Patent Application
20200347396
This application incorporates by reference the Sequence Listing contained in the following ASCII text file being submitted concurrently herewith:
Cyclic peptides are an emerging source of pharmaceutical and agrochemical innovation in order to treat human and plant diseases, respectively [1]. Cyclic peptide natural products can be used in medicine as immunosuppressants, antibiotics and anti-cancer agents [2] and in agriculture for plant pathogen control [3]. The emergence of resistance against many current antibiotics and pest control agents in human pathogens and plant pathogens [5], respectively, demands new discovery and metabolic engineering platforms for peptide-based drug and agrochemical development. Additionally, plant peptides are important endogenous chemicals to modulate the rhizosphere during plant development [6] and under abiotic stresses [7], and, thus, they have potential in optimizing plant fitness in changing climates through rhizosphere engineering.
Described herein is a method of producing one or more lyciumin cyclic peptides. In some embodiments, the method of producing one or more lyciumin cyclic peptides can include providing a host cell that includes a transgene encoding a lyciumin precursor peptide, or a biologically-active fragment thereof, and expressing the transgene in the host cell to thereby produce a lyciumin precursor peptide, or biologically-active fragment thereof. The lyciumin precursor peptide, or biologically-active fragment thereof, can include one or more core lyciumin peptide domains. In some embodiments, the lyciumin precursor peptide, or biologically-active fragment thereof, can be converted to one or more lyciumin cyclic peptides in the host cell.
In some embodiments, the transgene is operably linked to a heterologous promoter in the host cell. In some embodiments, the transgene is introduced in a vector. In some embodiments, the method includes introducing the transgene into the host cell. In some embodiments, the method includes introducing a vector that includes the transgene into the host cell. In some embodiments, the lyciumin precursor peptide includes a plurality of core lyciumin peptide domains. In some embodiments, the core lyciumin peptide domains can encode two or more different lyciumin cyclic peptides.
The host cell can express one or more of: an enzyme that cyclizes the lyciumin precursor peptide; an endopeptidase; a glutamine cyclotransferase; and/or an exopeptidase. In some embodiments, arginine is immediately N-terminal to the core lyciumin peptide domain. In some embodiments, the endopeptidase is an arginine endopeptidase. In some embodiments tyrosine is immediately C-terminal to the core lyciumin peptide domain.
In some embodiments, the host cell is a plant cell. In some embodiments, the plant cell is an Amaranthaceae family plant cell. In some embodiments, the plant cell is an Amaranthus genus plant cell, such as an Amaranthus hypochondriacus plant cell. In some embodiments, the plant cell is a Beta genus plant cell, such as a Beta vulgaris plant cell. In some embodiments, the plant cell is a Chenopodium genus plant cell, such as a Chenopodium quinoa plant cell. In some embodiments, the plant cell is a Fabaceae family plant cell. In some embodiments, the plant cell is a Glycine genus plant cell, such as a Glycine max plant cell. In some embodiments, the plant cell is a Medicago genus plant cell, such as a Medicago truncatula plant cell. In some embodiments, the plant cell is a Solanaceae family plant cell. In some embodiments, the plant cell is a Solanum genus plant cell, such as a Solanum melongena plant cell or a Solanum tuberosum plant cell. In some embodiments, the plant cell is a Nicotiana genus plant cell, such as a Nicotiana benthamiana plant cell. In some embodiments, the plant cell is a Capsicum genus plant cell, such as a Capsicum annuum plant cell.
In some embodiments, the lyciumin precursor peptide includes SEQ ID NO: 1. In some embodiments, the lyciumin precursor peptide consists of SEQ ID NO: 1. In some embodiments, the lyciumin precursor peptide consists essentially of SEQ ID NO: 1. In some embodiments, the lyciumin precursor peptide includes SEQ ID NO: 2. In some embodiments, the lyciumin precursor peptide consists of SEQ ID NO: 2. In some embodiments, the lyciumin precursor peptide consists essentially of SEQ ID NO: 2. In some embodiments, the lyciumin cyclic peptide is Lyciumin A, Lyciumin B, Lyciumin C, or Lyciumin D, or a combination thereof.
Described herein also is a method of generating a library of nucleic acids encoding lyciumin precursor peptides, or biologically-active fragments thereof. The method can include constructing a plurality of vectors, each vector comprising a nucleic acid encoding a different lyciumin precursor peptide, or biologically-active fragment thereof, operably linked to a heterologous promoter for expression in a host cell. In some embodiments, the library can include at least at least hundreds of nucleic acids, e.g., at least 103 nucleic acids, at least 104 nucleic acids, at least 105 nucleic acids, at least 106 nucleic acids, or at least 10′ nucleic acids.
In some embodiments, the method of generating a library of nucleic acids can include introducing the plurality of vectors into host cells. In certain embodiments, the lyciumin precursor peptide, or biologically-active fragments thereof, can be converted to one or more lyciumin cyclic peptides in the host cell. In some embodiments, the host cell is a plant cell. In some embodiments, the plant cell is a Solanaceae family plant cell. In some embodiments, the plant cell is a Nicotiana genus plant cell, such as a Nicotiana benthamiana plant cell.
In some embodiments, the method can include isolating a lyciumin cyclic peptide from the host cell. In some embodiments, the method can include assaying for an activity of interest either crude extract from the host cell or a lyciumin peptide isolated from the host cell.
In some embodiments, the method of generating a library of nucleic acids can include introducing a nucleic acid encoding a lyciumin peptide having an activity of interest into a second host cell. In some embodiments, the second host cell is a plant cell. In some embodiments, the plant cell is an Amaranthaceae family plant cell. In some embodiments, the plant cell is an Amaranthus genus plant cell, such as an Amaranthus hypochondriacus plant cell. In some embodiments, the plant cell is a Beta genus plant cell, such as a Beta vulgaris plant cell. In some embodiments, the plant cell is a Chenopodium genus plant cell, such as a Chenopodium quinoa plant cell. In some embodiments, the plant cell is a Fabaceae family plant cell. In some embodiments, the plant cell is a Glycine genus plant cell, such as a Glycine max plant cell. In some embodiments, the plant cell is a Medicago genus plant cell, such as a Medicago truncatula plant cell. In some embodiments, the plant cell is a Solanaceae family plant cell. In some embodiments, the plant cell is a Solanum genus plant cell, such as a Solanum melongena plant cell or a Solanum tuberosum plant cell. In some embodiments, the plant cell is a Nicotiana genus plant cell, such as a Nicotiana benthamiana plant cell. In some embodiments, the plant cell is a Capsicum genus plant cell, such as a Capsicum annuum plant cell.
Also described herein are isolated nucleic acids comprising a nucleotide sequence encoding a lyciumin precursor peptide, or a biologically-active fragment thereof, operably linked to a heterologous promoter. In some embodiments, the lyciumin precursor peptide includes a plurality of core lyciumin peptide domains. In some embodiments, the core lyciumin peptide domains encode two or more different lyciumin cyclic peptides. In some embodiments, the lyciumin precursor peptide comprises SEQ ID NO: 1. In some embodiments, the lyciumin precursor peptide comprises SEQ ID NO: 2. In some embodiments, the nucleic acid is a cDNA.
Described herein are vectors that include any of the nucleic acids described herein.
Described herein are host cells that include any of the nucleic acids or vectors described herein. In some embodiments, the host cell is a plant cell. In some embodiments, the plant cell is an Amaranthaceae family plant cell. In some embodiments, the plant cell is an Amaranthus genus plant cell, such as an Amaranthus hypochondriacus plant cell. In some embodiments, the plant cell is a Beta genus plant cell, such as a Beta vulgaris plant cell. In some embodiments, the plant cell is a Chenopodium genus plant cell, such as a Chenopodium quinoa plant cell. In some embodiments, the plant cell is a Fabaceae family plant cell. In some embodiments, the plant cell is a Glycine genus plant cell, such as a Glycine max plant cell. In some embodiments, the plant cell is a Medicago genus plant cell, such as a Medicago truncatula plant cell. In some embodiments, the plant cell is a Solanaceae family plant cell. In some embodiments, the plant cell is a Solanum genus plant cell, such as a Solanum melongena plant cell or a Solanum tuberosum plant cell. In some embodiments, the plant cell is a Nicotiana genus plant cell, such as a Nicotiana benthamiana plant cell. In some embodiments, the plant cell is a Capsicum genus plant cell, such as a Capsicum annuum plant cell.
Further described herein is a library that includes a plurality of nucleic acid molecules, each nucleic acid molecule including a nucleotide sequence encoding a lyciumin precursor peptide, or a biologically-active fragment thereof. In some embodiments, the nucleotide sequence encoding a lyciumin precursor peptide, or a biologically-active fragment thereof, is operably linked to a heterologous promoter in each nucleic acid molecule. In some embodiments, the nucleic acid molecules are complementary DNA (cDNA) molecules.
In addition, described herein are lyciumin cyclic peptides produced by a method described herein.
Described herein is a method of producing one or more lyciumin cyclic peptides. The method can include: a) providing a host cell that includes a transgene encoding a polypeptide that includes one or more core lyciumin peptide domains; and b) expressing the transgene in the host cell to thereby produce a polypeptide that comprises one or more core lyciumin peptide domains. In some embodiments, the polypeptide is converted to one or more lyciumin cyclic peptides in the host cell.
The methods and products described herein can be used to produce a platform for lyciumin expression and diversification, which can be used to create a library of lyciumin cyclic peptides. The lyciumins precursor peptides described herein can be expressed in planta. The lyciumin cyclic peptides described herein can be used in agrochemical and pharmaceutical applications that aim to increase plant fitness towards abiotic and biotic stresses and treat human diseases, respectively.
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
The foregoing will be apparent from the following more particular description of example embodiments, as illustrated in the accompanying drawings in which like reference characters refer to the same parts throughout the different views. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating embodiments.
A description of example embodiments follows.
Ribosomally synthesized and post-translationally modified peptides (RiPPs) have been rapidly expanded in defined classes in the era of whole genome sequencing. While most RiPPs have been characterized from bacteria and fungi, few examples are known from plants. Described herein are lyciumins as a plant RiPP class. A lyciumin precursor gene was identified from the lyciumin producer Lycium barbarum. A precursor gene-guided genome mining approach was used to show that lyciumin genotypes and chemotypes are widely distributed in crop and forage plants. The promiscuity of the lyciumin pathway led to the discovery of peptide macrocyclization chemistry in lyciumin-type peptides from pepper seeds and suggests a largely untapped peptide chemical space in the plant kingdom. Based on the physical connection of lyciumin core peptides to protein domains associated with abiotic stress responses in plants, a platform for lyciumin expression and diversification was developed, which can be used to create a library of lyciumin cyclic peptides. The lyciumins described herein can be expressed in planta. The lyciumins cyclic peptides described herein can be used in agrochemical and pharmaceutical applications that aim to increase plant fitness towards abiotic and biotic stresses and treat human diseases, respectively.
Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a rapidly growing class of natural products, since whole genome sequencing enabled the discovery of many RiPP precursor genes and corresponding biosynthetic pathways [8]. While most RiPPs have been discovered from bacteria and fungi, few examples are known from plants. The two biosynthetically defined classes of characterized plant RiPPs are cyclotides and orbitides, which are “head-to-tail” cyclic peptides with or without disulfide bonds, respectively [8-10]. Beyond “head-to-tail” cyclic peptides, the phytochemical repertoire of cyclic peptides suggests that there is a largely untapped diversity of branched cyclic plant peptide chemistry and underlying biochemistry to be discovered [11].
Discovery of peptide natural products and their biosynthetic pathways from microbes and fungi has been revolutionized by genome mining approaches using a vast resource of microbial and fungal genome sequences and biosynthetic knowledge of peptide natural product biosynthesis [12-16]. During the past decade, the number of publicly available plant genomes has exponentially increased due to the improvement of sequencing technologies and lowered genome sequencing costs [17]. In addition, characterization of plant biosynthetic pathways has accelerated due to synthetic biology approaches [18,19]. In analogy to microbial and fungal genomics, the growing plant genomic resource has inspired genome mining approaches for known classes of plant natural products [20-22] based on the current knowledge of plant natural product biosynthesis [23]. Generally, in a genome mining experiment predicted biosynthetic genotypes are connected with corresponding chemotypes based on applied biosynthetic knowledge in three steps: (A) Genotype prediction (the prediction of biosynthetic genes), (B) chemotype prediction (the prediction of structural features of a natural product from its biosynthetic genes) and (C) structure-guided chemotyping (the connection of an analyte structure with the predicted natural product structure) [13]. However, there are several challenges associated with plant natural product discovery by genome mining: (A) Genotype prediction is complicated by knowledge gaps in plant natural product biosynthesis and no or only partial clustering of biosynthetic genes in some plant natural product pathways, (B) chemotype prediction from biosynthetic genes can be difficult for certain natural product classes, and (C) structure-guided chemotyping can be problematic in terms of identification of structural information that can be connected to a predicted chemotype from given biosynthetic genes for a successful genome mining experiment. RiPPs have advantages to circumvent these general problems of plant genome mining because the peptide sequence is directly encoded in the genome as a core peptide within a precursor peptide [8]. Thus, identification of a precursor peptide gene specific to a chemically defined RiPP class yields structural information via the core peptide to enable the prediction of a RiPP structure for subsequent structure-guided chemotyping, for example by mass spectrometry [24]. Such a precursor gene-guided genome mining approach for plant RiPPs should not require knowledge of other biosynthetic genes encoding post-translationally modifying enzymes or proteases.
A precursor gene-guided genome mining approach was employed to identify a candidate class of plant RiPPs: the branched cyclic lyciumin peptides [25]. Lyciumins were originally isolated as inhibitors of the angiotensin-converting enzyme and renin from the roots of Lycium barbarum (Solanaceae,
Described herein is the discovery of lyciumin cyclic peptides from crop and forage plants by precursor gene-guided genome mining. Based on the physical connection of lyciumin biosynthesis and abiotic stress response reception in lyciumin precursor peptides, a platform is established to metabolically engineer and produce lyciumin peptide libraries, e.g., in planta. Such lyciumin peptide libraries can be used for, e.g., future engineering applications towards crops with increased stress tolerance. The discovery of lyciumins is a blueprint for peptide discovery by genome mining in the plant kingdom and lyciumin metabolic engineering sets the stage for their potential application as agrochemicals or pharmaceuticals to increase crop fitness and treat human diseases, respectively.
