap penalties so that, for the same number of identical molecules in the compared sequences, a sequence alignment with as few gaps as possible, reflecting a higher relatedness between the two compared sequences, will achieve a higher score than one with more gaps.The calculation of maximum percent identity involves generating an optimal alignment, taking into account gap penalties. As mentioned above, the percentage sequence identity may be determined using the Needleman-Wunsch Global Sequence Alignment tool, using default parameter settings. The Needleman-Wunsch algorithm was published in J. Mol. Biol. (1970) Vol. 48: 443-453.

[0068] One aspect of the present invention relates to a yeast cell capable of producing glutathione-polyamine conjugates, wherein the yeast cell is capable of producing at least one polyamine and comprises a polyamine:glutathione ligase-encoding gene and at least one polyamine synthase-encoding gene, but lacks a polyamine oxidase-encoding gene or comprises a disrupted polyamine oxidase-encoding gene.

[0069] In one embodiment, the yeast cells are modified for overexpression of polyamine:glutathione ligase.

[0070] In one embodiment, overexpression of polyamine:glutathione ligase is achieved by placing the polyamine:glutathione ligase-encoding gene under the transcriptional control of a highly active promoter in yeast cells. Suitable promoters for use in yeast cells include, but are not limited to, the promoters PDC, GPD, GPD1, TEF1, PGK1, TDH, and TDH3. Other suitable promoters include the promoters GAL1, GAL2, GAL10, GAL7, CUP1, HIS3, CYC1, ADH1, PGL, GAPDH, ADC1, URA3, TRP1, LEU2, TPI, AOX1, and ENO1.

[0071] The yeast cells may contain one or more copies, i.e., at least two copies, of the polyamine:glutathione ligase-encoding gene, thereby increasing the copy number of mRNA for the polyamine:glutathione ligase and thereby increasing the amount of polyamine:glutathione ligase produced by the yeast cells. In such cases, the multiple copies of the polyamine:glutathione ligase-encoding gene may be under the transcriptional control of a single promoter, or each polyamine:glutathione ligase-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:glutathione ligase-encoding gene, or different types of promoters may be used.

[0072] In one embodiment, the glutathione-polyamine conjugate is trypanothione (N 1 ,N 10 -bis(glutathionyl)spermidine), N 1 -Glutathionylspermidine, N 10 -Glutathionylspermidine, N 1 ,N 10 -Bis(glutathionyl)spermine, N 1 ,N 5 ,N 10 -tri(glutathionyl)spermine, N 1 ,N 5 ,N 10 ,N 14 -tetra(glutathionyl)spermine. Thus, the conjugate can be a conjugate of one polyamine, such as spermidine or spermine, with one, two, or more, for example, three or four, glutathione molecules.

[0073] In one embodiment, the polyamine:glutathione ligase-encoding gene is selected from the group consisting of trypanothione synthase (EC 6.3.1.9), glutathionyl spermidine synthase (EC 6.3.1.8), and combinations thereof.

[0074] In one embodiment, the trypanothione synthase (TryS)-encoding gene is selected from the group consisting of a nucleotide sequence encoding a trypanothione synthase having at least 80% sequence identity to Trypanosoma brucei brucei trypanothione synthase (TbbTryS) and a nucleotide sequence encoding a trypanothione synthase having at least 85%, or even 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to Trypanosoma brucei brucei TrrS. In one embodiment, the trypanothione synthase having at least 80% sequence identity is capable of catalyzing the conversion of glutathione and polyamines to glutathionylpolyamines and / or the conversion of glutathione and glutathionylpolyamines to bis(glutathionyl)polyamines, preferably the conversion of glutathione and spermidine to glutathionylspermidine and glutathione and glutathionylspermidine to N-(N-) ... 1 ,N 10 -bis(glutathionyl)spermidine. The enzymatic efficiency of the trypanothione synthase having at least 80% sequence identity can be less than, substantially equal to, or greater than the corresponding enzymatic efficiency of TbbTrrS, preferably at least substantially equal to or greater than the enzymatic efficiency of TbbTrrS.

[0075] The amino acid sequence of TbbTryS is shown in SEQ ID NO:34, and the nucleotide sequence of TbbTrrS is shown in SEQ ID NO:35.

