Patent Application
20240035060
The XML file submitted herewith via Patent Center, named “TIER-003-02US.xml” created on Oct. 17, 2023, having a size of 60 KB, is hereby incorporated by reference in its entirety.
The disclosure relates to cell-free compositions and use thereof, particularly in the biodiscovery of natural products.
There is extensive biological data encoded in DNA that is of unknown function. This data varies from naturally derived proteins, peptides, and molecular control components to semi-synthetic and engineered variants thereof. With the advent of high-throughput sequencing, as of 2017, more than 2.7 trillion bases of information are known, and only a small fraction are expressed. Tools that are able to determine the products of this DNA will be essential to understanding this information.
Separately, synthetic biology has emerged as an important field for which essential processes can be understood and engineered. Within this field there are both natural, semi-synthetic, and engineered genes, regulatory parts, and other components that are in need of testing.
Despite efforts and progresses, current approaches are limited to conduct high-throughput functional genomics to determine products from DNA and to promote synthetic biology approaches. Challenges still remain in developing engineering-driven approaches and systems to accelerate the design-build-test cycles required for reprogramming existing biological systems, constructing new biological systems and testing genetic circuits for transformative future applications in diverse areas including biology, engineering, green chemistry, agriculture and medicine.
An in vitro transcription-translation cell-free system (Shin & Noireaux, 2012; Sun et al., 2013) has been developed which allows for the rapid prototyping of genetic constructs (Sun, Yeung, Hayes, Noireaux, & Murray, 2014) in an environment that behaves similarly to a cell (Niederholtmeyer, Sun, Hori, & Yeung, 2015; Takahashi et al., 2015). One of the main purposes of working in vitro is to be able to generate fast speeds—in vitro, reactions can take 8 hours and can scale to thousands of reactions a day, a multi-fold improvement over similar reactions in cells (Sun et al., 2014). Despite the potential of this cell-free system, it needs be fine-tuned when used in different applications to achieve optimal results.
Natural products have played key roles over the past century in advancing our understanding of biology and in the development of medicine. Research in the 20th century identified many classes of natural products with four groups being particularly prevalent: terpenoids, alkaloids, polyketides, and non-ribosomal peptides. The genome sequencing efforts of the first decade of the 21st century have revealed that another major class is formed by ribosomally synthesized and post-translationally modified peptides. These molecules are produced in all three domains of life, their biosynthetic genes are ubiquitous in the currently sequenced genomes and transcriptomes, and their structural diversity is vast. The extensive post-translational/co-translational modifications endow these peptides with structures not directly accessible for natural ribosomal peptides, typically restricting conformational flexibility to allow better target recognition, to increase metabolic and chemical stability, and to augment chemical functionality.
Thus, a need exists for tools that allow the rapid, efficient discovery of national products, such as the cell-free systems disclosed herein.
In one aspect, provided herein is a composition for in vitro transcription and translation, comprising:
In some embodiments, the cell lysate is substantially free of protease.
The plurality of supplements can include reagents for transcription and translation, and optionally can include one or more non-canonical amino acids.
The stabilizing domain in some embodiments is linked to the propeptide via a linker, preferably a peptide linker comprising Gly and Ser, more preferably Gly-Gly-Gly-Gly-Ser-Ser (SEQ ID NO.: 22), Gly-Gly-Ser-Gly (SEQ ID NO.: 23), or Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly (SEQ ID NO.: 24).
In some embodiments, the engineered propeptide contains one or more protease sites that allow the stabilizing domain to be cleaved away, such as Tobacco Etch Virus (TEV) sites, PreScission Protease sites, Thrombin Protease sites, Factor Xa protease sites, and Enterokinase protease sites; and wherein the engineered propeptide contains a tag for detection by small molecule interactions, antibodies, affinity purification, or other reagents, such as FLASH/REASH sites, MBP, NusA, GST, His6, CBP, FLAG, HA, HBH, Myc, S-tag, SUMO, TAP, TRX, V5.
In some embodiments, the engineered propeptide is separately synthesized and added exogenously to the composition. In certain embodiments, the engineered propeptide contains a modification so as to resist proteolysis, preferably one or more non-canonical amino acids or a post-translation modification of an existing amino acid, or a stapled peptide.
In some embodiments, the composition can further include an engineered nucleic acid, such as DNA and/or mRNA, designed to express the engineered propeptide in the composition.