As used herein, the term “lyciumin precursor peptide” refers to a peptide that includes an N-terminal leader domain, one or more core lyciumin peptide domains, and, optionally, a C-terminal BURP domain or C-terminal DUF2775 domain. In some instances, one or more core lyciumin peptide domains can be within a BURP domain. In some instances, one or more core lyciumin peptide domains can be within a DUF2775 domain. In some instances, one or more core lyciumin peptide domains are not within (e.g., outside) a BURP domain. In some instances, one or more core lyciumin peptide domains can be within the N-terminal leader domain. In some instances, one or more core lyciumin peptide domains are not within (e.g., outside) the N-terminal leader domain. In some embodiments, a lyciumin precursor peptide includes from one to twenty core lyciumin peptide domains. In some embodiments, a lyciumin precursor peptide includes from one to ten core lyciumin peptide domains. In some instances, lyciumin precursor peptides can include more than twenty core lyciumin peptide domains. In some embodiments, the lyciumin precursor peptide includes a C-terminal BURP domain. In some embodiments, the lyciumin precursor peptide, or biologically-active fragment thereof, can include a signal peptide sequence. For example, a signal peptide sequence can direct a lyciumin precursor peptide, or biologically-active fragment thereof, through a portion of the secretory pathway and can facilitate localization to a particular organelle, such as a vacuole, which can be relevant for subsequent processing or conversion from a lyciumin precursor peptide to a lyciumin cyclic peptide. A signal peptide can be endogenous for a particular host cell or plant cell, or it can be heterologous. Typically, a signal peptide is located N-terminal to one or more core lyciumin peptide domains. In some instances, a signal peptide can be part of an N-terminal leader domain. In certain host cells (e.g., mammalian or plant host cells), expression and/or secretion of a protein can be increased by using a signal sequence, such as a heterologous signal sequence. Therefore, in some embodiments, the lyciumin precursor peptide includes a heterologous signal sequence at its N-terminus.
As used herein, the term “core lyciumin peptide domain” refers to a peptide domain that includes eight amino acids. The peptide is of the form QXX(G/A/T/S/P/E/F/L/R)XXX(Y/W), where X is any amino acid. For example, in some embodiments of interest, the peptide is of the form QXX(G/A/T/S/P)XXX(Y/W), where X is any amino acid. For example, in some embodiments of interest, the peptide is of the form QXX(G/A/T/S)XXX(Y/W), where X is any amino acid. For example, in some embodiments of interest, the peptide is of the form QXX(G/A/T)XXX(Y/W), where X is any amino acid. In particular embodiments, X is any of the twenty-two naturally occurring amino acids. In particular embodiments, X is any of the twenty amino acids encoded by the universal genetic code. In some embodiments, a core lyciumin peptide domain is a sequence listed in Table 1, Table 2, Table 3, or Table 4. In some embodiments, a core lyciumin peptide domain differs in sequence from a sequence listed in Table 1, Table 2, Table 3, or Table 4. For example, a core lyciumin peptide domain can have at least one substitution (e.g., 2, 3, 4, 5, etc. substitutions) relative to a sequence listed in Table 1, Table 2, Table 3, or Table 4. In some embodiments, the core lyciumin peptide domain differs in sequence from a naturally occurring core lyciumin peptide domain. In some embodiments, the sequence of the lyciumin precursor peptide, or biologically-active fragment thereof, differs from a naturally occurring sequence. In particular embodiments, as described herein, the variable X in the peptide QXX(G/A/T/S/P/E/F/L/R)XXX(Y/W), the peptide QXX(G/A/T/S/P)XXX(Y/W), the peptide QXX(G/A/T/S)XXX(Y/W), or the peptide QXX(G/A/T)XXX(Y/W) may be further restricted at individual positions, as described in the following paragraphs. A wide variety of core lyciumin peptide domains can be created. For example, in some embodiments, one of the X positions can be restricted. In other embodiments, two of the X positions can be restricted. In other embodiments, three of the X positions can be restricted. In other embodiments, four of the X positions can be restricted. In other embodiments, five of the X positions can be restricted.
In some embodiments of the core lyciumin peptide, the second position is proline or alanine. In some embodiments, the second position is proline. In some embodiments, the second position is not proline. In some embodiments, the second position is alanine. In some embodiments, the second position is not alanine.
In some embodiments of the core lyciumin peptide, the third position is tryptophan, alanine, tyrosine, phenylalanine, leucine, isoleucine, or serine. In some embodiments, the third position is tryptophan. In some embodiments, the third position is not tryptophan. In some embodiments, the third position is alanine. In some embodiments, the third position is not alanine. In some embodiments, the third position is tyrosine. In some embodiments, the third position is not tyrosine. In some embodiments, the third position is phenylalanine. In some embodiments, the third position is not phenylalanine. In some embodiments, the third position is leucine. In some embodiments, the third position is not leucine. In some embodiments, the third position is isoleucine. In some embodiments, the third position is not isoleucine. In some embodiments, the third position is serine. In some embodiments, the third position is not serine.
In some embodiments of the core lyciumin peptide, the fourth position is glycine. In some embodiments, the fourth position is not glycine. In some embodiments, the fourth position is alanine. In some embodiments, the fourth position is not alanine. In some embodiments, the fourth position is threonine. In some embodiments, the fourth position is not threonine. In some embodiments, the fourth position is serine. In some embodiments, the fourth position is not serine. In some embodiments, the fourth position is proline. In some embodiments, the fourth position is not proline. In some embodiments, the fourth position is glutamic acid. In some embodiments, the fourth position is not glutamic acid. In some embodiments, the fourth position is phenylalanine. In some embodiments, the fourth position is not phenylalanine. In some embodiments, the fourth position is leucine. In some embodiments, the fourth position is not leucine. In some embodiments, the fourth position is arginine. In some embodiments, the fourth position is not arginine.
In some embodiments of the core lyciumin peptide, the fifth position is valine, alanine, phenylalanine, serine, glycine, threonine, isoleucine, glutamine, or leucine. In some embodiments, the fifth position is valine. In some embodiments, the fifth position is not valine. In some embodiments, the fifth position is alanine. In some embodiments, the fifth position is not alanine. In some embodiments, the fifth position is phenylalanine. In some embodiments, the fifth position is not phenylalanine. In some embodiments, the fifth position is serine. In some embodiments, the fifth position is not serine. In some embodiments, the fifth position is glycine. In some embodiments, the fifth position is not glycine. In some embodiments, the fifth position is threonine. In some embodiments, the fifth position is not threonine. In some embodiments, the fifth position is isoleucine. In some embodiments, the fifth position is not isoleucine. In some embodiments, the fifth position is glutamine. In some embodiments, the fifth position is not glutamine. In some embodiments, the fifth position is leucine. In some embodiments, the fifth position is not leucine.
In some embodiments of the core lyciumin peptide, the sixth position is glycine, tyrosine, alanine, threonine, serine, phenylalanine, leucine, cysteine, methionine, isoleucine, arginine, histidine, asparagine, valine, or aspartate. In some embodiments, the sixth position is glycine. In some embodiments, the sixth position is not glycine. In some embodiments, the sixth position is tyrosine. In some embodiments, the sixth position is not tyrosine. In some embodiments, the sixth position is alanine. In some embodiments, the sixth position is not alanine. In some embodiments, the sixth position is threonine. In some embodiments, the sixth position is not threonine. In some embodiments, the sixth position is serine. In some embodiments, the sixth position is not serine. In some embodiments, the sixth position is phenylalanine. In some embodiments, the sixth position is not phenylalanine. In some embodiments, the sixth position is leucine. In some embodiments, the sixth position is not leucine. In some embodiments, the sixth position is cysteine. In some embodiments, the sixth position is not cysteine. In some embodiments, the sixth position is methionine. In some embodiments, the sixth position is not methionine. In some embodiments, the sixth position is isoleucine. In some embodiments, the sixth position is not isoleucine. In some embodiments, the sixth position is arginine. In some embodiments, the sixth position is not arginine. In some embodiments, the sixth position is histidine. In some embodiments, the sixth position is not histidine. In some embodiments, the sixth position is asparagine. In some embodiments, the sixth position is not asparagine. In some embodiments, the sixth position is valine. In some embodiments, the sixth position is not valine. In some embodiments, the sixth position is aspartate. In some embodiments, the sixth position is not aspartate.
In some embodiments of the core lyciumin peptide, the seventh position is serine, isoleucine, threonine, alanine, phenylalanine, glycine, tyrosine, methionine, lysine, valine, or arginine. In some embodiments, the seventh position is serine. In some embodiments, the seventh position is not serine. In some embodiments, the seventh position is isoleucine. In some embodiments, the seventh position is not isoleucine. In some embodiments, the seventh position is threonine. In some embodiments, the seventh position is not threonine. In some embodiments, the seventh position is alanine. In some embodiments, the seventh position is not alanine. In some embodiments, the seventh position is phenylalanine. In some embodiments, the seventh position is not phenylalanine. In some embodiments, the seventh position is glycine. In some embodiments, the seventh position is not glycine. In some embodiments, the seventh position is tyrosine. In some embodiments, the seventh position is not tyrosine. In some embodiments, the seventh position is methionine. In some embodiments, the seventh position is not methionine. In some embodiments, the seventh position is lysine. In some embodiments, the seventh position is not lysine. In some embodiments, the seventh position is valine. In some embodiments, the seventh position is not valine. In some embodiments, the seventh position is arginine. In some embodiments, the seventh position is not arginine.
In some embodiments of the core lyciumin peptide, the eighth position is tyrosine. In some embodiments, the eighth position is not tyrosine. In some embodiments, the eighth position is tryptophan. In some embodiments, the eighth position is not tryptophan.
As used herein, the term “biologically-active fragment,” when referring to a lyciumin precursor peptide, refers to a fragment of a lyciumin precursor peptide that includes at least one core lyciumin peptide domain and that can be converted to a lyciumin cyclic peptide (e.g., in a host cell). Typically, the biologically-active fragment is cyclized in the host cell. In some instances, the biologically-active fragment may have shorter N-terminal or C-terminal domains compared to a lyciumin precursor peptide. In some instances, biologically-active fragments can be fragments of naturally-occurring lyciumin precursor peptides. In some instances, a biologically-active fragment can be a portion of a lyciumin precursor peptide having at least one core lyciumin peptide, which is embedded in, or linked to (e.g., at the N-terminus of, at the C-terminus of), a heterologous amino acid sequence that is not generally found in a lyciumin precursor peptide.
In some embodiments, the invention provides a method of producing one or more lyciumin cyclic peptides that includes: (a) providing a host cell that includes a transgene encoding a polypeptide that comprises one or more core lyciumin peptide domains; (b) expressing the transgene in the host cell to thereby produce a polypeptide that includes one or more core lyciumin peptide domains. In some embodiments, the polypeptide is converted to one or more lyciumin cyclic peptides in the host cell.
As used herein, the term “lyciumin cyclic peptide” refers to a branched cyclic peptide with an N-terminal pyroglutamate and a macrocyclic linkage involving a C-terminal tryptophan or tyrosine residue. A lyciumin cyclic peptide typically consists of the eight amino acids of the core lyciumin peptide domain. Typically, the C-terminal residue cyclizes with the α-carbon of the fourth residue. An example is a macrocyclic linkage between a C-terminal tryptophan indole-nitrogen and a glycine-α-carbon.
The BURP domain (Pfam 03181) is around 230 amino acid residues and has the following conserved features: two phenylalanine residues at its N-terminus; two cysteine residues; and four repeated cysteine-histidine motifs, arranged as: CH-X(10)-CH-X(25-27)-CH-X(25-26)-CH, where X can be any amino acid.
The DUF2775 domain (Pfam 10950) is a eukaryotic protein family which includes a number of plant organ-specific proteins. Their predicted amino acid sequence is often repetitive and suggests that these proteins could be exported and glycosylated. Multiple sequence alignment shows a highly conserved motif of 135 amino acids. This motif includes approximately 20 amino acids from the non-repeating area of the peptide, 2 tandem repeats and 1 truncated tandem repeat (Albornos et al., 2012). The first seven amino acids of the DUF2775 domain are typically KDXYXGW, where X can be any amino acid.
Embodiments described herein also include engineered nucleic acids that encode engineered lyciumin precursor peptides (and engineered lyciumin precursor peptides encoded by such engineered nucleic acids). An example is an engineered nucleic acid that encodes n number of core lyciumin peptide domains, wherein n is an integer. The core lyciumin peptide domains within an engineered lyciumin precursor peptide can be identical or non-identical. Multiple identical core lyciumin peptide domains can allow for increased production of a homogenous population of core lyciumin peptides and lyciumin cyclic peptides. Typically, n is an integer from 1 to 10, preferably from 5 to 10. In some instances, n can be greater than 10. In some instances, an engineered nucleic acid encodes from 5 to 10 identical lyciumin precursor peptides. The core lyciumin peptides domains are typically separated by an intervening sequence.
In the example shown in
As used herein, “converting the lyciumin precursor peptide, or biologically-active fragment thereof, to one or more lyciumin cyclic peptides in a host cell,” “converted to one or more lyciumin cyclic peptides in a host cell,” and similar phrases refer to one or more enzymatic reactions that convert a lyciumin precursor peptide, or biologically-active fragment thereof, to one or more lyciumin cyclic peptides. In some instances, conversion is facilitated by one or more enzymes that cyclizes the lyciumin precursor peptide, or biologically-active fragment thereof. In some instances, conversion is catalyzed, in part, by one or more endopeptidases, such as an arginine endopeptidase, which acts N-terminal to a core lyciumin peptide domain. In some instances, conversion is catalyzed by one or more glutamine cyclotransferases, which cyclize an N-terminal glutamine in a core lyciumin peptide domain. In some instances, conversion is catalyzed by one or more exopeptidases. Conversion to a lyciumin cyclic peptide can, but need not, occur within in a host cell.
Host cells include cells that are capable of converting a lyciumin precursor peptide to a lyciumin cyclic peptide, as well as cells that are incapable of converting a lyciumin precursor peptide to a lyciumin cyclic peptide. For example, a host cell can express a lyciumin precursor peptide but lack one or more enzymes required to convert the lyciumin precursor peptide to a lyciumin cyclic peptide. In such circumstances, the lyciumin precursor peptide can be isolated or obtained from the host cell and then converted to a lyciumin cyclic peptide in another environment (e.g., in a cell free system, such as in a cell lysate (or fractionated cell lysate) from a source that is capable of converting a lyciumin precursor peptide to a lyciumin cyclic peptide).
In some embodiments, a lyciumin precursor peptide can include a tag, which can be used to isolate the lyciumin precursor peptide from a cell that expresses it. Such a tag can be useful for a manufacturing process that involves recombinant expression of a lyciumin precursor peptide and subsequent cyclization using purified enzyme. In some embodiments, a nucleotide sequence encoding a lyciumin precursor peptide is fused in-frame with a nucleotide sequence encoding an epitope tag, also known as an affinity tag, which can be useful for, e.g., protein purification. Examples of suitable epitope tags are known in the art and include FLAG, HA, His, GST, CBP, MBP, c-Myc, DHFR, GFP, CAT and others.
As used herein, the term “nucleic acid” refers to a polymer comprising multiple nucleotide monomers (e.g., ribonucleotide monomers or deoxyribonucleotide monomers). “Nucleic acid” includes, for example, DNA (e.g., genomic DNA and cDNA), RNA, and DNA-RNA hybrid molecules. Nucleic acid molecules can be naturally occurring, recombinant, or synthetic. In addition, nucleic acid molecules can be single-stranded, double-stranded or triple-stranded. In certain embodiments, nucleic acid molecules can be modified. In the case of a double-stranded polymer, “nucleic acid” can refer to either or both strands of the molecule.