[0076] In one embodiment, the glutathionyl spermidine synthase (GSS)-encoding gene is selected from the group consisting of Escherichia coli glutathionyl spermidine synthase (EcGSS) and a nucleotide sequence encoding a glutathionyl spermidine synthase having at least 80% sequence identity to glutathionyl spermidine synthase EcGSS. In one embodiment, the nucleotide sequence encodes a glutathionyl spermidine synthase having at least 85%, or even 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to Escherichia coli EcGSS. In one embodiment, the glutathioneyl spermidine synthase with at least 80% sequence identity can catalyze the conversion of glutathione and polyamines to glutathionyl polyamines, preferably the conversion of glutathione and spermidine to glutathionyl spermidine. The enzymatic efficiency of the glutathione spermidine synthase with at least 80% sequence identity can be lower, substantially equal to, or higher than the corresponding enzymatic efficiency of EcGSS, preferably at least substantially equal to or higher than the enzymatic efficiency of EcGSS.

[0077] The amino acid sequence of EcGSS is set forth in SEQ ID NO:259, and the nucleotide sequence of EcGSS is set forth in SEQ ID NO:260.

[0078] In one embodiment, 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.

[0079] The yeast cells of the present invention lack a polyamine oxidase (EC 1.5.3.17) encoding gene or contain a disrupted polyamine oxidase encoding gene, and also contain at least one polyamine synthase encoding gene.

[0080] The at least one polyamine synthase expressed by the yeast cell catalyzes the production of at least one polyamine within the yeast cell. Polyamine oxidase is an enzyme that catalyzes the conversion of spermine back to spermidine. Thus, the yeast cell lacks any polyamine oxidase-encoding gene or contains a disrupted polyamine oxidase-encoding gene. This means that the yeast cell preferably lacks any polyamine oxidase, or if such a polyamine oxidase is expressed within the yeast cell, the polyamine oxidase is preferably enzymatically inactive, or at least has significantly reduced enzymatic efficiency compared to native polyamine oxidase.

[0081] In one embodiment, the yeast cells are modified for overexpression of at least one polyamine synthase.

[0082] 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. Suitable promoters for use in yeast cells include, but are not limited to, the promoters PDC, GPD, GPD1, TEF1, PGK1, TDH, and TDH3. Other suitable promoters include the promoters GAL1, GAL2, GAL10, GAL7, CUP1, HIS3, CYC1, ADH1, PGL, GAPDH, ADC1, URA3, TRP1, LEU2, TPI, AOX1, and ENO1.

[0083] The yeast cell may contain one or more copies of a polyamine synthase-encoding gene, thereby increasing the copy number of mRNA for the polyamine synthase and thereby increasing the amount of polyamine synthase produced by the yeast cell. In such cases, the multiple copies of the polyamine synthase-encoding gene may be under the transcriptional control of a single 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.

[0084] In one embodiment, the polyamine synthase-encoding gene is selected from the group consisting of a spermine synthase (EC 2.5.1.22)-encoding gene, a thermospermine synthase (EC 2.5.1.79)-encoding gene, and a homospermidine synthase (EC 2.5.1.44 or EC 2.5.1.45)-encoding gene.

[0085] In one embodiment, the spermine synthase-encoding gene is selected from the group consisting of a Saccharomyces cerevisiae spermine synthase, preferably ScSPE4, an Arabidopsis thaliana spermine synthase (AtSPMS), and a nucleotide sequence encoding a spermine synthase having at least 80% sequence identity to the spermine synthase ScSPE4 or the spermine synthase AtSPMS. In one embodiment, the nucleotide sequence encodes a spermine synthase having at least 85%, or even 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to ScSPE4 or AtSPMS. In one embodiment, the spermine synthase having at least 80% sequence identity can catalyze the conversion of spermidine to spermine. The enzymatic efficiency of a spermine synthase having at least 80% sequence identity may be lower than, substantially equal to, or higher than the corresponding enzymatic efficiency of ScSPE4 or AtSPES, preferably at least substantially equal to or higher than the enzymatic efficiency of ScSPE4 or AtSPES.

[0086] The amino acid sequence of ScSPE4 is shown in SEQ ID NO: 1, and the nucleotide sequence of ScSPE4 is shown in SEQ ID NO: 2. The corresponding amino acid sequence of AtSPMS is shown in SEQ ID NO: 3, and the nucleotide sequence of AtSPMS is shown in SEQ ID NO: 4.