In various embodiments, the engineered propeptide can be provided from a variant library. The variant library can be pre-designed to include a plurality of propeptide variants, each having one or more mutations. The mutations can be randomized amino acid mutations, or targeted mutations designed to introduce a desired change in function, activity, stability, etc.
The composition can, in some embodiments, further include an unstructured peptide provided at no less than 0.1 mg/ml concentration in the composition.
In various embodiments, the composition is designed to produce a natural product, preferably a ribosomal natural product, more preferably a amatoxin, phallotoxin, bottromycin, cyanobactin, lanthipeptide, lasso peptide, linear azol(in)e-containing peptide, microcin, thiopeptide, autoinducing peptide, bacterial head-to-tail cycized peptide, conopeptide, cyclotide, glyocin, linearidin, microviridin, orbitide, proteusin, sactipeptide, toxin, or venom.
The composition may comprise one or more enzymes for modifying the natural product to produce a modified variant thereof. At least a portion of the one or more enzymes can be provided in the cell lysate. The composition can additionally or alternatively further comprise an engineered genetic circuit designed to express at least a portion of the one or more enzymes.
In some embodiments, the natural product can be further modified outside of the composition to produce a modified variant thereof.
The composition in some embodiments, is engineered to produce an antibiotic, herbicide, pesticide, insecticide, animal feed additive, signaling molecule, receptor agonist, receptor antagonist, activator, inhibitor, quorum sensing molecule, or anticancer therapeutic, toxin, or venom.
In some embodiments, the engineered nucleic acid or engineered genetic circuit can be derived from a microbiome, preferably human gut, animal, oral, skin, vaginal, soil, ocean, rhizosome, umbilical, vaginal, conjunctival, intestinal, stomach, nasal, gastrointestinal tract, or urogenital tract microbiomes.
The composition in some embodiments, can further include a crowding agent, preferably present at no less than 0.1% (w/v), wherein more preferably the crowding agent is polyethylene glycol present at no greater than 0.2% (w/v).
Also provided herein is a method of synthesizing a propeptide in vitro, comprising:
Another aspect relates to a method of preparing a composition for in vitro transcription and translation, comprising:
In some embodiments, the composition is substantially free of proteases due to the presence of an unstructured peptide at no less than 0.1 mg/ml concentration to competitively deplete proteases, or due to genetic engineering of the organisms to remove proteases either directly or through application of tags against which to remove proteases during lysate production, or due to presence of reagents that specifically or non-specifically targets proteases. The cell lysate can be derived from any cells, such as is Rhodococcus jostii, Vibrio natriegens, Clostridium acetobutylicum, or HeLa cells.
In certain embodiments, the determining step comprises mixing the composition with an effective amount (e.g., 1 ug) of a test, unstructured peptide and determining that at least 10% of the test peptide remains after incubation for about 60 minutes.
While the above-identified drawings set forth presently disclosed embodiments, other embodiments are also contemplated, as noted in the discussion. This disclosure presents illustrative embodiments by way of representation and not limitation. Numerous other modifications and embodiments can be devised by those skilled in the art which fall within the scope and spirit of the principles of the presently disclosed embodiments.
Disclosed herein, in one aspect, is a cell-free system that can be used to explore natural products and their variants that result from metagenomic as well as synthetic sources. One reservoir of natural products are actinomycetes, a group of gram-positive bacteria, typically with high genomic G+C content, that are a source of diverse bioactive secondary metabolites. Of all antibiotics known, 66% are produced by actinomycetes. Representative marketed natural products include streptomycin, erythromycin, clauvanic acid, chloramphenicol, and amorpha-1,4-diene. Two members of the group, Streptomycetales and Pseudonocardiales, were found to have a per-genome average of ˜20 secondary metabolites (defined as PKS Types I and II, NRPS, lanthipeptides, thiazole-oxazole modified microcins, and NRPS-independent siderophores). Currently characterized metabolites represent only 10% of bioinformatically detected secondary metabolites, indicating a large unexplored database. Compounding the difficulty of secondary metabolite detection is low native production rates—for example, daptomycin (a lipopeptide antibiotic) was identified after fermentation of 107 strains. By removing the hurdle of producing normally silent secondary metabolites in cells, one can remove native negative regulatory methods and the costs and time to conduct experiments in vivo.