The terms “nucleotide” and “nucleotide monomer” refer to naturally occurring ribonucleotide or deoxyribonucleotide monomers, as well as non-naturally occurring derivatives and analogs thereof. Accordingly, nucleotides can include, for example, nucleotides comprising naturally occurring bases (e.g., adenosine, thymidine, guanosine, cytidine, uridine, inosine, deoxyadenosine, deoxythymidine, deoxyguanosine, or deoxycytidine) and nucleotides comprising modified bases known in the art.
As used herein, the term “sequence identity,” refers to the extent to which two nucleotide sequences, or two amino acid sequences, have the same residues at the same positions when the sequences are aligned to achieve a maximal level of identity, expressed as a percentage. For sequence alignment and comparison, typically one sequence is designated as a reference sequence, to which a test sequences are compared. The sequence identity between reference and test sequences is expressed as the percentage of positions across the entire length of the reference sequence where the reference and test sequences share the same nucleotide or amino acid upon alignment of the reference and test sequences to achieve a maximal level of identity. As an example, two sequences are considered to have 70% sequence identity when, upon alignment to achieve a maximal level of identity, the test sequence has the same nucleotide or amino acid residue at 70% of the same positions over the entire length of the reference sequence.
Alignment of sequences for comparison to achieve maximal levels of identity can be readily performed by a person of ordinary skill in the art using an appropriate alignment method or algorithm. In some instances, the alignment can include introduced gaps to provide for the maximal level of identity. Examples include the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), and visual inspection (see generally Ausubel et al., Current Protocols in Molecular Biology).
When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequent coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters. A commonly used tool for determining percent sequence identity is Protein Basic Local Alignment Search Tool (BLASTP) available through National Center for Biotechnology Information, National Library of Medicine, of the United States National Institutes of Health. (Altschul et al., 1990).
In various embodiments, two nucleotide sequences, or two amino acid sequences, can have at least, e.g., 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, sequence identity. When ascertaining percent sequence identity to one or more sequences described herein, the sequences described herein are the reference sequences.
For many of the nucleotide sequences described herein, additional 5′- and 3′-nucleotides can be appended to the nucleotide sequence in order to perform Gibson cloning of the sequence into an expression vector. Gibson cloning utilizes Gibson assembly, an exonuclease-based method for joining DNA fragments. For example, a 5′ adapter (see SEQ ID NO: 123) and a 3′ adapter (see SEQ ID NO: 124) can be appended 5′ and 3′, respectively, to SEQ ID NOS: 9 through 33 for Gibson cloning and assembly into tobacco expression vector pEAQ-HT.
The terms “vector”, “vector construct” and “expression vector” mean the vehicle by which a DNA or RNA sequence (e.g. a foreign gene) can be introduced into a host cell, so as to transform the host and promote expression (e.g. transcription and translation) of the introduced sequence. Vectors typically comprise the DNA of a transmissible agent, into which foreign DNA encoding a protein is inserted by, e.g., restriction enzyme technology. Some viral vectors comprise the RNA of a transmissible agent. A common type of vector is a “plasmid”, which generally is a self-contained molecule of double-stranded DNA that can readily accept additional (foreign) DNA and which can readily introduced into a suitable host cell. A large number of vectors, including plasmid and fungal vectors, have been described for replication and/or expression in a variety of eukaryotic and prokaryotic hosts.
The terms “express” and “expression” mean allowing or causing the information in a gene or DNA sequence to become manifest, for example producing a protein by activating the cellular functions involved in transcription and translation of a corresponding gene or DNA sequence. A DNA sequence is expressed in or by a cell to form an “expression product” such as a protein. The expression product itself, e.g. the resulting protein, may also be said to be “expressed” by the cell. A polynucleotide or polypeptide is expressed recombinantly, for example, when it is expressed or produced in a foreign host cell under the control of a foreign or native promoter, or in a native host cell under the control of a foreign promoter.
Gene delivery vectors generally include a transgene (e.g., nucleic acid encoding an enzyme) operably linked to a promoter and other nucleic acid elements required for expression of the transgene in the host cells into which the vector is introduced. Suitable promoters for gene expression and delivery constructs are known in the art. For bacterial host cells, suitable promoters, include, but are not limited to promoters obtained from the E. coli lac operon, Streptomyces coelicolor agarase gene (dagA), Bacillus subtilis levansucrase gene (sacB), Bacillus licheniformis alpha-amylase gene (amyL), Bacillus stearothermophilus maltogenic amylase gene (amyM), Bacillus amyloliquefaciens alpha-amylase gene (amyQ), Bacillus licheniformis penicillinase gene (penP), Bacillus subtilis xy1A and xy1B genes, and prokaryotic beta-lactamase gene (See e.g., Villa-Kamaroff et al., Proc. Natl. Acad. Sci. USA 75: 3727-3731, 1978), as well as the tac promoter (See e.g., DeBoer et al., Proc. Natl. Acad. Sci. USA 80: 21-25, 1983). Examples of promoters for filamentous fungal host cells, include, but are not limited to promoters obtained from the genes for Aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus niger neutral alpha-amylase, Aspergillus niger acid stable alpha-amylase, Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Rhizomucor miehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triose phosphate isomerase, Aspergillus nidulans acetamidase, and Fusarium oxysporum trypsin-like protease (See e.g., WO 96/00787), as well as the NA2-tpi promoter (a hybrid of the promoters from the genes for Aspergillus niger neutral alpha-amylase and Aspergillus oryzae triose phosphate isomerase), and mutant, truncated, and hybrid promoters thereof. Examples of yeast cell promoters can be from the genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae galactokinase (GAL1), Saccharomyces cerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP), and Saccharomyces cerevisiae 3-phosphoglycerate kinase. Other useful promoters for yeast host cells are known in the art (See e.g., Romanos et al., Yeast 8:423-488, 1992). For plant host cells, examples of suitable promoters include the cauliflower mosaic virus 35S promoter (CaMV 35S), and promoters (e.g., constitutive promoters) of genes that are highly expressed in plants (e.g., plant housekeeping genes, genes encoding Ubiquitin, Actin, Tubulin, or EIF (eukaryotic initiation factor)). Plant virus promoters can also be used. Additional useful plant promoters include those discussed in [50, 51], the entire contents of which are incorporated herein by reference. The selection of a suitable promoter is within the skill in the art. The recombinant plasmids can also comprise inducible, or regulatable, promoters for expression of a lyciumin precursor peptide, or biologically-active fragment thereof, in cells.
Various gene delivery vehicles are known in the art and include both viral and non-viral (e.g., naked DNA, plasmid) vectors. Viral vectors suitable for gene delivery are known to those skilled in the art. Such viral vectors include, e.g., vector derived from the herpes virus, baculovirus vector, lentiviral vector, retroviral vector, adenoviral vector and adeno-associated viral vector (AAV). Vectors derived from plant viruses can also be used, such as the viral backbones of the RNA viruses Tobacco mosaic virus (TMV), Potato virus X (PVX) and Cowpea mosaic virus (CPMV), and the DNA geminivirus Bean yellow dwarf virus. The viral vector can be replicating or non-replicating.
Non-viral vectors include naked DNA and plasmids, among others. Non-limiting examples include pKK plasmids (Clonetech), pUC plasmids, pET plasmids (Novagen, Inc., Madison, Wis.), pRSET or pREP plasmids (Invitrogen, San Diego, Calif.), or pMAL plasmids (New England Biolabs, Beverly, Mass.), and such vectors may be introduced into many appropriate host cells, using methods disclosed or cited herein or otherwise known to those skilled in the relevant art.
In certain embodiments, the vector comprises a transgene operably linked to a promoter. The transgene encodes a biologically-active molecule, such as a lyciumin precursor peptide described herein.
To facilitate the introduction of the gene delivery vector into host cells, the vector can be combined with different chemical means such as colloidal dispersion systems (macromolecular complex, nanocapsules, microspheres, beads) or lipid-based systems (oil-in-water emulsions, micelles, liposomes).
Some embodiments relate to a vector comprising a nucleic acid encoding lyciumin precuror peptide, or biologically-active fragment thereof, described herein. In certain embodiments, the vector is a plasmid, and includes any one or more plasmid sequences such as, e.g., a promoter sequence, a selection marker sequence, or a locus-targeting sequence. Suitable plasmid vectors include p423TEF 2μ, p425TEF 2μ, and p426TEF 2μ. Another suitable vector is pHis8-4 (Whitehead Institute, Cambridge, Mass., United States of America). Another suitable vector is pEAQ-HT [50].
Although the genetic code is degenerate in that most amino acids are represented by multiple codons (called “synonyms” or “synonymous” codons), it is understood in the art that codon usage by particular organisms is nonrandom and biased towards particular codon triplets. Accordingly, in some embodiments, the vector includes a nucleotide sequence that has been optimized for expression in a particular type of host cell (e.g., through codon optimization). Codon optimization refers to a process in which a polynucleotide encoding a protein of interest is modified to replace particular codons in that polynucleotide with codons that encode the same amino acid(s), but are more commonly used/recognized in the host cell in which the nucleic acid is being expressed. In some aspects, the polynucleotides described herein are codon optimized for expression in a bacterial cell, e.g., E. coli. In some aspects, the polynucleotides described herein are codon optimized for expression in a yeast cell, e.g., S. cerevisiae. In some aspects, the polynucleotides described herein are codon optimized for expression in a tobacco cell, e.g., N. benthamiana.
A wide variety of host cells can be used in the present invention, including fungal cells, bacterial cells, plant cells, insect cells, and mammalian cells.
In some embodiments, the host cell is a fungal cell, such as a yeast cell and an Aspergillus spp cell. A wide variety of yeast cells are suitable, such as cells of the genus Pichia, including Pichia pastoris and Pichia stipitis; cells of the genus Saccharomyces, including Saccharomyces cerevisiae; cells of the genus Schizosaccharomyces, including Schizosaccharomyces pombe; and cells of the genus Candida, including Candida albicans.
In some embodiments, the host cell is a bacterial cell. A wide variety of bacterial cells are suitable, such as cells of the genus Escherichia, including Escherichia coli; cells of the genus Bacillus, including Bacillus subtilis; cells of the genus Pseudomonas, including Pseudomonas aeruginosa; and cells of the genus Streptomyces, including Streptomyces griseus.
In some embodiments, the host cell is a plant cell. A wide variety of cells from a plant are suitable, including cells from a Nicotiana benthamiana plant. In some embodiments, the plant belongs to a genus selected from the group consisting of Arabidopsis, Beta, Glycine, Helianthus, Solanum, Triticum, Oryza, Brassica, Medicago, Prunus, Malta, Hordeum, Musa, Phaseolus, Citrus, Piper, Sorghum, Daucus, Manihot, Capsicum, and Zea. In some embodiments, the host cell is a plant cell from the Amaranthaceae family. In some embodiments, the plant cell is an Amaranthus genus plant cell, such as an Amaranthus hypochondriacus plant cell. In some embodiments, the plant cell is a Beta genus plant cell, such as a Beta vulgaris plant cell. In some embodiments, the plant cell is a Chenopodium genus plant cell, such as a Chenopodium quinoa plant cell. In some embodiments, the plant cell is a Fabaceae family plant cell. In some embodiments, the plant cell is a Glycine genus plant cell, such as a Glycine max plant cell. In some embodiments, the plant cell is a Medicago genus plant cell, such as a Medicago truncatula plant cell. In some embodiments, the plant cell is a Solanaceae family plant cell. In some embodiments, the plant cell is a Solanum genus plant cell, such as a Solanum melongena plant cell or a Solanum tuberosum plant cell. In some embodiments, the plant cell is a Nicotiana genus plant cell, such as a Nicotiana benthamiana plant cell. In some embodiments, the plant cell is a Capsicum genus plant cell, such as a Capsicum annuum plant cell.
In some embodiments, the host cell is an insect cell, such as a Spodoptera frugiperda cell, such as Spodoptera frugiperda Sf9 cell line and Spodoptera frugiperda Sf21
In some embodiments, the host cell is a mammalian cell.
In some embodiments, the host cell is an Escherichia coli cell. In some embodiments, the host cell is a Nicotiana benthamiana cell. In some embodiments, the cell is a Saccharomyces cerevisiae cell.
As used herein, the term “host cell” encompasses cells in cell culture and also cells within an organism (e.g., a plant). In some embodiments, the host cell is part of a transgenic plant.
Some embodiments relate to a host cell comprising a vector as described herein. In certain embodiments, the host cell is an Escherichia coli cell, a Nicotiana benthamiana cell, or a Saccharomyces cerevisiae cell.
In some embodiments, the host cells are cultured in a cell culture medium, such as a standard cell culture medium known in the art to be suitable for the particular host cell.
Described herein are methods of making a transgenic host cell. The transgenic host cells can be made, for example, by introducing one or more of the vector embodiments described herein into the host cell.
In some embodiments, the method comprises introducing into a host cell a vector that includes a nucleic acid transgene that encodes a lyciumin precursor peptide, or a biologically-active fragment thereof. The lyciumin precursor peptide, or biologically-active fragment thereof, can include one or more core lyciumin peptide domains.
In some embodiments, one or more of the nucleic acids are integrated into the genome of the host cell. In some embodiments, the nucleic acids to be integrated into a host genome can be introduced into the host cell using any of a variety of suitable methodologies known in the art, including, for example, CRISPR-based systems (e.g., CRISPR/Cas9; CRISPR/Cpf1), TALEN systems and Agrobacterium-mediated transformation. However, as those skilled in the art would recognize, transient transformation techniques can be used that do not require integration into the genome of the host cell. In some embodiments, nucleic acid (e.g., plasmids) can be introduced that are maintained as episomes, which need not be integrated into the host cell genome.
In certain embodiments, the nucleic acid is introduced into a tissue, cell, or seed of a plant cell. Various methods of introducing nucleic acid into the tissue, cell, or seed of plants are known to one of ordinary skill in the art, such as protoplast transformation. The particular method can be selected based on several considerations, such as, e.g., the type of plant used. For example, a floral dip method is a suitable method for introducing genetic material into a plant. In other embodiments, agroinfiltration can be useful for transient expression in plants. In certain embodiments, the nucleic acid can be delivered into the plant by an Agrobacterium.
In some embodiments, a host cell is selected or engineered to have increased activity of the synthesis pathway.
Some of the methods described herein include assaying for an activity of interest. For example, crude extract from a host cell that expresses a lyciumin precursor peptide and/or lyciumin cyclic peptide, or a lyciumin cyclic peptide isolated from the host cell, can be assayed for an activity of interest. An example of an activity of interest is modulation (enhancement or inhibition) of fungal or bacterial growth, such as the ability to inhibit growth of a pathogenic fungal or bacterial species or the ability to promote growth of a potentially desirable fungal or bacterial species. Another example of an activity of interest is a protease inhibitor activity, which can include inhibition of a viral, bacterial, fungal, or mammalian protease.