[0087] In one embodiment, the thermospermine synthase-encoding gene is selected from the group consisting of an Arabidopsis thaliana thermospermine synthase, preferably AtACL5, and a nucleotide sequence encoding a thermospermine synthase having at least 80% sequence identity to the 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 to AtACL5. In one embodiment, this thermospermine synthase having at least 80% sequence identity can catalyze the conversion of spermidine to thermospermine. The enzymatic efficiency of a thermospermine synthase having at least 80% sequence identity may be lower, substantially equal to, or higher than the corresponding enzymatic efficiency of AtACL5, preferably at least substantially equal to or higher than the enzymatic efficiency of AtACL5.

[0088] The amino acid sequence of AtACL5 is shown in SEQ ID NO:5, and the nucleotide sequence of AtACL5 is shown in SEQ ID NO:6.

[0089] In one embodiment, the homospermidine synthase (HSS)-encoding gene is selected from the group consisting of Senecio vernalis homospermidine synthase (SvHSS), Blastochloris viridis homospermidine synthase (BvHSS), and a nucleotide sequence encoding a homospermidine synthase having at least 80% sequence identity to the homospermidine synthase SvHSS or the 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 to the SvHSS or BvHSS. In one embodiment, the 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 enzymatic efficiency of the homospermidine synthase having at least 80% sequence identity can be less than, substantially equal to, or greater than the corresponding enzymatic efficiency of SvHSS or BvHSS, and is preferably at least substantially equal to or greater than the enzymatic efficiency.

[0090] The amino acid sequence of SvHSS is shown in SEQ ID NO: 7, and the nucleotide sequence of SvHSS is shown in SEQ ID NO: 8. The corresponding amino acid sequence of BvHSS is shown in SEQ ID NO: 9, and the nucleotide sequence of BvHSS is shown in SEQ ID NO: 10.

[0091] In one embodiment, the yeast cell is selected from the group consisting of Saccharomyces, Kluyveromyces, Zygosaccharomyces, Candida, Hanseniaspora, Pichia, Hansenula, Schizosaccharomyces, Trigonopsis, Brettanomyces, Debaromyces, and the like. The fungus is selected from the group consisting of the genera Rhodotorula, Rhodotorula, 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, and Schizosaccharomyces malidevorans. malidevorans).Saccharomyces cerevisiae is the preferred yeast species.

[0092] In one embodiment, the yeast cell is a Saccharomyces cerevisiae cell and the polyamine oxidase is FMS1. Thus, in one embodiment, the S. cerevisiae cell lacks FMS1 or contains a disrupted FMS1.

[0093] Another aspect of the present invention relates to a yeast cell capable of producing a glutathione-polyamine conjugate, wherein the yeast cell is capable of producing at least one polyamine and the yeast cell comprises a gene encoding a polyamine:glutathione ligase.

[0094] Additionally, the various embodiments of yeast cells listed above may be utilized in this aspect of the invention.

[0095] A further aspect of the invention relates to a method for producing a glutathione-polyamine conjugate, the method comprising culturing yeast cells according to the invention in a medium under culture conditions suitable for production of the glutathione-polyamine conjugate by the yeast cells, and collecting the glutathione-polyamine conjugate from the medium and / or from the yeast cells.

[0096] The medium in this aspect of the invention can be any medium in which yeast cells can be cultured to produce glutathione-polyamine conjugates. The culture can be, for example, in the form of batch, fed-batch, or perfusion culture, or fermentation, bioreactor fermentation, etc.

[0097] example Example 1: Improving spermidine production by systematic rewiring of native metabolism in yeast In Example 1, we systematically refactored the metabolism of 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, we also introduced additional potential positive gene targets. This yeast strain was constructed using a novel modular genetic design. Specifically, the de novo Spd biosynthetic pathway was divided into multiple genetic modules containing coding sequences for multiple biosynthetic enzymes to divert a larger carbon flux from sugar carbon sources to Spd.