The compositions and methods disclosed herein relieve the hurdle of expressing natural products and synthetic variants thereof by utilizing cell-free expression system in lieu of cellular expression. This allows for the expression of otherwise silent natural products, or natural product variants that cannot be catalyzed by traditional heterologous expression or by isolating natural products. The focus of the compositions and methods proposed is on expressing ribosomal natural products and synthetic variants. Techniques to express ribosomal natural products, including the stabilization of propeptide/prepeptide starting precursors, are disclosed herein.
For convenience, certain terms employed in the specification, examples, and appended claims are collected here. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.
The articles “a” and “an” are used herein to refer to one or to more than one (i.e., at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.
As used herein, the term “about” means within 20%, more preferably within 10% and most preferably within 5%. The term “substantially” means more than 50%, preferably more than 80%, and most preferably more than 90% or 95%.
As used herein, “a plurality of” means more than 1, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more, e.g., 25, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, or more, or any integer therebetween.
The term “natural product” refers to biological products that can be found in nature. The compositions and systems disclosed herein can be used as effective tools to discover unknown natural products. In some embodiments, the natural product can be a ribosomal natural product (also referred to as a Ribosomally synthesized and post translationally modified peptide or RiPP). Examples include but are not limited to, amatoxin, phallotoxin, bottromycin, cyanobactin, lanthipeptide, lasso peptide, linear azol(in)e-containing peptide, microcin, thiopeptide, autoinducing peptide, bacterial head-to-tail cycized peptide, conopeptide, cyclotide, glyocin, linearidin, microviridin, orbitide, proteusin, sactipeptide, toxin, and/or venom.
The term “modified variant” of a natural product refers to a non-naturally existing product that is a variant of the natural product. Such variant can contain one or more modifications such as non-canonical amino acids, post-translational modifications, methylation, glycosylation, prenylation, dehydration, cyclodehydration, macrocyclization, thioester linkages, crosslinks, chelation, thioamide linkages, heterocycles. Such variants may also be rationally engineered by utilizing known information about the production of a natural product and varying components that are not necessary for the natural product processing. Examples include scaffolding of propeptide regions for processing by native machinery to produce variants of natural products capable of binding receptors, or providing modified inputs (eg. decorated side chains) that are then processed to produce modified natural products with unique properties. Modified variants of natural products can have favorable properties beyond the original natural product (e.g., cancer therapeutic, anti-proteolysis, receptor binding, increased specificity, etc.). The production of modified variants is similar to the process of diversifying scaffolds in synthetic chemistry, but utilizes biological techniques.
As used herein, the terms “nucleic acid,” “nucleic acid molecule” and “polynucleotide” may be used interchangeably and include both single-stranded (ss) and double-stranded (ds) RNA, DNA and RNA:DNA hybrids. These terms are intended to include, but are not limited to, a polymeric form of nucleotides that may have various lengths, including deoxyribonucleotides and/or ribonucleotides, or analogs or modifications thereof. A nucleic acid molecule may encode a full-length polypeptide or RNA or a fragment of any length thereof, or may be non-coding.
Nucleic acids can be naturally-occurring or synthetic polymeric forms of nucleotides. The nucleic acid molecules of the present disclosure may be formed from naturally-occurring nucleotides, for example forming deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) molecules. Alternatively, the naturally-occurring oligonucleotides may include structural modifications to alter their properties, such as in peptide nucleic acids (PNA) or in locked nucleic acids (LNA). The terms should be understood to include equivalents, analogs of either RNA or DNA made from nucleotide analogs and as applicable to the embodiment being described, single-stranded or double-stranded polynucleotides. Nucleotides useful in the disclosure include, for example, naturally-occurring nucleotides (for example, ribonucleotides or deoxyribonucleotides), or natural or synthetic modifications of nucleotides, or artificial bases. Modifications can also include phosphorothioated bases for increased stability.