The requirement for precursor gene-guided genome mining of a class of plant ribosomal peptides is the identification of a peptide-specific precursor gene, which provides the peptide sequence information via the core peptide. In order to identify the lyciumin precursor gene, a de novo transcriptome was generated of the roots of a Lycium barbarum plant, which produced lyciumin A, B and D based on liquid chromatography-mass spectrometry (LC-MS). Tblastn search of predicted core peptide sequences of lyciumin A (SEQ ID NO: 148; QPYGVGSW), lyciumin B (SEQ ID NO: 50; QPWGVGSW) and lyciumin D (SEQ ID NO: 174; QPYGVGIW) yielded three partial transcripts of candidate lyciumin precursor genes and a full length sequence of a candidate lyciumin precursor gene was obtained by cloning guided by these transcripts (
In order to test whether LbaLycA is a precursor peptide for lyciumin biosynthesis, LbaLycA was expressed heterologously in Nicotiana benthamiana via infiltration of Agrobacterium tumefaciens LBA4404 pEAQ-HT-LbaLycA. LC-MS analysis of an organic extract of N. benthamiana leaves six days after inoculation with A. tumefaciens LBA4404 pEAQ-HT-LbaLycA showed mass signals for lyciumin A, B and D, as detected in Lycium barbarum root extracts (
Amaranthus
hypochondriacus
Amborella
trichopoda (v1.0)
Anacardium
occidentale (v0.9)
Ananas comosus
Aquilegia coerulea
Arabidopsis halleri
Arabidopsis lyrata
Arabidopsis
thaliana (TAIR10)
Arachis duranensis
Arachis ipaensis
Aegilops tauschii
Asparagus
officinalls (V1.1)
Beta vulgaris
Boechera stricta
Brachypodium
distachyon (v3.1)
Brachypodium
stacei (v1.1)
Brachypodium
sylvaticum (v1.1)
Brassica oleracea
capitata (v1.0)
Brassica rapa FPsc
Cajanus cajan
Camelina sativa
Capsella
grandiflora (v1.1)
Capsella rubella
Capsicum annuum
Capsicum
baccatum
Capsicum chinense
Carica papaya
Chenopodium
quinoa (v1.0)
Cicer arietinum
Citrus clementina
Citrus sinensis
Coffea arabica
Cucumis melo
Cucumis sativus
Cucurbita
moschata
Daucus carota
Dichanthelium
oligosanthes
Durio zibethinus
Elaeis guineensis
Erythranthe guttata
Eucalyptus grandis
Eutrema
salsugineum (v1.0)
Fragaria vesca
Glycine max
Gossypium
hirsutum (v1.1)
Gossypium
raimondii (v2.1)
Hevea brasiliensis
Helianthus annuus
Hordeum vulgare
Ipomoea nil
Jatropha curcas
Juglans regia
Kalanchoe
fedtschenkoi (v1.1)
Kalanchoe laxiflora
Lactuca sativa (V8)
Linum
usitatissimum
Lupinus
angustifolius
Malus domestica
Manihot esculenta
Marchantia
polymorpha (v3.1)
Medicago
truncatula
Mimulus guttatus
Miscanthus sinensis
Morus notabilis
Musa acuminata
Nelumbo nucifera
Nicotiana attenuata
Nicotiana
benthamiana
Nicotiana sylvestris
Nicotiana tabacum
Nicotiana
tomentosiformis
Olea europaea var.
sylvestris (v1.0)
Oropetium
thomaeum (v1.0)
Oryza sativa
Panicum hallii
Panicum virgatum
Petunia axillaris
Petunia inflata
Phalaenopsis
equestris
Phaseolus vulgaris
Phoenix dactylifera
Physcomitrella
patens (v3.3)
Populus deltoides
Populus euphratica
Populus
trichocarpa (v3.0)
Prunus avium
Prunus persica
Pyrus ×
bretschneideri
Raphanus sativus
Ricinus communis
Salix purpurea
Selaginella
moellendorffii
Sesamum indicum
Setaria italica
Setaria viridis
Solanum
lycopersicum
Solanum
melongena (v2.5.1)
Solanum pennellii
Solanum
pimpinellifolium
Solanum tuberosum
Sorghum bicolor
Sphagnum fallax
Spinacia oleracea
Spirodela polyrhiza
Tarenaya
hassleriana
Theobroma cacao
Trifolium pratense
Triticum aestivum
Vicia faba
Vigna angularis
Vigna radiata
Vigna unguiculata
Vitis vinifera
Zea mays PH207
Ziziphus jujuba
Zostera marina
With a precursor gene for lyciumin biosynthesis in hand, lyciumin genotypes and chemotypes in genome sequenced plants were identified. The precursor gene-guided genome mining approach (
Genome mining of LbaLycA homologs revealed that 21 of 116 analyzed plant genomes have a candidate lyciumin precursor peptide gene (Table 1). The putative lyciumin producing plants were Amaranthaceae, Fabaceae, Rosaceae or Solanaceae plants. Bioinformatic analysis of identified BURP domain proteins yielded 71 distinct core peptide sequences with 60 of them being species-specific (Table 2), indicating a large untapped diversity of lyciumin chemotypes, and several core peptide sequences, which are present in multiple species and families (QPYGVYTW (SEQ ID NO: 43) and QPWGVGSW (SEQ ID NO: 50)), indicating functional selection of their products. Subsequently, ten plants with candidate lyciumin genotypes were selected and their organic extracts were analyzed for predicted lyciumin chemotypes by LC-MS. For seven of those plants, predicted lyciumin analytes could be detected and verified by MS and MS/MS analysis including economically important crop and forage plants such as Amaranthus hypochondriacus (amaranth), Beta vulgaris (beet), Chenopodium quinoa (quinoa), Glycine max (soy), Solanum melongena (eggplant), and Medicago truncatula (
For Solanum tuberosum (potato), several lyciumin peptides could be characterized by LC-MS analysis. However, none of the detected peptides matched the predicted core peptide sequences from the genome derived lyciumin precursor. Analysis of the corresponding genome location showed that the 5′-region of the lyciumin precursor gene PGSC0003DMG400047074 was incomplete (
Amaranthus
hypochondriacus
Amaranthus
hypochondriacus
Arachis duranensis,
Arachis ipaensis,
Glycine max,
Medicago
truncatula,
Solanum
melongena,
Trifolium pratens
Beta vulgaris
Beta vulgaris
Beta vulgaris
Beta vulgaris
Capsicum annuum
Capsicum annuum
Capsicum annuum,
Capsicum chinense,
Solanum
lycopersicum,
Solanum
melongena,
Solanum pennellii,
Solanum
pimpinellifolium,
Solanum tuberosum
Capsicum annuum,
Capsicum chinense
Capsicum chinense
Chenopodium
quinoa
Chenopodium
quinoa
Chenopodium
quinoa
Chenopodium
quinoa
Chenopodium
quinoa
Chenopodium
quinoa
Chenopodium
quinoa
Glycine max
Glycine max
Medicago
truncatula
Medicago
truncatula
Medicago
truncatula
Medicago
truncatula
Medicago
truncatula
Medicago
truncatula,
Trifolium pratense
Medicago
truncatula
Medicago
truncatula
Medicago
truncatula
Medicago
truncatula
Medicago
truncatula
Medicago
truncatula
Medicago
truncatula
Medicago
truncatula
Medicago
truncatula
Nicotiana attenuata
Petunia axillaris,
Solanum tuberosum
Petunia axillaris
Petunia inflata
Petunia inflata
Petunia inflata,
Solanum tuberosum
Petunia inflata,
Solanum tuberosum
Petunia inflata
Prunus avium,
Prunus persica
Prunus persica
Solanum
lycopersicum,
Solanum pennellii,
Solanum
pimpinellifolium,
Solanum tuberosum
Solanum
lycopersicum
Solanum
lycopersicum,
Solanum
pimpinellifolium,
Solanum tuberosum
Solanum
lycopersicum,
Solanum
pimpinellifolium
Solanum melongena
Solanum melongena
Solanum melongena
Solanum pennellii
Solanum pennellii
Solanum
pimpinellifolium
Solanum tuberosum
Solanum tuberosum
Solanum tuberosum
Solanum tuberosum
Solanum tuberosum
Solanum tuberosum
Solanum tuberosum
Solanum tuberosum
Solanum tuberosum
Solanum tuberosum
Solanum tuberosum
Trifolium pratense
Trifolium pratense
Trifolium pratense
Vigna unguiculata
Next, the observed lyciumins were analyzed in their structural diversity (
The discovery of lyciumin biosynthesis in three plant families via genome mining motivated a more detailed exploration of the distribution of lyciumin genotypes in the plant kingdom. To do this, plant transcriptomes were targeted as an alternative source for the discovery of lyciumins in plants, as the analysis of the sequenced plant genomes only represented 42 of the estimated 667 plant families. Given success in improving BURP domain precursor gene assembly for lyciumin discovery from the potato transcriptome, large-scale de novo re-assembly of transcriptomes of 793 plants species representing 317 plant families was performed using rnaSPAdes [35], starting from raw sequencing reads generated as part of the 1 kp project. Subsequently, tblastn searches of type 1 and type 2 lyciumin precursors in the assembled transcriptomes identified candidate lyciumin precursors in multiple plant families, including Aizoaceae, Mollunginaceae, Nyctaginaceae, Petiveriaceae, Phytolaccaceae (all Caryophyllales) and Selaginellaceae (Table 3). Corresponding to these transcriptome predictions, a lyciumin chemotype was detected in the peptide extract of Selaginella uncinata roots, showcasing that lyciumins can also be discovered by mining transcriptomes. The putative Selaginella peptide lyciumin P is derived from the core peptide QPYSVFAW and features a serine as the putative cyclization site (
Several observations support independent diversification of lyciumin peptides in Selaginellaceae, Fabaceae, Solanaceae and Caryophyllales families with a few cases of parallel evolution of the identical lyciumin peptide natural products in distantly related plant families. First, the phylogenetic analysis of the BURP domains of the predicted and characterized lyciumin precursor proteins reveals five well-defined clades of sequences from Caryophyllales, Fabaceae, Rosaceae, Solanaceae, and Selaginellaceae (
willdenowii
Selaginella
bryopteris
Selaginella
moellendorffii
Selaginella
uncinata
Acacia
argyrophylla
Acacia
pycnantha
Apios
americana
Astragalus
membranaceus
Bituminaria
bituminosa
Glycine soja
Glycyrrhiza
lepidota
Lathyrus
sativus
Senna
hebecarpa
Xanthocercis
zambesiaca
Delosperma
echinatum
Alternanthera
brasiliana
Alternanthera
sessilis
Amaranthus
tricolor
Atriplex
hortensis
Atriplex
prostrata
Chenopodium
quinoa
Hypertelis
cerviana
Bougainvillea
spectabilis
Petiveria
alliacea
Phytolacca
bogotensis
Microtea
debilis
Hilleria
latifolia
Atropa
belladonna
Lycium
barbarum
Solanum
cheesmaniae
Solanum
dulcamara
Solanum
lasiophyllum
Solanum
ptychanthum
Solanum
sisymbriifolium
Solanum
virginianum
Amaranthus
Amaranthaceae,
Caryophyllales,
hypochondriacus,
Phytolaccaceae,
Selaginellales,
Amaranthus
Nyctaginaceae,
Solanales
tricolor, Atropa
Selaginellaceae,
belladonna,
Solanaceae
Bougainvillea
spectabilis, Hilleria
latifolia, Lycium
barbarum,
Selaginella
bryopteris
Amaranthus
Amaranthaceae,
Caryophyllales,
hypochondriacus,
Phytolaccaceae,
Solanales
Phytolacca
Solanaceae
bogotensis, Atropa
belladonna, Lycium
barbarum,
Solanum
dulcamara,
Arachis
Fabaceae,
Fabales,
duranensis,
Solanaceae
Solanales
Arachis ipaensis,
Glycine max,
Glycine soja,
Glycyrrhiza
lepidota, Medicago
truncatula,
Solanum
cheesmaniae,
Solanum
melongena,
Trifolium pratens
Beta vulgaris
Amaranthaceae
Caryophyllales
Beta vulgaris
Amaranthaceae
Caryophyllales
Beta vulgaris
Amaranthaceae
Caryophyllales
Beta vulgaris
Amaranthaceae
Caryophyllales
Capsicum annuum
Solanaceae
Solanales
Capsicum annuum
Solanaceae
Solanales
Capsicum annuum,
Solanaceae
Solanales
Capsicum
chinense, Lycium
barbarum,
Solanum
cheesmaniae,
Solanum
dulcamara,
Solanum
lasiophyllum,
Solanum
lycopersicum,
Solanum
melongena,
Solanum pennellii,
Solanum
pimpinellifolium,
Solanum
tuberosum
Capsicum annuum,
Solanaceae
Solanales
Capsicum chinense
Capsicum chinense
Solanaceae
Solanales
Chenopodium
Amaranthaceae
Caryophyllales
quinoa
Chenopodium
Amaranthaceae
Caryophyllales
quinoa
Chenopodium
Amaranthaceae
Caryophyllales
quinoa
Chenopodium
Amaranthaceae
Caryophyllales
quinoa
Chenopodium
Amaranthaceae,
Caryophyllales,
quinoa, Solanum
Solanaceae
Solanales
virginianum
Chenopodium
Amaranthaceae,
Caryophyllales,
quinoa, Hypertelis
Molluginaceae,
Solanales
cerviana, Petunia
Solanaceae
inflata, Solanum
tuberosum
Chenopodium
Amaranthaceae,
Caryophyllales,
quinoa, Solanum
Solanaceae
Solanales
tuberosum
Glycine max,
Fabaceae,
Fabales,
Petunia axillaris,
Solanaceae
Solanales
Senna hebecarpa
Glycine max,
Fabaceae
Fabales
Acacia
argyrophylla,
Acacia pycnantha,
Glycine soja
Medicago
Fabaceae
Fabales
truncatula
Medicago
Fabaceae
Fabales
truncatula
Medicago
Fabaceae
Fabales
truncatula
Medicago
Fabaceae
Fabales
truncatula
Medicago
Fabaceae
Fabales
truncatula
Medicago
Fabaceae
Fabales
truncatula,
Trifolium pratense
Medicago
Fabaceae
Fabales
truncatula
Medicago
Fabaceae
Fabales
truncatula
Medicago
Fabaceae
Fabales
truncatula
Medicago
Fabaceae
Fabales
truncatula
Medicago
Fabaceae
Fabales
truncatula
Medicago
Fabaceae
Fabales
truncatula
Medicago
Fabaceae
Fabales
truncatula
Atriplex prostrata,
Amaranthaceae,
Caryophyllales,
Medicago
Fabaceae
Fabales
truncatula
Medicago
Fabaceae
Fabales
truncatula
Nicotiana
Solanaceae
Solanales
attenuata
Microtea debilis,
Phytolaccaceae,
Caryophyllales,
Petunia axillaris,
Fabaceae,
Fabales,
Phytolacca
Solanaceae
Solanales
bogotensis, Senna
hebecarpa,
Solanum
ptychanthum,
Solanum
tuberosum
Petunia inflata
Solanaceae
Solanales
Petunia inflata
Solanaceae
Solanales
Petunia inflata,
Solanaceae
Solanales
Solanum
tuberosum
Petunia inflata
Solanaceae
Solanales
Prunus avium,
Rosaceae
Rosales
Prunus persica
Prunus persica
Rosaceae
Rosales
Solanum
Solanaceae
Solanales
lasiophyllum,
Solanum
lycopersicum,
Solanum pennellii,
Solanum
pimpinellifolium,
Solanum
tuberosum
Solanum
Solanaceae
Solanales
lasiophyllum,
Solanum
lycopersicum
Solanum
Solanaceae
Solanales
lasiophyllum,
Solanum
lycopersicum,
Solanum
pimpinellifolium,
Solanum
tuberosum
Solanum
Fabaceae,
Fabales,