[0098] The precursor overproduction module (I), designed to increase L-ornithine (Orn) accumulation, consists of eight proteins: NADP from Saccharomyces cerevisiae (S. cerevisiae); (+) Dependent glutamate dehydrogenase (GDH1) [SEQ ID NO: 11], mitochondrial aspartate glutamate carrier protein (AGC1) from Saccharomyces cerevisiae (S. cerevisiae) [SEQ ID NO: 12], mitochondrial L-ornithine carrier protein (ORT1) from Saccharomyces cerevisiae (S. cerevisiae) [SEQ ID NO: 13], glutamate N-acetyltransferase (EcargA) from Escherichia coli (E. coli) [SEQ ID NO: 14], acetylglutamate kinase (EcargB) from Escherichia coli (E. coli) [SEQ ID NO: 15], Corynebacterium The overexpression of N-acetyl-gamma-glutamyl-phosphate reductase (CgargC) from C. glutamicum (SEQ ID NO: 16), acetylornithine aminotransferase (CgargD) from C. glutamicum (SEQ ID NO: 17), and ornithine acetyltransferase (CgargJ) from C. glutamicum (SEQ ID NO: 18) was also included. Additionally, the attenuation or removal of two proteins: the native promoter P ARG3 , a weaker promoter P KEX2Attenuation of yeast native ornithine carbamoyltransferase (ARG3) [SEQ ID NO: 19] by replacing it with and elimination of L-ornithine transaminase (CAR2) [SEQ ID NO: 20] activity by knocking out CAR2 were also included in this module (I).

[0099] The putrescine (Put) module (II), designed to overproduce Put from L-ornithine, contained two genetic modifications: overexpression of ornithine decarboxylase (SPE1) [SEQ ID NO: 21] from Saccharomyces cerevisiae and deletion of native ornithine decarboxylase antienzyme (OAZ1) [SEQ ID NO: 22].

[0100] The spermidine biosynthesis module (III) was designed for the overproduction of spermidine (Spd) from putrescine (featuring overexpression of two proteins from S. cerevisiae: adenosylmethionine decarboxylase (AdoMetDC; SPE2) [SEQ ID NO: 23] and spermidine synthase (SpdSyn; SPE3) [SEQ ID NO: 24]). This module also contained deletions of two native proteins to avoid spermidine consumption or degradation: SPE4 [SEQ ID NO: 2], encoding spermine synthase, and FMS1 [SEQ ID NO: 25], encoding a nonspecific polyamine oxidase.

[0101] The S-adenosyl-L-methionine (AdoMet) module (IV) was designed to increase the accessibility of the cofactor AdoMet. The modifications included overexpression of multiple proteins: 5'-methylthioadenosine phosphorylase (MEU1) from S. cerevisiae [SEQ ID NO: 26], branched-chain amino acid aminotransferase (BAT2) from S. cerevisiae [SEQ ID NO: 27], adenine phosphoribosyltransferase (APT1) from S. cerevisiae [SEQ ID NO: 28], ribose-phosphate pyrophosphokinase (PRS5) from S. cerevisiae [SEQ ID NO: 29], and S-adenosylmethionine synthase (LiMAT) from Leishmania infantum [SEQ ID NO: 30]. This module also contained deletion of adenine deaminase activity (AAH1) [SEQ ID NO: 31].

[0102] The polyamine efflux module (V) was designed to alleviate cytotoxicity to cells or inhibition of polyamine biosynthesis and included overexpression of the yeast native polyamine transporter encoded by TPO5 [SEQ ID NO: 32].

[0103] Finally, and more importantly, an additional spermidine biosynthesis module (VI) was designed for the overproduction of spermidine from putrescine and AdoMet, which involved overexpression of the AdoMetDC-SpdSyn fusion protein [SEQ ID NO: 33] encoded by SPE2-SPE3.

[0104] Overexpression of the genes in Example 1 was achieved by chromosomal integration into regions predicted by the inventors to lack growth defects and active expression at the integration locus via the CRISPR / Cas9 system or traditional genetic marker-based methods. CRISPR / Cas9-based genome editing was performed according to the protocol developed by Mans et al. (2015). Specifically, the S. cerevisiae strain CEN.PK113-11C, carrying the plasmid pL-CAS9-HIS with a HIS3 marker, which enables constitutive expression of Cas9, was the starting strain for all genetic modifications. Multiple guide RNA (gRNA) plasmids were constructed to enable efficient genome editing at selected loci. Genetic modules containing various combinations of promoters, terminators, ORFs, and homology arms were assembled as integration cassettes using overlap extension PCR (OE-PCR) procedures. 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; TEF1 p-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.