As used herein, unless otherwise stated, the term “transcription” refers to the synthesis of RNA from a DNA template; the term “translation” refers to the synthesis of a polypeptide from an mRNA template. Translation in general is regulated by the sequence and structure of the 5′ untranslated region (5′-UTR) of the mRNA transcript. One regulatory sequence is the ribosome binding site (RBS), which promotes efficient and accurate translation of mRNA. The prokaryotic RBS is the Shine-Dalgarno sequence, a purine-rich sequence of 5′-UTR that is complementary to the UCCU core sequence of the 3′-end of 16S rRNA (located within the 30S small ribosomal subunit). Various Shine-Dalgarno sequences have been found in prokaryotic mRNAs and generally lie about 10 nucleotides upstream from the AUG start codon. Activity of a RBS can be influenced by the length and nucleotide composition of the spacer separating the RBS and the initiator AUG. In eukaryotes, the Kozak sequence lies within a short 5′ untranslated region and directs translation of mRNA. An mRNA lacking the Kozak consensus sequence may also be translated efficiently in an in vitro system if it possesses a moderately long 5′-UTR that lacks stable secondary structure. While E. coli ribosome preferentially recognizes the Shine-Dalgarno sequence, eukaryotic ribosomes (such as those found in retic lysate) can efficiently use either the Shine-Dalgarno or the Kozak ribosomal binding sites.
As used herein, the term “host” or “host cell” refers to any prokaryotic or eukaryotic single cell (e.g., yeast, bacterial, archaeal, etc.) cell or organism. The host cell can be a recipient of a replicable expression vector, cloning vector or any heterologous nucleic acid molecule. Host cells may be prokaryotic cells such as species of the genus Escherichia or Lactobacillus, or eukaryotic single cell organism such as yeast. The heterologous nucleic acid molecule may contain, but is not limited to, a sequence of interest, a transcriptional regulatory sequence (such as a promoter, enhancer, repressor, and the like) and/or an origin of replication. As used herein, the terms “host,” “host cell,” “recombinant host” and “recombinant host cell” may be used interchangeably. For examples of such hosts, see Green & Sambrook, 2012, Molecular Cloning: A laboratory manual, 4th ed., Cold Spring Harbor Laboratory Press, New York, incorporated herein by reference.
One or more nucleic acid sequences can be targeted for delivery to target prokaryotic or eukaryotic cells via conventional transformation techniques. As used herein, the term “transformation” is intended to refer to a variety of art-recognized techniques for introducing an exogenous nucleic acid sequence (e.g., DNA) into a target cell, including calcium phosphate or calcium chloride co-precipitation, conjugation, electroporation, sonoporation, optoporation, injection and the like. Suitable transformation media include, but are not limited to, water, CaCl2), cationic polymers, lipids, and the like. Suitable materials and methods for transforming target cells can be found in Green & Sambrook, 2012, Molecular Cloning: A laboratory manual, 4th ed, Cold Spring Harbor Laboratory Press, New York, incorporated herein by reference, and other laboratory manuals.
As used herein, the term “selectable marker” or “reporter” refers to a gene, operon, or protein that upon expression in a host cell or organism, can confer certain characteristics that can be relatively easily selected, identified and/or measured. Reporter genes are often used as an indication of whether a certain gene has been introduced into or expressed in the host cell or organism. Examples, without limitation, of commonly used reporters include: antibiotic resistance (“abR”) genes, fluorescent proteins, auxotropic selection modules, β-galactosidase (encoded by the bacterial gene lacZ), luciferase (from lightning bugs), chloramphenicol acetyltransferase (CAT; from bacteria), GUS (β-glucuronidase; commonly used in plants) green fluorescent protein (GFP; from jelly fish), and red fluorescent protein (RFP). Typically host cells expressing the selectable marker are protected from a selective agent that is toxic or inhibitory to cell growth.
The term “engineer,” “engineering” or “engineered,” as used herein, refers to genetic manipulation or modification of biomolecules such as DNA, RNA and/or protein, or like technique commonly known in the biotechnology art.
A “circuit” or “genetic circuit” as used herein refers to a collection of parts (e.g., genes or other genetic elements) that undergo transcription and/or translation to produce mRNA or proteins, respectively (each an “output” of the part). The part output can interact with other parts (for example to regulate transcription or translation) or can interact with other molecules in the cell-free system (e.g., small molecules, DNA, RNA or propeptides). For example, a circuit can be a metabolic pathway or a genetic cascade, which can be naturally occurring or non-naturally occurring, artificially engineered. Each part in the circuit can include a set of components or genetic modules, e.g., a promoter, ribosome binding site (RBS), coding sequence (CDS) and/or terminator. These components may be interconnected or assembled in different ways to implement different parts, and the resultant parts may be combined in different ways to create different circuits or pathways. In addition to these parts, the circuit may contain additional molecular species that are present in a cell or in the cell's environment that the components interact with. In one example, a genetic circuit can be designed to express one or more enzymes that modify the propeptides.