lasiophyllum,
Solanaceae
Solanales
Solanum
lycopersicum,
Solanum
pimpinellifolium,
Solanum
virginianum,
Xanthocercis
zambesiaca
Solanum
Solanaceae
Solanales
melongena
Delosperma
Aizoaceae
Caryophyllales
echinatum
Solanum
Solanaceae
Solanales
melongena
Solanum
Solanaceae
Solanales
melongena
Solanum pennellii
Solanaceae
Solanales
Solanum pennellii
Solanaceae
Solanales
Solanum
Solanaceae
Solanales
tuberosum
Solanum
Solanaceae
Solanales
tuberosum
Solanum
Solanaceae
Solanales
tuberosum
Solanum
Solanaceae
Solanales
tuberosum
Solanum
Solanaceae
Solanales
tuberosum
Solanum
Solanaceae
Solanales
tuberosum
Solanum
Solanaceae
Solanales
tuberosum
Solanum
Solanaceae
Solanales
tuberosum
Solanum
Solanaceae
Solanales
tuberosum
Trifolium pratense
Fabaceae
Fabales
Trifolium pratense
Fabaceae
Fabales
Trifolium pratense
Fabaceae
Fabales
Vigna unguiculata
Fabaceae
Fabales
Selaginella
Selaginellaceae
Selaginellales
bryopteris
Alternanthera
Amaranthaceae,
Caryophyllales,
brasiliana,
Nyctaginaceae,
Selaginellales
Bougainvillea
Petiveriaceae,
spectabilis,
Selaginellaceae
Petiveria alliacea,
Selaginella
moellendorffii
Apios americana
Fabaceae
Fabales
Astragalus
Fabaceae
Fabales
membranaceus
Astragalus
Fabaceae
Fabales
membranaceus
Astragalus
Fabaceae
Fabales
membranaceus
Astragalus
Fabaceae
Fabales
membranaceus
Astragalus
Fabaceae
Fabales
membranaceus
Bituminaria
Fabaceae
Fabales
bituminosa
Lathyrus sativus
Fabaceae
Fabales
Delosperma
Aizoaceae
Caryophyllales
echinatum
Atriplex hortensis,
Amaranthaceae
Caryophyllales
Atriplex prostrata,
Alternanthera
sessilis
Hypertelis cerviana
Molluginaceae
Caryophyllales
Bougainvillea
Nyctaginaceae
Caryophyllales
spectabilis
Bougainvillea
Nyctaginaceae
Caryophyllales
spectabilis
Atropa belladonna
Solanaceae
Solanales
Atropa belladonna
Solanaceae
Solanales
Amaranthus
Solanaceae
Solanales
tricolor,
Bougainvillea
spectabilis, Lycium
barbarum
Solanum
Solanaceae
Solanales
dulcamara
Solanum
Solanaceae
Solanales
dulcamara
Solanum
Solanaceae
Solanales
virginianum
Selaginella
Selaginellaceae
Selaginellales
uncinata,
Selaginella
willdenowii
Solanum
Solanaceae
Solanales
sisymbriifolium
A biosynthetic proposal for lyciumins was established from heterologous expression, sequences and genome locations of precursor peptide genes. Following the general dogma of RiPP biosynthesis, lyciumin biosynthesis starts with translation of a precursor peptide gene such as LbaLycA by the ribosome (
Chenopodium quinoa (v1.0) locus
vulgaris subsp. vulgaris]
vulgaris) [80/71]
Beta vulgaris locus (RefBeet-1.2.2)
vulgaris subsp. vulgaris)
vulgaris subsp. vulgaris)
Despite the lack of a characterized lyciumin cyclase, the promiscuity of the lyciumin biosynthetic pathway in N. benthamiana was investigated. In order to generate lyciumin core peptide mutants, a lyciumin precursor from Glycine max with only one core peptide (QPYGVYTW (SEQ ID NO: 43)) in the N-terminal domain was characterized. Heterologous expression of this precursor, namely Sali3-2 (Glyma. 12G217400) [39], in N. benthamiana resulted in the formation of its predicted lyciumin product, lyciumin I (
Next, it was tested whether the length of the linear N-terminus or the size of the peptide macrocycle could be modified in the N. benthamiana heterologous expression system (
In order to investigate whether the lyciumin pathway can produce unknown peptide macrocyclizations, the BURP domain sequences from genome sequenced plants for core peptides with the motif QP(X)5Y, i.e. a C-terminal tyrosine instead of a tryptophan, were revisited. This search identified a candidate precursor peptide from Capsicum annuum, CanBURP, with a predicted QPYGVYFY core peptide was transiently expressed in N. benthamiana, which suggested a lyciumin derivative with a tyrosine macrocyclization. In a parallel experiment, Sali3-2 was transiently expressed with the same core peptide sequence. Furthermore, peptide metabolic profiling was also conducted with C. annuum seed extract. In all three experiments, analytes were detected suggesting cyclic peptide chemistry derived from the tyrosine-terminal core peptide (
Taken together, these structure-function relationship studies varying precursor core peptide sequence in heterologously reconstituted lyciumin pathway in N. benthamiana suggest restriction in peptide length but promiscuity in peptide sequence and macrocyclization, presenting tremendous opportunity for branched cyclic RiPP diversification via metabolic engineering. Several lyciumins have been produced, such as lyciumin H and K (
Heterologous Lyciumin Production in Tobacco Vs. Source Plant (Lycium barbarum)
Transient expression was measured for engineered lyciumin precursor from Lycium barbarum (LbaLycA) with one, five or ten repeats of a single core peptide (QPWGVGSW=lyciumin B (SEQ ID NO: 50)) after infiltration of six week old tobacco leaves with A. tumefaciens LBA4404 pEAQ-HT-LycA-1×/5×/10× (three plants per construct). See SEQ ID NOS: 112-117,
Peptide extraction of freeze-dried, infiltrated leaf samples of each plant (0.1 g) occurred six days after infiltration and peptide extraction of freeze-dried Lycium barbarum roots was done with plant material from 6 month old plants (0.1 g).
LC-MS analysis for lyciumin B mass signal (single ion monitoring: 896.3-897.3 m/z) and manual peak integration was performed in QualBrowser (Thermo).
The results are shown in
Here, lyciumins are described as a branched cyclic plant RiPP class that is characterized by an N-terminal pyroglutamate and a macrocyclic bond between a C-terminal tryptophan or tyrosine residue, e.g., C-terminal tryptophan-indole-nitrogen and a glycine-α-carbon. Lyciumins were determined to be RiPPs by identification of their precursor peptide from Lycium barbarum. The characterized precursor peptide of this RiPP class enabled successful genome mining of other lyciumin genotypes and chemotypes in genome sequenced crop and forage plants. Biosynthetic investigation of the lyciumin pathway in tobacco indicates promiscuity in peptide sequence, but not in peptide architecture. The pathway tolerates mutation of the C-terminal tryptophan to a tyrosine to form a putative new peptide macrocyclization found in lyciumin-[QPYGVYFY] from pepper seeds by genome mining. The connection of lyciumin core peptides with BURP domains suggests a physical connection of abiotic stress responses via heavy metal-binding BURP domains in plant vacuoles and lyciumin peptide signaling from roots and young plants for potential rhizosphere modulation to alleviate stress such as drought and acidic soil. As rhizosphere engineering has potential to increase crop and forage plant fitness in changing climates and growth conditions, a platform was engineered to produce lyciumin peptide libraries in planta for potential agricultural applications of the lyciumin pathway as transgenes or gene editing targets. Lyciumin cyclic peptide libraries may be used for other purposes, such as development of peptide based-drugs such as protease inhibitors.
Genome mining for plant natural product discovery has been realized for multiple natural product classes on a single-pathway scale [20] and on a multi-pathway scale [21,22,41,42]. Increasing biosynthetic knowledge catalyzed by synthetic biology [18,23] and increasing genomic plant resources prime the field of plant natural products for automated natural product discovery by genome mining. Plant RiPPs such as the lyciumins are a suitable class to be added to plant genome mining pipelines as they could be readily connected from a genotype to a chemotype in a similar fashion as microbial peptide natural products [43].
Overall, described herein is a blueprint for genome mining of branched cyclic RiPPs in plants by identification of pathway-specific precursor peptides. These cyclic lyciumin peptides have potential utility for increasing crop fitness and developing peptide-based drugs.
All chemicals were purchased from Sigma-Aldrich, unless otherwise specified. Oligonucleotide primers and synthetic genes were purchased as gBlocks® from Integrated DNA Technologies, Inc. Solvents for liquid chromatography high-resolution mass spectrometry were Optima® LC-MS grade (Fisher Scientific) or LiChrosolv® LC-MS grade (Millipore). High resolution mass spectrometry analysis was performed on a Thermo ESI-Q-Exactive Orbitrap MS coupled to a Thermo Ultimate 3000 UHPLC system. Low-resolution mass spectrometry analysis was done on a Thermo ESI-QQQ MS coupled to a Thermo Ultimate 3000 UHPLC system. NMR analysis was performed on a Bruker Avance II 600 MHz NMR spectrometer equipped with a High Sensitivity Prodigy Cryoprobe. Preparative HPLC was performed on a Shimadzu LC-20AP liquid chromatograph equipped with a SPD-20A UV/VIS detector and a FRC-10A fraction collector.
Lycium barbarum was purchased as three year-old plants for extraction and cultivation. Amaranthus hypochondriacus seeds for cultivation were purchased from Strictly Medicinal® Seeds. Amaranth grain for extraction was Arrowhead Mills® amaranth. Chenopodium quinoa seeds for cultivation were purchased from Earthcare Seeds. Quinoa for extraction was Trader Joe's® Tricolor quinoa. Beta vulgaris seeds (Detroit Dark Red cultivar) for cultivation and extraction were purchased from David's Garden Seeds. Glycine max seeds (Chiba green soybean) for cultivation and extraction were purchased from High Mowing Organic Seeds®. Seeds of wild-type Medicago truncatula for cultivation were a gift by Prof. Dong Wang (U Mass Amherst). Capsicum annuum seeds (Jalapeno Early) for cultivation and extraction were purchased from EdenBrothers®. Solanum lycopersicum seeds (cultivar Heinz 1706-BG) for cultivation were provided by the Tomato Genetics Resource Center (UC Davis). Solanum melongena seeds for cultivation were purchased from Seedz®. Solanum tuberosum tubers for cultivation (Russett or Red potato) were purchased from Trader Joe's®. Trifolium pratense seeds were purchased from OutsidePride.com®. Nicotiana benthamiana seeds for cultivation were a gift from the Lindquist lab (Whitehead Institute, MIT). Selaginella uncinata plant was purchased from Plant Delights Nursery®.
Lycium barbarum was grown from three year-old live roots in MiracleGro® potting soil as a potted plant in full sun with occasional application of organic fertilizer. Lycium barbarum seeds from fruits of the three year-old plant, were grown in Sun Gro® Propagation Mix soil with added vermiculite (Whittemore Inc.) and added fertilizer in a greenhouse with a 16 h light/8 h dark cycle for six months. Amaranthus hypochondriacus, Chenopodium quinoa, Beta vulgaris, Glycine max, Medicago truncatula, Capsicum annuum, Solanum lycopersicum, Solanum melongena and Trifolium pratense were grown from seeds in Sun Gro® Propagation Mix soil with added vermiculite (Whittemore Inc.) and added fertilizer in a greenhouse with a 16 h light/8 h dark cycle for six months. Nicotiana benthamiana was grown from seeds in Sun Gro® Propagation Mix soil with added vermiculite (Whittemore Inc.) and added fertilizer in a greenhouse with a 16 h light/8 h dark cycle for three months. Solanum tuberosum tubers were sprouted under natural light for three weeks.
Transcriptomic Analysis of Lycium barbarum and Identification of Candidate Precursor Gene LbaLycA
Lycium barbarum roots were removed from a three year-old plant, washed with sterile water, and total RNA was extracted with the QIAGEN RNeasy Plant Mini kit. RNA quality was assessed by Agilent Bioanalyzer. A strand-specific mRNA library was prepared (TruSeq Stranded Total RNA with Ribo Zero Library Preparation Kit, Illumina) and sequenced with a HiSeq2000 Illumina sequencer in HISEQRAPID mode (100×100). Illumina sequence raw-files were combined and assembled by the Trinity package [34]. Gene expression was estimated by mapping raw sequencing reads to the assembled transcriptomes using RSEM [37]. The Lycium barbarum root transcriptome was analyzed for lyciumin precursors by searching predicted core peptide sequences for known lyciumin A (SEQ ID NO: 148; QPYGVGSW), lyciumin B (SEQ ID NO: 50; QPWGVGSW), lyciumin C (SEQ ID NO: 175; QPYGVFSW), and lyciumin D (SEQ ID NO: 174; QPYGVGIW) by blastp algorithm on an internal Blast server. In order to clone and sequence a lyciumin precursor gene from Lycium barbarum, cDNA was prepared from root total RNA with SuperScript® III First-Strand Synthesis System (Invitrogen). Transcripts with lyciumin core peptide sequences were used to design cloning primers (LbaLycA-pEAQ-AgeI (SEQ ID NO: 118): AGACCGGTATGGAGTTGCATCACCATTAC, LbaLycA-pEAQ-XhoI (SEQ ID NO: 119): AGCTCGAGTTAGTTTTCAGACACTTGAGTTGCG) for amplification of precursor peptide gene LbaLycA with Phusion® High-Fidelity DNA polymerase (New England Biolabs) and directional cloning with restriction enzymes AgeI and XhoI (New England Biolabs) and T4 DNA ligase (New England Biolabs) into pEAQ-HT, which was linearized by restriction enzymes AgeI and XhoI [38]. Cloned LbaLycA was sequenced by Sanger sequencing from pEAQ-HT-LbaLycA.