[0105] All native genetic segments, i.e., native promoter, terminator, ORF, and homology 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 standard LiAc / SS DNA / PEG transformation methods. 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 without amino acids (YNB), 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 on 5-fluoroorotic acid (5'-FOA) plates. Additionally, CRISPR / cas9-based systems were used to perform deletions of AAH1, SPE4, and FMS1. Other gene knockout experiments were performed by conventional methods. All primers used herein 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

[0106] The resulting strain JQSPD_AA was evaluated using an assay combining deep-well scale fermentation with high-performance liquid chromatography (HPLC). Specifically, 24 deep-well batch fermentations of the resulting strain JQSPD_AA for polyamine production were performed in minimal medium developed by Verduyn et al. (1992). Cultures from 24-hour precultures were grown to an initial OD of 0.2. 600The strain was inoculated into 2 ml of minimal medium in a 24-deep-well plate 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, if necessary), 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, we used minimal medium for deep-well plate fermentation as the extraction context. Tubes containing 0.9 ml of fermentation medium were preheated in a 100°C water bath for 10 minutes. The hot fermentation medium was then quickly poured onto 0.1 ml of liquid culture; the mixture was immediately vortexed, and the sample was placed in a water bath. After 30 minutes, each tube was placed on ice for 5 minutes. After centrifugation, the supernatant was directly used 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 sample. The reaction mixture was then incubated in the dark at 40°C 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), which was used for HPLC detection. The following chromatographic conditions were used: C18 (100 mm x 4.6 mm i.d., 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 start at 4.0 min. The mobile phase was water and methanol at a rate of 1 ml / min. The elution program was as follows: 50% to 65% methanol (0-5 min), 65% to 75% methanol (5-7.5 min), 75% to 87.5% methanol (7.5-9.5 min), 87.5% to 100% methanol (9.5-10.5 min), 100% methanol (10.5-11.5 min), 100% to 50% methanol (11.5-13.5 min), and 50% methanol (13.5-16 min).

[0107] Strain JQSPD_AA produced Spd titers at concentrations of >400 mg / l, a significantly increased Spd titer compared to strains with only partial modifications used herein (see examples in WO 2016 / 144247 and WO 2019 / 013696).

[0108] Example 2: Higher polyamine production in yeast Life has evolved diverse pathways to synthesize structural variants of polyamines. Indeed, while Put and Spd are typically found in most cells as common polyamines, unusual polyamines such as sym-homospermidine (Hspd), thermospermine (Tspm), spermine (Spm), branched-chain polyamines, and long-chain polyamines (LCPAs) have also been identified in nature. Example 2 investigated the biosynthesis of sym-homospermidine (Hspd), thermospermine (Tspm), and spermine (Spm) by designing and introducing genetic module VII into the Spd platform strain JQSPD_AA of Example 1.

[0109] We first set out to heterologously synthesize the triamine Hspd, which is present in both plants and bacteria. In plants, Hspd is the first pathway-specific intermediate in pyrrolizidine alkaloid biosynthesis, 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). This is because the latter cannot use Put as the aminobutyl group donor. To explore the potential of both plant and bacterial HSSs for microbial production of Hspd, genetic submodules (VII-a) and (VII-b) were designed for the biosynthesis of Hspd in yeast, encoding the expression of Senecio vernalis SvHSS and Blastochloris viridis BvHSS13, respectively. The submodules were introduced as high-copy plasmids SvHSS_p426GPD and BvHSS_p426GPD ordered from GenScipt to carry the yeast-codon-optimized SvHSS gene [SEQ ID NO: 8] and BvHSS gene [SEQ ID NO: 10], respectively, in the Spd platform strain JQSPD_AA. Transformation experiments followed the same procedures as in Example 1. The resulting strains JQSPD_AA(SvHSS_p426GPD) and JQSPD_AA(BvHSS_p426GPD) were assayed for Hspd production using the same procedures as described in Example 1.

[0110] We found that overexpression of both HSSs enabled the biosynthesis of Hspd. Specifically, SvHSS enabled an Hspd titer of 40.9 mg / L, whereas BvHSS enabled an Hspd titer of 31.1 mg / L (Fig. 1a and 1d).