As described herein, “genetic module” and “genetic element” may be used interchangeably and refer to any coding and/or non-coding nucleic acid sequence. Genetic modules may be operons, genes, gene fragments, promoters, exons, introns, regulatory sequences, tags, or any combination thereof. In some embodiments, a genetic module refers to one or more of coding sequence, promoter, terminator, untranslated region, ribosome binding site, polyadenlylation tail, leader, signal sequence, vector and any combination of the foregoing. In certain embodiments, a genetic module can be a transcription unit as defined herein.
As used herein, the term “operably linked” means a first genetic element (e.g., propeptide encoding DNA) is engineered to be in the same nucleic acid molecule, and is in a functional relationship, with a second genetic element (e.g., a stabilizing domain encoding DNA) such that both can be, e.g., expressed as intended.
Other terms used in the fields of recombinant nucleic acid technology and molecular and cell biology as used herein will be generally understood by one of ordinary skill in the applicable arts.
The in vitro transcription and translation system is a system that is able to conduct transcription and translation outside of the context of a cell. In some embodiments, this system is also referred to as “cell-free system”, “cell-free transcription and translation”, “TX-TL”, “lysate systems”, “in vitro system”, “ITT”, or “artificial cells.” In vitro transcription and translation systems can be either purified protein systems, that are not made from hosts, as referenced by (Shimizu et al., 2001), or can be made from a host strain that is formed as a “lysate.” Those skilled in the art will recognize that an in vitro transcription and translation requires transcription and translation to occur, and therefore does not encompass reactions with purified enzymes.
Cell-free transcription-translation is described in
Directions on how to make the lysate component of cell-free systems, particularly from E. coli, can be found in (Sun et al., 2013), which is incorporated herein by reference in its entirety. While this procedure is adapted for E. coli cell-free systems, it can be used to produce other cell-free systems from other organisms and hosts (prokaryotic, eukaryotic, archaea, fungal, etc.) Examples, without limitation, of the production of other cell-free systems include Streptomyces spp. (Thompson, Rae, & Cundliffe, 1984), Bacillus spp. (Kelwick, Webb, MacDonald, & Freemont, 2016), and Tobacco BY2 (Buntru, Vogel, Spiegel, & Schillberg, 2014), where directions are incorporated herein by reference in its entirety. The process for producing lysates in this disclosure involves growing a host in a rich media to mid-log phase, followed by washes, lysis by French Press and/or Bead Beating Homogenization, and clarification. A lysate that has been processed as such can be referred to as a “lysate”, a “treated cell lysate”, or an “extract”.
The extract can be made from one host, multiple hosts, or mixes of multiple hosts. It is obvious to those that are skilled in the art that mixing extracts of multiple hosts, as described in U.S. Pat. No. 9,469,861 for producing carbapenems in cell-free systems and incorporated herein by reference in its entirety, may be necessary to supply cofactors that are necessary to produce different natural products.
A plurality of supplements is supplied along-side an extract to maintain gene expression. This includes necessary items for transcription and translation, such as amino acids, nucleotides, salts (Magnesium and Potassium), and buffers. A review of supplements can be found in (Chiao, Murray, & Sun, 2016), incorporated herein by reference in its entirety. This can also include optional items that assist transcription and translation, such as phage polymerases, T7 RNA polymerase, SP6 phage polymerase, cofactors, elongation factors, nanodiscs, vesicles, and antifoaming agents.
An energy recycling system is necessary to drive synthesis of mRNA and proteins by providing ATP (adenosine triphosphate) to a system and by maintaining system homoeostasis by recycling ADP (adenosine diphosphate) to ATP, by maintaining pH, and generally supporting a system for transcription and translation. A review of energy recycling systems can be found in (Chiao et al., 2016), incorporated herein by reference in its entirety. Examples, without limitation, of energy recycling systems that can be used include 3-PGA (Sun et al., 2013), PANOx (D.-M. Kim & Swartz, 2001), and Cytomim (Jewett & Swartz, 2004).