Cloning of Lyciumin Precursor Gene StuBURP from Solanum tuberosum
Tuber sprout tissue was removed from a sprouting potato tuber and total RNA was extracted with the QIAGEN RNeasy Plant Mini kit. cDNA was prepared from sprout total RNA with SuperScript® III First-Strand Synthesis System (Invitrogen). A de novo transcriptome was assembled from a Russett potato RNA-seq dataset (NCBI SRA: SRR5970148) and transcripts homologous to target lyciumin precursor PGSC0003DMG400047074 were used to design cloning primers (StuBURP-pEAQ-fwd (SEQ ID NO: 120): TGCCCAAATTCGCGACCGGTATGGAGTTGCATCACCAATA; StuBURP-pEAQ-rev (SEQ ID NO: 121): CCAGAGTTAAAGGCCTCGAGTTAGTTTTCAGCCACTTGAAGAACTG) for amplification of precursor gene StuBURP with Phusion® High-Fidelity DNA polymerase (New England Biolabs). StuBURP was cloned into pEAQ-HT, which was linearized by restriction enzymes AgeI and XhoI, by Gibson cloning assembly (New England Biolabs). Cloned StuBURP (SEQ ID NO. 126) was sequenced by Sanger sequencing from pEAQ-HT-StuBURP.
Heterologous Expression of Lyciumin Precursor Genes in Nicotiana benthamiana
Agrobacterium tumefaciens LBA4404 was transformed with pEAQ-HT-LbaLycA, other pEAQ-HT constructs with lyciumin precursor genes (pEAQ-HT-StuBURP, pEAQ-HT-CanBURP, pEAQ-HT-Sali3-2, pEAQ-HT-Sali-3-2-mutants) or pEAQ-HT-LbaQC by electroporation (2.5 kV), plated on YM agar (0.4 g yeast extract, 10 g mannitol, 0.1 g sodium chloride, 0.2 g magnesium sulfate (heptahydrate), 0.5 g potassium phosphate, (dibasic, trihydrate), 15 g agar, ad 1 L Milli-Q Millipore water, adjusted pH 7) with 100 μg/mL rifampicin, 50 μg/mL kanamycin and 100 μg/mL streptomycin and incubated for two days at 30° C. A 5 mL starter culture of YM medium with 100 μg/mL rifampicin, 50 μg/mL, kanamycin and 100 μg/mL streptomycin was inoculated with a clone of Agrobacterium tumefaciens LBA4404 pEAQ-HT-LbaLycA and incubated for 24-36 h at 30 on a shaker at 225 rpm. Subsequently, the starter culture was used to inoculate a 50 mL culture of YM medium with 100 μg/mL rifampicin, 50 μg/mL kanamycin and 100 μg/mL streptomycin, which was incubated for 24 h at 30° C., on a shaker at 225 rpm. The cells from the 50 mL culture were centrifuged for 30 min at 3000 g, the YM medium was discarded and cells were resuspended in MMA medium (10 mM MES KOH buffer (pH 5.6), 10 mM magnesium chloride, 100 μM acetosyringone) to give a final optical density of 0.8. The Agrobacterium suspension was infiltrated into the bottom of Nicotiana benthamiana plants (six weeks old). N. benthamiana plants were placed in the shade two hours before infiltration. After infiltration, N. benthamiana plants were grown as described above for six days. Subsequently, infiltrated leaves were collected and subjected to chemotyping.
Chemotyping of Lyciumin Peptides from Plant Material
For peptide chemotyping, 0.2 g plant material (fresh weight) were frozen in liquid nitrogen and ground with mortar and pestle. Ground plant material was extracted with 10 mL methanol for 1 h at 37° C. in a glass vial. Plant methanol extract was dried under nitrogen gas in a separate glass vial. Dried plant methanol extract was resuspended in water (10 mL) and partitioned with hexane (2×10 mL) and ethyl acetate (2×10 mL), and subsequently extracted with n-butanol (10 mL). The n-butanol extract was dried in vacuo and resuspended in 2 mL methanol for liquid chromatography-mass spectrometry (LC-MS) analysis. Peptide extracts were subjected to high resolution MS analysis with the following LC-MS parameters: LC—Phenomenex Kinetex® 2.6 μm C18 reverse phase 100 Å 150×3 mm LC column, LC gradient: solvent A—0.1% formic acid, solvent B—acetonitrile (0.1% formic acid), 0-2 min—5% B, 2-23 min—5-95% B, 23-25 min—95% B, 25-30 min—5% B, 0.5 mL/min, MS—positive ion mode, Full MS: Resolution 70000, mass range 425-1250 m/z, dd-MS2 (data-dependent MS/MS): resolution 17500, Loop count 5, Collision energy 15-35 eV (stepped), dynamic exclusion 1 s. LC-MS data was analyzed with QualBrowser in the Thermo Xcalibur software package (version 3.0.63, ThermoScientific).
For comparative chemotyping of lyciumin concentrations in different plant tissues, peptides were extracted from plant tissues as described above from three different plants of the same age. Analyzed tissues of Amaranthus hypochondriacus and Chenopodium quinoa (three month old) were flower, leaf, root, seed and stem. Analyzed tissues of Beta vulgaris (three month old) were leaf, root, seed and stem. Analyzed tissues of Glycine max (three month old) were bean, leaf, pod, root and stem. Analyzed tissues for Solanum tuberosum (three week old) were sprout and tuber. Peptide extracts were subjected to low resolution MS analysis by selected-ion monitoring (SIM) of lyciumin masses specific to each plant and the following LC-MS parameters: LC—Phenomenex Kinetex® 2.6 μm C18 reverse phase 100 Å 150×3 mm LC column, LC gradient: solvent A—0.1% formic acid, solvent B—acetonitrile (0.1% formic acid), 0-1 min: 5% B, 1-8 min: 5-95% B, 8-10 min: 95% B, 10-15 min: 5% B, MS—positive ion mode, SIM (Amaranthus hypochondriacus: 872.8-873.8 m/z and 963.8-964.8 m/z, Chenopodium quinoa: 869.8-870.8 m/z and 972.8-973.8 m/z, Beta vulgaris: 894.8-895.8 m/z, Glycine max: 910.8-911.8 m/z and 993.8-994.8 m/z, Solanum tuberosum: 880.8-881.8 m/z, 896.8-897.8 m/z, 913.8-914.8 m/z, 922.8-923.8 m/z, 947.8-948.8 m/z, 972.8-973.8 m/z and 1048.8-1049.8 m/z). Lyciumin ion abundance values were determined by peak area integration from each lyciumin SIM chromatogram in QualBrowser in the Thermo Xcalibur software package (version 3.0.63, ThermoScientific).
Prediction of lyciumin genotypes: For prediction of lyciumin precursor peptide genes in a plant genome, LbaLycA homologs were searched by tblastn search in the 6-frame translated genome sequence (JGI Phytozome v12.1 and pre-release genomes) or by blastp of Refseq protein sequences (NCBI genomes, Table 1). In addition, annotated BURP domains were identified by ‘BURP domain’ Keyword search (JGI Phytozome v12.1 and pre-release genomes). All identified BURP domain proteins from a plant genome were then searched for lyciumin core peptide sequences with the search criteria of a glutamine and proline as the first and second amino acid, respectively, in the core peptide sequence and a tryptophan at the eighth position of the core peptide sequence. A BURP domain protein, which contained one or multiple sequences matching these lyciumin core peptide criteria, was a candidate lyciumin precursor peptide and, thus, its gene a predicted lyciumin genotype in the target plant genome.
In order to complement missing core peptide sequences from a lyciumin precursor gene with a sequence gap in the potato genome (PGSC0003DMG400047074), a Russett potato tuber transcriptome (NCBI SRA: SRR5970148) was assembled by Trinity (v2.4) [34] and rnaSPAdes (v1.0, kmer 25,75) [35]. Precursor peptide transcripts with missing core peptide sequences were searched in both de novo transcriptome assemblies by LbaLycA tblastn search.
Prediction of lyciumin chemotypes: A lyciumin structure was predicted from a putative lyciumin core peptide sequence by transformation of the glutamine at the first position to a pyroglutamate and formation of a covalent bond between the indole-nitrogen of the tryptophan at the eighth position with the α-carbon of the residue at the fourth position by loss of two hydrogens.
Lyciumin chemotyping: LC-MS data of peptide extracts from a predicted lyciumin producing plant was analyzed for lyciumin mass signals by (a) parent mass search (base peak chromatogram of calculated [M+H]+ of predicted lyciumin structure, Δm=5 ppm), (b) fragment mass search of pyroglutamate-proline-b-ion in MS/MS data (C10H13N2O+, 209.09207 m/z, Δm=5 ppm), and (c) iminium ion mass search of specific amino acids of predicted structure in MS/MS data (for example, pyroglutamate iminium ion [M+H]+ 84.04439 m/z) with QualBrowser in the Thermo Xcalibur software package (version 3.0.63, ThermoScientific). Putative mass signals of predicted lyciumin structures were confirmed by MS/MS data analysis.
For lyciumin transcriptome mining, transcriptomes of terrestrial plants from the 1 kp database were assembled by rnaSPAdes (v1.0, kmer 25,75 or, if failed, default kmer 55). De novo assembled transcriptomes were searched for LbaLycA homologs (type 1 lyciumin precursor) and Sali3-2 (Glycine max, AAB66369.1, type 2 lyciumin precursor) by tblastn search on an internal Blast server. Candidate lyciumin precursors were predicted with the same core peptide search criteria as for lyciumin genome mining with some precursors being partial sequences due to failed complete de novo assembly (Table 3). In order to verify lyciumin genotype prediction in Selaginellaceae (1 kp dataset: ERR2040880—Selaginella willdenowii), other Selaginella transcriptomes from the NCBI SRA were de novo assembled (rnaSPAdes, v1.0, kmer 25,75, SRR3136708—Selaginella bryopteris, SRR4762537—Selaginella martensii, SRR5499403—Selaginella uncinata, SRR7132763 —Selaginella uncinata, SRR7132764—Selaginella rupestris, SRR7132766—Selaginella moellendorffii, SRR7132767—Selaginella peruviana, SRR7132768—Selaginella borealis, SRR7132769—Selaginella braunii), and searched for lyciumin genotypes as described above. For core peptide analysis of predicted lyciumin genotypes (Table 4,
Protein sequences of characterized and predicted lyciumin precursors from genomes and transcriptomes (except 3′-partial sequences) and four founding members of the BURP domain family (NP_001303011.1—BURP domain-containing protein BNM2A precursor [Brassica napus], NP 001234835.1—Polygalacturonase-1 non-catalytic subunit beta precursor [Solanum lycopersicum], CAA31603.1/CAA31602.1—Embryonic abundant protein USP87/Embryonic abundant protein USP92 [Vicia faba], NP 197943.1—BURP domain protein RD22 [Arabidopsis thaliana]) [30-33] were reduced to their BURP domain (Pfam PF03181) and aligned using Muscle algorithm in MEGA (ver. 7.0.9). A neighbor-joining phylogenetic tree was generated with 2000 bootstrap generations using the p-distance method in MEGA.
Lyciumin Metabolic Engineering in Nicotiana benthamiana
Predicted lyciumin precursor Sali3-2 (Glyma.12G217400) was synthesized as an IDT gBlock® with a 5′-adapter (TGCCCAAATTCGCGACCGGT (SEQ ID NO: 252)) and a 3′-adapter (CTCGAGGCCTTTAACTCTGG (SEQ ID NO: 253) for Gibson assembly. pEAQ-HT was digested by AgeI and XhoI restriction enzymes and the Sali3-2 gBlock® was cloned into the digested pEAQ-HT with Gibson Assembly Master Mix (New England Biolabs). pEAQ-HT-Sali3-2 was verified by Sanger sequencing and transformed into Agrobacterium tumefaciens LBA4404 for heterologous expression as described above. Constructs for metabolic engineering of lyciumins were Sali3-2 mutants of its core peptide sequence (SEQ ID NOS: 8-33). Sali3-2 mutants were synthesized as gBlocks® and cloned into pEAQ-HT for heterologous expression in N. benthamiana as described above. Chemotyping of infiltrated N. benthamiana leafs for lyciumins was done as described above.
For lyciumin A, B and I) isolation, Lycium barbarum roots (100 g wet weight) were ground with a tissue homogenizer and extracted for 16 h with methanol shaking at 225 rpm and 37 T. For lyciumin C isolation, amaranth grain (4.5 kg) was ground in a tissue homogenizer and extracted for 16 h with methanol shaking at 22.5 rpm and 37° C. Methanol extracts were filtered and dried in vacuo. Dried methanol extracts were resuspended in water and partitioned twice with hexane and twice with ethylacetate and then extracted twice with n-butanol. n-butanol extracts were dried in vacuo. Dried n-butanol extracts were resuspended in 10% methanol and separated by flash liquid chromatography with Sephadex LH20 as a stationary phase and a gradient of 10-100% methanol as a mobile phase. Fractions were collected with a fraction collector and analyzed for lyciumin content by LC-QQQ-MS with the following LC-MS settings: LC—Phenomenex Kinetex® 2.6 μm C18 reverse phase 100 Å 150×3 mm LC column, LC gradient: solvent A—0.1% formic acid, solvent B—acetonitrile (0.1% formic acid), 0.5 mL/min, 0-1 min: 5% B, 1-8 min: 5-95% B, 8-10 min: 95% B, 10-15 min: 5% B, MS—positive ion mode, Full MS: Lyciumin A/B/D—860-920 m/z, Lyciumin C/I—950-1010 m/z. LH20 fractions with lyciumins were combined, dried in vacuo, resuspended in 10% acetonitrile (0.1°/o trifluoroacetic acid) and subjected to preparative HPLC with a Phenomenex Kinetex® 5 μm C18 reverse phase 100 Å 150×21.2 mm LC column as a stationary phase for two rounds of separation. LC settings were as follows: solvent A—0.1% trifluoroacetic acid, solvent B—acetonitrile (0.1% trifluoroacetic acid), 10 mL/min, Lyciumin A (20 mg)—1.LC: 0-3 min: 10% B, 3-43 min: 10-50% B, 43-45 min: 50-95% B, 45-48 min: 95% B, 48-49 min: 95-10% B, 49-69 min: 10% B, 2.LC: 0-5 min: 35% B, 5-35 min: 35-50% B, 35-38 min: 50-95% B, 38-40 min: 95% B, 40-40.1 min: 95-35% B, 40.1-60 min: 35% B, Lyciumin B (13 mg)—1.LC: 0-3 min: 20% B, 3-48 min: 20-40% B, 48-50 min: 40-95% B, 50-54 min: 95% B, 54-55 min: 95-20% B, 55-70 min: 20% B, 2.LC: 0-3 min: 30% B, 3-35 min: 30-45% B, 35-38 min: 45-95% B, 38-40 min: 95% B, 40-40.1 min: 95-30% B, 40.1-60 min: 30% B, Lyciumin C—1.LC: 0-3 min: 10% B, 3-43 min: 10-50% 13, 43-45 min: 50-95% B, 45-48 min: 95% B, 48-49 min: 95-10% B, 49-69 min: 10% B, 2.LC: 0-3 min: 40% B, 3-48 min: 40-55% B, 48-50 min: 55-95% B, 50-54 min 95% B, 54-55 min: 95-40%, 55-70 min: 40% B, Lyciumin D (5 mg)—1.LC: 0-3 min: 20% B, 3-48 min: 20-40% B, 48-50 min: 40-95% B, 50-54 min: 95% B, 54-55 min: 95-20% B, 55-70 min: 20% B, 2.LC: 0-3 min: 30% B, 3-48 min: 30-50% B, 48-50 min: 50-95% B, 50-54 min: 95% B, 54-55 min: 95-30% B, 55-70 min: 30% B, Lyciumin I—1.LC: 0-3 min: 20% B, 3-48 min: 20-50% B, 48-50 min: 50-95% B, 50-54 min: 95% B, 54-55 min: 95-20% B, 55-70 min: 20% B. 2.LC: 0-3 min: 25% B, 3-48 min: 25-45% B, 48-50 min: 45-95% B, 50-54 min: 95% B, 54-55 min: 95-25% B, 55-70 min: 25% B. Preparative HPLC fractions with lyciumin C and lyciumin I, respectively, were combined, dried in vacuo, resuspended in 30% acetonitrile (0.1% trifluoroacetic acid) and subjected to semipreparative HPLC with a Phenomenex Kinetex® 5 μm C18 reverse phase 100 Å 250×10 mm LC column as a stationary phase. LC settings were as follows: Solvent A—0.1% trifluoroacetic acid, solvent B—acetonitrile (0.1% trifluoroacetic acid), 1.5 mL/min, Lyciumin C (25 mg)—0-5 min: 40% B, 5-15 min: 40-42% B, 15-17 min: 42-95% B, 17-20 min, 95% B, 20-20.1 min: 95-40% B, and lyciumin I (2.5 mg)—0-5 min: 30% B, 5-30 min: 30-35% B, 30-32 min: 35-95% B, 32-36 min: 95% B, 36-40 min: 95-30% B, 40-60 min: 30% B. For NMR analysis, lyciumin A, B, C, D and I were each dissolved in DMSO-d6. Lyciumin A was analyzed for 1H and 13C NMR data, lyciumin B, D and C were analyzed for 1H NMR data. Lyciumin I was analyzed for 1H NMR, 1H-1H COSY, 1H-1H TOCSY, HSQC, HMBC and ROESY data. NMR data was analyzed with TopSpin software (v3.5) from Broker. Stereochemistry of crosslinked glycine α-carbons at the fourth position of lyciumms was inferred as (R) based on lyciumin A analysis [52] and same ROESY correlations of lyciumin I glycine-Ha as reported for lyciumin A [52]. Stereochemistry of other amino acids of lyciumin I was inferred as (L)-amino acids because of its ribosomal biosynthesis and (L)-amino acid stereochemistry in all reported lyciumins [25,26,52].