[0111] Subsequently, we also utilized the Spd platform (Example 1) for the production of the 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, spermine synthase (SpmSyn; EC 2.5.1.22), is responsible for Spm biosynthesis. We first investigated the yeast native SpmSyn Spe4p for Spm overproduction.

[0112] When the codon-optimized yeast SPE4 [SEQ ID NO: 2] was overexpressed as a high-copy plasmid SPE4_p426GPD (submodule (VII-c)) in JQSPD_AA, 53.1 mg / L of Spm was obtained (see Figure 1c and Figure 1f). We also demonstrated that AtSPMS [SEQ ID NO: 4] overexpressed as a high-copy plasmid AtSPMS_p426GPD (submodule (VII-d)) in JQSPD_AA strain increased the production of Arabidopsis thaliana ( SpmSyn from Arabidopsis thaliana was tested, resulting in the production of Spm (41.8 mg / L; see Figures 1c and 1f). Plant ACL5 aminopropyltransferase (TspmSyn; EC 2.5.1.79) from Arabidopsis thaliana was shown to synthesize the Spm isomer Tspm. Accordingly, we also overexpressed AtACL5 [SEQ ID NO: 6] as a high-copy plasmid, AtACL5_p426GPD (submodule (VII-e)), in strain JQSPD_AA. This strategy enabled the production of 43.8 mg / L Tspm (see Figures 1b and 1e). All plasmids carrying yeast codon-optimized genes were purchased from GenScript. In Example 2, the same transformation and product assays as those used in Example 1 were used. All plasmids are listed in Table 2, and all strains are listed in Table 3.

[0113] FIG. 3a shows a modified pathway for the biosynthesis of spermidine and higher polyamines in yeast.

[0114] Example 3: Biosynthesis of trypanothione in yeast The amide bond is undoubtedly one of the most important structural motifs in nature. Approximately one-quarter of all marketed drugs and two-thirds of all drug candidates contain at least one amide bond, and acylation of amines is one of the most widely performed reactions in the pharmaceutical industry. Polyamines present unique scaffolds for attaching other moieties and are often incorporated into specialized metabolisms, leading to the biosynthesis of a variety of amide bond-containing natural products with complex structures. For example, trypanothione (N 1 ,N 10 -Bis(glutathionyl)spermidine (T(SH)2) is the major low-molecular-weight thiol in trypanosomatids of the genera Crithidia, Trypanosoma, and Leishmania. The latter two are the causative agents of fatal or disabling diseases, such as African sleeping sickness, kala-azar, Chagas disease, and espunzia or eastern bronchus. Meanwhile, abundant polyamine-containing hydroxycinnamic acid amides, which are hypothesized to be involved in flower, pollen, and seed development as well as pathogen resistance, can be synthesized in plants. However, obtaining polyamine-containing hydroxycinnamic acid amides either through traditional synthetic chemistry or extraction from natural sources is difficult. First, these polyamine conjugates exhibit low abundance in nature (Li et al. 2018). On the other hand, establishing an efficient catalytic system for direct amidation reactions has remained a formidable challenge in organic chemistry for many years (Wang 2019). In Example 3, we utilized the polyamine platform of Example 1 for the biosynthesis of polyamine conjugates by introducing module (VIII).

[0115] We first undertook the heterologous biosynthesis of [T(SH)2] in yeast. [T(SH)2], a major redox mediator in pathogenic trypanosomes, is synthesized in a stepwise manner in Crithidia fasciculata by two distinct enzymes, glutathione (GSH)- and spermidine-derived enzymes, glutathione synthase (GspS; EC 6.3.1.8) and trypanothione synthase (TryS; EC 6.3.1.9). In Trypanosoma brucei brucei, both steps are catalyzed by a specialized TryS enzyme with broad substrate specificity (EC 6.3.1.9). To investigate TbbTryS for microbial production of [T(SH)2], the genetic submodule (VIII-a) designed to synthesize [T(SH)2] in yeast encoded the expression of T. brucei brucei TryS (TbbTryS). The submodule was introduced into the Spd platform strain JQSPD_AA as a high-copy plasmid, TbbTryS_p426GPD, carrying the yeast codon-optimized TbbTryS gene [SEQ ID NO: 35], ordered from GenScript. Transformation experiments were performed according to the same procedure as described in Example 1. The resulting strain, JQSPD_AA(TbbTryS_p426GPD), was used for 24-deep-well fermentation using the same procedure as described in Example 1. Fermentation samples were prepared by taking 0.1 ml of liquid culture. The fermentation samples were subjected to hot water (HW) extraction. In our method, extraction was performed using the fermentation medium. Tubes containing 0.9 ml of fermentation medium were preheated in a water bath at 100°C for 10 minutes. The hot fermentation medium was then quickly poured onto 0.1 ml of 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 directly used for detection.