Gene expression of characterized lyciumin precursors was estimated by mapping raw sequencing reads to de novo assembled transcriptomes using RSEM [37]. For Solanum tuberosum, gene expression of lyciumin precursor peptide gene (PGSC0003DMG400047074, SEQ ID NO: 38) was analyzed in 16 tissue samples (NCBI SRA datasets: ERR029909, ERR029910, ERR029911, ERR029912, ERR029913, ERR029914, ERR029915, ERR029916, ERR029917, ERR029918, ERR029919, ERR029920, ERR029921, ERR029922, ERR029923, ERR029924) by RSEM against the combined de novo Trinity-assembled transcriptome of all 16 samples (
Glutamine Cyclotransferase Co-Expression Assays with LbaLycA in Nicotiana benthamiana
Glutamine cyclotransferase LbaQC was characterized as the closest homolog of Chenopodium quinoa glutamine cyclotransferase (AUR62017096, Phytozome 12.1) by blastp search oft barbarum root transcriptome on an internal Blast server. LbaQC was synthesized as a gBlock® (ATGGTTTCTTCTACTTCATATCTACCTACCAATCACACAAAAATGCCTCTGCTAA ATCCAAGGTTTCTAGTCATAAGCTTGATTGTTCTACTGAGCATCACCGTATTCAGA GAAGCTGAAGCATCATATAGAGTTTACAAAGTCAAAGTAGTCAATGAATTCCCTC ACGACCCCCAAGCCTACACTCAGGGGCTTCTCTATGCAGAAAATAATACACTCTT TGAATCAACTGGACTTTACGGACGTTCATCTGTTCGAAAAGTTGCATTGCTGGAC GGTAAGGTTGAGAGACTTCATGAAATGGAGTCTTCTTACTTTGGAGAGGGTCTAA CTCTTCTTGGTGAGAGGTTGTTCCAACTAACATGGTTGCTGGATACAGGTTTCATA TATGATCGATACAACTTCAGCAAATTCAAAAAGTTTACTCATCACATGCAAGATG GTTGGGGATTGGCAACCGATGGGAAAGTACTTITTGGAAGTGATGGAACATCAA CATTATATAAGATTGACCCTAAAACAATGAAAGTCATCAGAAAACAAGTTGTCAA GTCTCAAGGGCATGAAGTGCGCTACCTGAATGAGCTGGAGTATGTGAAAGCTGA AGTCTGGGCAAATGTTTATGTGACTGATTGCATTGCTAGAATTTCACCAAAAGAT GGCACTGTGATCGGGTGGATTCTCCTTCAATCTCTAAGAGAAGAGTTAATATCAA GAGGATATAAGGACTTCGAGGTCCTGAATGGAATCGCATGGGACAGAGATGGTG ACCGTATTTTTGTGACAGGGAAACTATGGCCAAAGCTCTTTGAGATCAAGTTGCT CCCCCTCACACCGAATGATCCATTGGCTGGAGAAATCAATAACTTGTGCATCCCG AAAACCAGTTTTCTCTTGGAAATTTAG (SEQ ID NO: 122)) with a 5′-adapter (TGCCCAAATTCGCGACCGGT) (SEQ ID NO: 123) and a 3′-adapter (CTCGAGGCCTTTAACTCTGG) (SEQ ID NO: 124) for Gibson assembly. pEAQ-HT was digested by AgeI and XhoI restriction enzymes and the LbaQC gBlock@ was cloned into linearized pEAQ-HT with Gibson Assembly Master Mix (New England Biolabs). pEAQ-HT-LbaQC was verified by Sanger sequencing. For glutamine cyclotransferase co-expression assays. pEAQ-HT-LbaQCand pEAQ-HT-LbaLycA were transformed into Agrobacterium tumefaciens LBA4404 for heterologous expression as described above. For co-expression assay, leaves of three plants of Nicotiana benthamiana (six week old) were infiltrated with a 1:1 mixture of resuspended A. tumefaciens LBA4404 pEAQ-HT-LbaQC (OD 0.8) and A. tumefaciens LBA4404 pEAQ-HT-LbaLycA (OD 0.8). For LbaLycA control expression without LbaQC, leaves of three plants of Nicotiana benthamiana (six week old) were infiltrated with resuspended A. tumefaciens LBA4404 pEAQ-HT-LbaLycA (OD 0.4). Infiltrated plants were cultivated as described before for six days for heterologous expression. After six days, leaves of three plants of the LbaQC-LbaLycA co-expression and leaves of three plants of LbaLycA expression control were collected and freeze-dried. For comparative chemotyping of [Gln1]-lyciumin B, [Gln1]-lyciumin D, lyciumin B and lyciumin D, peptides were extracted from 0.1 g of freeze-dried tobacco leaves as described above for peptide chemotyping from the LbaQC-LbaLycA co-expression plants and from the LbaLycA expression plants. Peptide extracts were subjected to low resolution MS analysis by selected-ion monitoring (SIM) of masses of [Gln1]-lyciumins and lyciumins with the following LC-MS parameters: LC—Phenomenex Kinetex@ 2.6 μm C18 reverse phase 100 Å 150×3 mm LC column, LC gradient: solvent A—0.1% formic acid, solvent B—acetonitrile (0.1% formic acid), 0.5 mL/min, 0-1 min: 5% B, 1-8 min: 5-95% B, 8-10 min: 95% B, 10-15 min: 5% B, MS—positive ion mode, SIM: 896.8-897.8 m/z (lyciumin B), 899.8-900.8 m/z (lyciumin D), 913.8-914.8 m/z ([Gln1]-lyciumin B), 916.8-917.8 m/z ([Gln1]-lyciumin D). Lyciumin and [Gin1]-lyciumin ion abundance values were determined by peak area integration from each peptide SIM chromatogram in QualBrowser in the Thermo Xcalibur software package (version 3.0.63, ThermoScientific).
This experiment investigates the taxonomic distribution of lyciumin-type RiPPs in the plant kingdom and further probes into the evolutionary mechanisms that could explain the observed distribution pattern. This endeavour was greatly facilitated by the extensive plant transcriptome sequencing effort in the recent years, e.g., the 1 kp project, which covers more than half of the extent plant families on earth (Matasci et al., 2014). This experiment establishes an evolutionary framework of how lyciumin-type RiPPs have emerged over the last 450 million years of land plant evolution.
Example 2 is an extension of the results described in Example 1 under the subheading “Parallel evolution of lyciumin biosynthesis in angiosperms and lycophytes.” Some of the results presented under that subheading of Example 1 may be repeated in Example 2 for clarity.
With the recently revealed knowledge of the lyciumin precursor protein (Kersten and Weng, 2018), the distribution of lyciumin genotypes in the plant kingdom was explored using available transcriptome resources generated by the plant community in recent years (Matasci et al., 2014). However, an apparent issue of identification of lyciumin precursor genes from plant transcriptomes is the repetitive nature of most of the lyciumin-precursor-peptide-encoding genes, which causes misassembly of these genes from short-read RNA-seq data using de novo transcriptome assembly programs. For example, known lyciumin precursor peptides from Amaranthaceae, Fabaceae and Solanaceae comprise repeating motifs of lyciumin core peptides either in the N-terminal domain (type 1 lyciumin precursor) or within the BURP domain (type 2 lyciumin precursor) (
Given the results of improved BURP-domain precursor gene assembly using rnaSPAdes at the time of the transcriptome analysis, de novo reassembly of transcriptomes of 793 plant species was performed using rnaSPAdes starting from raw sequencing reads generated as part of the 1 kp project (Matasci et al., 2014), which represent a total of 317 land plant families. Subsequently, lyciumin genotypes were searched for in these reassembled transcriptomes by tblastn using type 1 (LbaLycA, GenBank: MH124242) and type 2 (Sali3-2, GenBank: AAB66369) lyciumin precursors as queries. This exercise readily identified a battery of candidate lyciumin precursor genes distributed across diverse plant families that extend beyond the previously reported Amaranthaceae, Fabaceae and Solanaceae (Kersten and Weng, 2018). These newly identified lyciumin-genotype-containing plant families include Aizoaceae, Molluginaceae, Nyctaginaceae, Petiveriaceae and Phytolaccaceae, which are all under the order of Caryophyllales, as well as Selaginellaceae. It is noteworthy that Selaginellaceae is one of the three extant families of lycophytes which are basal vascular plants separated from all other euphyllophytes over 400 million years ago (Banks, 2009).
Since RiPPs have only been reported in angiosperms, we sought to confirm the predicted lyciumin production in Selaginella. To do this, several additional transcriptomes of Selaginella species were assembled starting from RNA-seq raw reads available from the NCBI SRA using rnaSPAdes, and searched for lyciumin genotypes. In addition to Selaginella willdenowii (1 kp dataset, NCBI SRA: ERR2040880) (Matasci et al., 2014), lyciumin precursor genes were found in three other Selaginella species: S. uncinata, S. moellendorffii and S. bryopteris (
Given the occurrence of lyciumins in multiple families of angiosperms and lycophytes, the evolutionary history of the characterized and predicted lyciumin precursor genes was examined (
To test these alternative evolutionary hypotheses, phylogenetic analyses were performed of BURP-domain proteins from several sequenced plant genomes together with BURP domains of the predicted and characterized lyciumin-producing precursor proteins or BURP domains of the predicted and characterized lyciumin-producing precursor proteins alone (
The core peptide motif sequences of all the predicted and characterized lyciumin precursors were systematically examined, and it was found that the lyciumin core-peptide motifs are mostly unique to each of the phylogenetic clades (75% of core peptides are found in one plant family,
From the perspective of lyciumin precursor gene structure, both Fabaceae and Selaginellaceae contain type 1 lyciumin precursors, while Caryophyllales and Solanaceae contain type 2 lyciumin precursors (
Given these results in the context of the taxonomic relationship of the predicted and characterized lyciumin-producing plants, homologous non-lyciumin-producing BURP-domain protein progenitors likely gave rise to independent occurrences of lyciumin biogenesis at least once in lycophytes and four times in eudicots followed by extensive divergent and parallel evolution to yield the extant lyciumin chemodiversity (
A Shared Lyciumin Chemotype in Hemp Plant Celtis occidentalis and Amaranth Plant Achyranthes Bidentata is Derived from Two Disparate Families of Precursor Peptides
When mining new lyciumin genotypes from diverse plant transcriptomes, a lyciumin core-peptide-containing gene from hackberry (Celtis occidentalis, Cannabaceae,
The emergence of lyciumins from precursor peptides unrelated to the BURP-domain proteins indicates a potential case of convergent evolution of lyciumins in plants. To probe this hypothesis further, all characterized and predicted BURP-domain lyciumin precursor peptides were queried for potential lyciumin-[QPFGVFGW] producers. This exercise identified several BURP-domain lyciumin precursor genes from Chenopodium quinoa (Amaranthaceae), Hypertelis (Kewa) cerviana (Molluginaceae), Petunia inflata and Solanum tuberosum (both Solanaceae) that harbor the capacity to produce lyciumin-[QPFGVFGW]. Because lyciumin-[QPFGVFGW] could not be detected in peptide extracts of greenhouse-grown C. quinoa and S. tuberosum plants, whereas H. cerviana and P. inflata plants were not available for chemical analysis, additional Amaranthaceae transcriptomes were further queried to search for BURP-domain precursor genes containing lyciumin-[QPFGVFGW] motifs. One such candidate gene was identified in the transcriptome of ox knee (Achyranthes bidentata) (
Knowledge of plant specialized metabolism is incomplete. The increase of genomic and transcriptomic data from taxonomically diverse plants can greatly accelerate the discovery of new plant chemotypes and unlock the trajectories of metabolic evolution towards these chemical adaptations. In Example 2, large-scale transcriptome mining followed by peptide chemotyping was carried out, which revealed that the branched cyclic lyciumin RiPPs most likely evolved independently at least once in lycophytes and four times in angiosperms. The results also suggest that lyciumins have emerged in BURP-domain precursor genes and DUF2775-domain precursor genes via convergent evolution.
Lyciumins share several similar features in terms of their biosynthetic origin and taxonomic distribution with head-to-tail cyclic RiPPs such as cyclotides and orbitides. Like other classes of plant cyclic RiPPS, lyciumins might have occurred through serendipitous emergence of a single lyciumin core peptide motif sequence in progenitor BURP domain proteins or DUF2775 domain proteins, which could be processed into small stable branched cyclic peptides by post-translationally modifying enzymes and proteases already present in the host plant cells. Lyciumins with favorable properties that render selective advantage to the plant host were more likely to be retained. Through core peptide mutagenesis and internal motif duplication, the ancestral lyciumins could undergo subsequent chemical optimization, amplification and diversification to yield extant lyciumins (Mylne et al., 2012). Indeed, lyciumin precursor peptides have diverse core peptide sequences that often exist as repetitive motifs in one precursor gene (
There are several unique aspects of lyciumins in the context of plant RiPP evolution. First, lyciumins occured in non-angiosperms, i.e., Selaginella plants, and, therefore, represent the only known RiPP family from non-angiosperms to date. Nevertheless, a report of a cyclotide-like protein domain with protease inhibitory activity in S. moellendorffii (James et al., 2017) implies that head-to-tail cyclic RiPPs may also exist in non-angiosperms and await to be discovered. Second, lyciumins have evolved from different precursor peptides than those that give rise to head-to-tail cyclic RiPPs. Whereas precursor peptides of cyclotides and orbitides are mainly stand-alone proteins (Jennings et al., 2001; Mylne et al., 2012) or seed storage albumins (Poth et al., 2011), lyciumin precursors are either BURP-domain proteins or DUF2775-domain proteins. The full elucidation of lyciumin biosynthetic steps such as proteolytic cleavage and cyclization in characterized lyciumin-producing plant families will further reveal additional differences and similarities between the evolutionary trajectories underlying the head-to-tail cyclic RiPPs and the branched cyclic RiPPs in plants.
This study reveals a complex history of lyciumin evolution within land plants. First, lyciumin genotypes and the corresponding chemotypes were identified in distantly related lycophytes (i.e., Selaginellaceae) and angiosperms (i.e., Cannabaceae, Caryophyllales families, Fabaceae, Solanaceae), whereas no lyciumin genotypes were found in taxa immediately sister to these lyciumin-producing plant lineages nor in any ferns and gymnosperms which are two major vascular plant lineages intermediate between lycophytes and angiosperms. Specifically, no BURP-domain proteins with lyciumin-like core peptide motifs were found in the transcriptomes of the families Lycopodiaceae and Isoetaceae, the only two other extant lycophyte families besides Selaginellaceae. Similarly, lyciumin-producing BURP-domain proteins are absent from plant orders neighboring Fabales (i.e., Malpighiales and Rosales), Caryophyllales (i.e., Berberidopsidales and Cornales), and Solanales (i.e., Gentianales and Lamiales). Moreover, no lyciumin-producing DUF2775-domain proteins were found in plant orders neighboring Rosales (i.e., Fabales and Cucurbitales). Based on these observations, it is most likely that the ability to produce lyciumins arose independently in lycophytes and angiosperms. Phylogenetic reconstruction of lyciumin-producing and non-lyciumin-producing BURP-domain sequences and analysis of the cyclization residues further suggest that the recruitment of BURP-domain proteins for lyciumin biogenesis occurred independently at least four times within angiosperms. Second, although lyciumin chemotypes vary greatly between lyciumin-producing plant families, a few overlapping lyciumin chemotypes were also found (
Lyciumins have been characterized as protease inhibitors, which implicates their potential physiological functions in host defense similar to other plant RiPP classes (Hernandez et al., 2000). While specific functions of DUF2775 proteins in plants are unknown (Albornos et al., 2012), BURP domains have been associated with plant responses to abiotic stresses such as drought (Wang et al., 2012). For example, the lyciumin I precursor peptide Sali3-2 is highly expressed in G. max roots under acidic soil conditions (Ragland and Soliman, 1997), and its overexpression in Arabidopsis alleviates heavy metal stress (Tang et al., 2014). Future research will help elucidate why lyciumins evolved from BURP-domain or DUF2775-domain proteins. It is possible that lyciymins first evolved to enhance host defense or metal-chelation when host plants were under certain abiotic stresses. A comprehensive understanding of the biosynthetic mechanism and evolution of lyciumin-type RiPPs in the plant kingdom will ultimately facilitate engineering of this cyclic peptide class for crop improvement and drug development.
All chemicals were purchased from Sigma-Aldrich, unless otherwise specified. Oligonucleotide primers and synthetic genes were purchased as gBlocks® from Integrated DNA Technologies, Inc. Solvents for liquid chromatography high-resolution mass spectrometry were Optima® LC-MS grade (Fisher Scientific) or LiChrosolv® LC-MS grade (Millipore). High resolution mass spectrometry analysis was performed on a Thermo ESI-Q-Exactive Orbitrap MS coupled to a Thermo Ultimate 3000 UHPLC system. Low-resolution mass spectrometry analysis was done on a Thermo ESI-QQQ MS coupled to a Thermo Ultimate 3000 UHPLC system.
Nicotiana benthamiana seeds for cultivation were a gift from the Lindquist lab (Whitehead Institute, MIT). Selaginella uncinata plants were purchased from Plant Delights Nursery, Inc. Celtis occidentalis leaves were collected from a living tree (Accession No. 7894*A) on Aug. 3, 2018 in the Arnold Arboretum of Harvard University (Project No. 25-2018). Achyranthes bidentata seeds for cultivation were purchased from Frozen Seed Capsules™. Chenopodium quinoa seeds for cultivation were purchased from Earthcare Seeds.
Achyranthes bidentata, Chenopodium quinoa and Nicotiana benthamiana were grown in Sun Gro® Propagation Mix soil with added vermiculite (Whittemore Inc.) and added fertilizer in a greenhouse with a 16 h light/8 h dark cycle for two to six months.
For comparative assembly of lyciumin precursor genes, selected plant transcriptome datasets from the NCBI Sequence Read Archive (Lycium barbarum—SRR6896657, Amaranthus hypochondriacus—SRR1598913, Chenopodium quinoa—ERR2040214, Solanum melongena—SRR1104129, Medicago truncatula—SRR5732302, Nicotiana attenuata—SRR1950612) were assembled in triplicate with Trinity (v2.6.6, Grabherr et al., 2011) or rnaSPAdes (v1.0, kmer=25,75 or 55, Bankevich et al., 2012; Bushmanova et al., 2018). Target lyciumin precursor peptides (Lycium barbarum—MH124242, Amaranthus hypochondriacus—AHYPO_007393, Chenopodium quinoa—XP_021740703.1, Solanum melongena—Sme2.5_02115.1 g00002.1, Medicago truncatula—-Medtr8g045890, Nicotiana attenuata—OIT08186.1) were searched in the corresponding de novo transcriptome assemblies by tblastn on an internal Blast server (Priyam et al., 2015).
For large-scale transcriptome mining of lyciumin precursor genes, land plant transcriptome datasets from the 1 kp database (Matasci et al. 2014, Table S1) were assembled by rnaSPAdes (v1.0, kmer=25,75). If an rnaSPAdes (kmer=25,75) assembly failed, the transcriptome was assembled with rnaSPAdes (kmer=55) if possible. Resulting rnaSPAdes-contig files of the 1 kp transcriptomes were searched on an internal Blast server by blastn for (A) homologs of type 1 lyciumin precursor Sali3-2 (GenBank: AAB66369) or (B) homologs of type 2 lyciumin precursor LbaLycA (GenBank: MH124242). Candidate lyciumin precursors were identified by the presence of a lyciumin core peptide motif defined as QP(X)5W, where X is any amino acid, in the N-terminal domain (type 2 lyciumin precursor) or within the BURP domain (type 1 lyciumin precursor).
Cloning of Selaginella uncinata Lyciumin Precursor Gene SunBURP
Illumina sequence raw-files of a Selaginella uncinata transcriptome (NCBI-SRA: SRR7132763) were combined and assembled by rnaSPAdes (v1.0, kmer 25,75, Bankevich et al., 2012; Bushmanova et al., 2018). The Selaginella uncinata root transcriptome was analyzed for lyciumin precursor genes by blastp algorithm on an internal Blast server (Priyam et al., 2015). Root tissue was removed from a Selaginella uncinata plant and total RNA was extracted with the QIAGEN RNeasy Plant Mini kit. cDNA was prepared from root total RNA with SuperScript® III First-Strand Synthesis System (Invitrogen). Transcripts homologous to target lyciumin precursor LbaLycA were used to design cloning primers (SunBURP-pEAQ-fwd (SEQ ID NO: 128): TGCCCAAATTCGCGACCGGTATGGCATCTAATCTCCTTTACTTGC, SunBURP-pEAQ-rev (SEQ ID NO: 129): CCAGAGTTAAAGGCCTCGAGTTACCACACAATGGTTTCGTACC) for amplification of precursor peptide gene SunBURP with Phusion® High-Fidelity DNA polymerase (New England Biolabs). SunBURP was cloned into pEAQ-HT (Sainsbury et al., 2009), which was linearized by restriction enzymes AgeI and XhoI, by Gibson cloning assembly (New England Biolabs) (Gibson et al., 2009). Cloned SunBURP was sequenced by Sanger sequencing from pEAQ-HT-SunBURP
For peptide chemotyping, 0.2 g plant material (fresh weight) were frozen and ground with mortar and pestle. Ground plant material was extracted with 10 mL methanol for 1 h at 37° C. in a glass vial. Plant methanol extract was dried under nitrogen gas in a separate glass vial. Dried plant methanol extract was resuspended in water (10 mL) and partitioned with hexane (2×10 mL) and ethyl acetate (2×10 mL), and subsequently extracted with n-butanol (10 mL). The n-butanol extract was dried in vacuo and resuspended in 2 mL methanol for liquid chromatography-mass spectrometry (LC-MS) analysis. Peptide extracts were subjected to high resolution MS analysis with the following LC-MS parameters: LC—Phenomenex Kinetex® 2.6 μm C18 reverse phase 100 Å 150×3 mm LC column, LC gradient: solvent A—0.1% formic acid, solvent B—acetonitrile (0.1% formic acid), 0-2 min: 5% B, 2-23 min: 5-95% B, 23-25 min: 95% B, 25-30 min: 5% B, 0.5 mL/min, MS—positive ion mode, Full MS: Resolution 70000, mass range 425-1250 m/z, dd-MS2 (data-dependent MS/MS): resolution 17500, Loop count 5, Collision energy 15-35 eV (stepped), dynamic exclusion 1 s. LC-MS data of peptide extracts from a predicted lyciumin producing plant was analyzed for lyciumin mass signals by (a) parent mass search (base peak chromatogram of calculated [M+H]+ of predicted lyciumin structure, Δm=5 ppm), (b) fragment mass search of pyroglutamate-proline-b-ion in MS/MS data (C10H13N2O3+, 209.09207 m/z, Δm=5 ppm), and (c) iminium ion mass search of specific amino acids of predicted structure in MS/MS data (for example, pyroglutamate iminium ion [M+H]+ 84.04439 m/z). Putative mass signals of predicted lyciumin structures were confirmed by MS/MS data analysis with QualBrowser in the Thermo Xcalibur software package (version 3.0.63, ThermoScientific).
Transient Expression of Lyciumin Precursor Genes Sali3-2-[QPFGVFGW] and Sali3-2-[QPYGVFAW] in Nicotiana benthamiana
Sali3-2-[QPFGVFGW] and Sali3-2-[QPYGVFAW] were cloned into pEAQ-HT (Sainsbury et al., 2009), which was linearized by restriction enzymes AgeI and XhoI, by Gibson cloning assembly (New England Biolabs) (Gibson et al., 2009). Agrobacterium tumefaciens LBA4404 was transformed with pEAQ-HT-Sali3-2-[QPFGVFGW] or pEAQ-HT-Sali3-2-[QPYGVFAW] by electroporation (2.5 kV), plated on YM agar (0.4 g yeast extract, 10 g mannitol, 0.1 g sodium chloride, 0.2 g magnesium sulfate (heptahydrate), 0.5 g potassium phosphate, (dibasic, trihydrate), 15 g agar, ad 1 L Milli-Q Millipore water, adjusted pH 7) with 100 μg/mL rifampicin, 50 μg/mL kanamycin and 100 μg/mL streptomycin and incubated for two days at 30° C. A 5 mL starter culture of YM medium with 100 μg/mL rifampicin, 50 μg/mL kanamycin and 100 μg/mL streptomycin was inoculated with a clone of Agrobacterium tumefaciens LBA4404 pEAQ-HT—Sali3-2-[QPFGVFGW] or pEAQ-HT-Sali3-2-[QPYGVFAW] and incubated for 24-36 h at 30° C. on a shaker at 225 rpm. Subsequently, the starter culture was used to inoculate a 50 mL culture of YM medium with 100 μg/mL rifampicin, 50 μg/mL kanamycin and 100 μg/mL streptomycin, which was incubated for 24 h at 30° C. on a shaker at 225 rpm. The cells from the 50 mL culture were centrifuged for 30 min at 3000 g, the YM medium was discarded and cells were resuspended in MMA medium (10 mM MES KOH buffer (pH 5.6), 10 mM magnesium chloride, 100 μM acetosyringone) to give a final optical density of 0.8. The Agrobacterium suspension was infiltrated into the bottom of leaves of Nicotiana benthamiana plants (six week old). N. benthamiana plants were placed in the shade two hours before infiltration. After infiltration, N. benthamiana plants were grown as described above for six days. Subsequently, infiltrated leaves were collected and subjected to chemotyping.
Protein sequences of characterized and predicted lyciumin precursors from genomes (Kersten et al., 2018, except 3′-partial sequences) and transcriptomes (
LC-MS datasets (MassIVE) (Wang et al., 2016): MSV000083215 (Celtis occidentalis leaf), MSV000083216 (Achyranthes bidentata seed), MSV000083217 (Selaginella uncinata root). GenBank: SunBURP—MK089798.
Glycine max: Glyma.12G217400 Org_Gmax peptide: Glyma.12G217400.1.p
53. Albornos, L., Martin, I., Iglesias, R., Jimenez, T., Labrador, E., and Dopico, B. (2012). ST proteins, a new family of plant tandem repeat proteins with a DUF2775 domain mainly found in Fabaceae and Asteraceae. BMC Plant Biol. 12:207.
64. Claeson, P., Goransson, U., Johansson, S., Luijendijk, T., and Bohlin, L. (1998). Fractionation Protocol for the Isolation of Polypeptides from Plant Biomass. J. Nat. Prod. 61:77-81.
The teachings of all patents, published applications and references cited herein are incorporated by reference in their entirety.
While example embodiments have been particularly shown and described, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the embodiments encompassed by the appended claims.
This application claims the benefit of U.S. Provisional Application No. 62/619,905, filed on Jan. 21, 2018. This application claims the benefit of U.S. Provisional Application No. 62/620,420, filed on Jan. 22, 2018. This application claims the benefit of U.S. Provisional Application No. 62/732,957, filed on Sep. 18, 2018. The entire teachings of the above applications are incorporated herein by reference.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2019/014430 | 1/21/2019 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62732957 | Sep 2018 | US | |
| 62620420 | Jan 2018 | US | |
| 62619905 | Jan 2018 | US |