[0116] Polyamine conjugates were detected by liquid chromatography-mass spectrometry (LC-MS) on 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 x 100 mm, 1.8 μm column maintained at 35°C. The flow rate was 0.350 mL / min using 0.1% formic acid (A) and 0.1% formic acid (B) in acetonitrile (mobile phase). The gradient started at 5% B for 1 min, followed by a linear gradient to 95% B for 5 min. This solvent composition was held for 1.5 min. It then changed to 5% B and held for 8 min. Samples (5 μl) were run in positive or negative ion mode on a MS equipped with a heated electrospray ionization source (HESI). The sheath gas was set at 50 au, the auxiliary gas at 10 au, and the sweep gas at 1 au. The cone and probe temperatures were 325°C and 380°C, respectively, and the spray voltage was 3500V. The scan range was 80 to 500 Da, and the time between scans was 50 ms. Consistent with the property of TbbTr catalyzing the two GSH moieties on spermidine, overexpression of this enzyme in the Spd platform JQSPD_AA of Example 1 resulted in the production of [T(SH)2]. Specifically, 722.2977 [M+H] + or 361.6524[M+H] 2+ A single new LC-MS peak was detected with an m / z value corresponding to [T(SH)2], indicating the presence of [T(SH)2] (see Figures 2a and 2b). All plasmids are listed in Table 2, and all strains are listed in Table 3.

[0117] FIG. 3b shows a modified pathway for the biosynthesis of glutathione-polyamine conjugates in yeast.

[0118] The above-described embodiments should be understood as some illustrative 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, different part solutions in different embodiments can be combined in other configurations where technically possible. However, the scope of the present invention is defined by the appended claims. The present invention includes the following preferred embodiments. (1) A yeast cell capable of producing a glutathione-polyamine conjugate, capable of producing at least one polyamine; Contains polyamine:glutathione ligase-encoding genes; at least one polyamine synthase-encoding gene; and A yeast cell lacking a polyamine oxidase-encoding gene or containing a disrupted polyamine oxidase-encoding gene. (2) The yeast cell according to (1), which has been modified for overexpression of the polyamine:glutathione ligase. (3) The glutathione-polyamine conjugate is trypanothione, N 1 -Glutathionylspermidine, N 10 -Glutathionylspermidine, N 1 ,N 10 -Bis(glutathionyl)spermine, N 1 ,N 5 ,N 10 -tri(glutathionyl)spermine, and N 1 ,N 5 ,N 10 ,N 14 -tetra(glutathionyl)spermine. (4) The yeast cell according to any one of (1) to (3), wherein the polyamine:glutathione ligase-encoding gene is selected from the group consisting of trypanothione synthase, glutathionylspermidine synthase, and a combination thereof. (5) The yeast cell according to (4), wherein the trypanothione synthase-encoding gene is selected from the group consisting of Trypanosoma brucei brucei TbbTryS and a nucleotide sequence encoding a trypanothione synthase having at least 80% sequence identity to the trypanothione synthase TbbTryS. (6) The yeast cell according to (4) or (5), wherein the glutathionyl spermidine synthase (GSS)-encoding gene is selected from the group consisting of Escherichia coli EcGSS and a nucleotide sequence encoding a glutathionyl spermidine synthase having at least 80% sequence identity with glutathionyl spermidine synthase EcGSS. (7) The yeast cell according to any one of (1) to (6), 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. (8) The yeast cell according to any one of (1) to (7), which has been modified for overexpression of the at least one polyamine synthase. (9) The yeast cell according to any one of (1) to (8), wherein the polyamine synthase-encoding gene is selected from the group consisting of a spermine synthase-encoding gene, a thermospermine synthase-encoding gene, and a homospermidine synthase-encoding gene. (10) The yeast cell according to (9), wherein the spermine synthase-encoding gene is selected from the group consisting of Saccharomyces cerevisiae SPE4, Arabidopsis thaliana AtSPMS, and a nucleotide sequence encoding a spermine synthase having at least 80% sequence identity to spermine synthase SPE4 or spermine synthase AtSPMS. (11) The yeast cell according to (9) or (10), wherein the thermospermine synthase-encoding gene is selected from the group consisting of Arabidopsis thaliana AtACL5 and a nucleotide sequence encoding a thermospermine synthase having at least 80% sequence identity to the thermospermine synthase AtACL5. (12) The yeast cell according to any one of (9) to (11), wherein the homospermidine synthase-encoding gene is selected from the group consisting of nucleotide sequences encoding homospermidine synthases having at least 80% sequence identity to the homospermidine synthase SvHSS or the homospermidine synthase BvHSS. (13) The yeast cell according to any one of (1) to (12), wherein the yeast cell is a Saccharomyces cerevisiae cell and the polyamine oxidase is FMS1. (14) A method for producing a glutathione-polyamine conjugate, comprising: Culturing the yeast cell according to any one of (1) to (13) in a medium under culture conditions suitable for producing the glutathione-polyamine conjugate by the yeast cell; collecting the glutathione-polyamine conjugate from the medium and / or from the yeast cells; A method comprising:

[0119] References JPEG0007742162000021.jpg176167 JPEG0007742162000022.jpg162167 [Sequence List Free Text]

[0120] Sequence list 36-258 <223> Primer

Claims

1. A yeast cell that produces a glutathione-polyamine conjugate, comprising: the yeast cell produces at least one polyamine; the at least one polyamine is selected from the group consisting of spermine, spermidine, and combinations thereof; the yeast cell contains and expresses a polyamine:glutathione ligase-encoding gene; the yeast cell contains and expresses at least one polyamine synthase-encoding gene; and the yeast cell lacks a polyamine oxidase-encoding gene or contains a disrupted polyamine oxidase-encoding gene; the polyamine:glutathione ligase-encoding gene is selected from the group consisting of a trypanothione synthase-encoding gene, a glutathionyl spermidine synthase-encoding gene, and a combination thereof; The yeast cells described above.

2. The glutathione-polyamine conjugate is trypanothione, N １ -glutathionylspermidine, N 8-glutathionylspermidine, and N １ , N 12-bis(glutathionyl)spermine.

3. The trypanothione synthase-encoding gene is A nucleotide sequence encoding Trypanosoma brucei brucei TbbTryS, and A nucleotide sequence encoding a trypanothione synthase having at least 90% sequence identity with the nucleotide sequence of trypanothione synthase TbbTryS set forth in SEQ ID NO:

35.

3. The yeast cell of claim 1 or 2, selected from the group consisting of:

4. The glutathionyl spermidine synthase (GSS) encoding gene is A nucleotide sequence encoding Escherichia coli EcGSS, and A nucleotide sequence encoding a glutathionyl spermidine synthase having at least 90% sequence identity with the nucleotide sequence of glutathionyl spermidine synthase EcGSS set forth in SEQ ID NO:

260. The yeast cell according to any one of claims 1 to 3, selected from the group consisting of:

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 and a spermidine synthase-encoding gene.

6. The spermine synthase-encoding gene is A nucleotide sequence encoding Saccharomyces cerevisiae SPE4, A nucleotide sequence encoding the Arabidopsis thaliana AtSPMS, and A nucleotide sequence encoding a spermine synthase having at least 90% sequence identity with the nucleotide sequence of spermine synthase SPE4 set forth in SEQ ID NO: 2 or the nucleotide sequence of spermine synthase AtSPMS set forth in SEQ ID NO:

4.

6. The yeast cell of claim 5, selected from the group consisting of:

7. The yeast cell according to any one of claims 1 to 6, wherein the yeast cell is a Saccharomyces cerevisiae cell and the polyamine oxidase is FMS1.

8. 1. A method for producing a glutathione-polyamine conjugate, comprising: The method comprises: Cultivating the yeast cell according to any one of claims 1 to 7 in a medium under culture conditions suitable for the production of the glutathione-polyamine conjugate by the yeast cell; collecting the glutathione-polyamine conjugate from the culture medium and / or from the yeast cells; Including, the polyamine is selected from the group consisting of spermine, spermidine, and combinations thereof; The above method.

Images