A polypeptide under 110 amino acids is supplied to the composition. A polypeptide of this size can also be referred to as a “prepeptide”, “propeptide”, “prepropeptide”, “structural peptide”, “pro-region”, “intervening region”, “precursor peptide”, or “leader peptide.” This polypeptide, typically between 20-110 residues, is characteristic of ribosomally-synthesized and post-translationally-modified peptides (RiPPs), or ribosomal natural product, as described in (Arnison et al., 2013), incorporated herein by reference in its entirety. In some embodiments, the polypeptide does not have significant tertiary structure and is therefore prone to proteolysis.
In some embodiments, a DNA is supplied that that can produce the polypeptide by utilizing transcription and translation machinery in the lysate and/or additions to the lysate. This DNA has regulatory regions, such as under the OR2-OR1-Pr promoter (Sun et al., 2014) the T7 promoter or T7-lacO promoter, along with a RBS region, such as the UTR1 from lambda phage (Sun et al., 2014), or BCD units (Mutalik et al., 2013). The DNA can be linear or plasmid. An example of a sequence is provided in SEQ ID NO.: 1.
In other embodiments, a mRNA is supplied that utilizes translational components in the lysate and/or additions to the lysate to produce the polypeptide. This mRNA can be from a purified natural source, or from a synthetically generated source, or can be generated in vitro, e.g., from an in-vitro transcription kit such as HiScribe™, MAXIscript™, MEGAscript™, mMESSAGE MACHINE™ MEGAshortscript™.
In other embodiments, the polypeptide is directly supplied. This polypeptide can be from a purified natural source, a synthetically generated source (custom peptide synthesis), or from another in vitro transcription and translation kit. In some embodiments, the polypeptide is directly supplied to introduce non-canonical or non-natural amino acids at high yield (Hong, Kwon, & Jewett, 2014). In other embodiments, the polypeptide is directly supplied to introduce non-naturally occurring polypeptides for applications in scaffolding for drug or receptor binding design, as described in (T. A. Knappe et al., 2011) and incorporated herein by reference in its entirety. Those skilled in the art will recognize that the polypeptide will need to be relatively devoid of contaminants, such as salts, to not interfere with the in vitro transcription and translation reaction.
The polypeptide under 110 amino acids is modified in the reaction by endogenous or exogenous factor to produce a product. In some embodiments, the factor is endogenous to the in vitro transcription and translation system, such as if the factor is supplied by the lysate. An exemplary example is provided for lariatin in (Inokoshi, Matsuhama, Miyake, Ikeda, & Tomoda, 2012; Iwatsuki, Uchida, & Takakusagi, 2007), incorporated herein by reference in its entirety, where the correct production of laraitin requires the presence of the R. jostii K01-B0171 strain and cannot be produced by heterologous expression in a E. coli strain, thereby implying that a factor endogenous to a lysate from R. jostii K01-B0171 would be required.
In some embodiments, the factor is exogenous to the in vitro transcription and translation system. Examples, without limitation, are ribosomal natural product pathways, such as those that produce microcin J25, klebsidin, and lactazole, as provided in examples herein, where factors necessary to modify the supplied polypeptide under 110 amino acids can be supplied as DNA, mRNA, or protein. In these cases, the factor may be part of the natural product biosynthetic machinery (e.g., (cyclizing enzyme, cleaving enzyme), or may conduct modifications on the supplied polypeptide under 110 amino acids or a variant (e.g., decorating enzyme, dehydration reactions, Michael-type additions). A review of potential factors are found in (Ortega & van der Donk, 2016), incorporated herein by reference in its entirety. In certain embodiments, the cell-free transcription and translation reactions, as described in WO2016134069A1, Niederholtmeyer et al., 2015, and Sun et al., 2014, each incorporated herein by reference in its entirety, can be utilized.
Unique to this application is the inclusion of a polypeptide of size under 110 amino acids that is then modified within the cell-free reaction. While polypeptides have been included into cell-free reactions, these polypeptides typically do not physically modify a externally supplied polypeptide. In cases were the polypeptide does modify an externally supplied polypeptide, the polypeptide is usually of larger size, such as an antibody or an established protein.
In some embodiments, the composition produces a natural product, or a ribosomal natural product, or “RiPP.” A description of the class is incorporated herein by reference in its entirety in (Arnison et al., 2013; Ortega & van der Donk, 2016).
In some embodiments, the composition produces a product that can be further modified to produce a non-naturally occurring Natural Product. This may be done through adding modifying enzymes, either directly or through DNA or mRNA that is translated/transcribed to produce the modifying enzyme, that do not naturally modify the intended product, but are promiscuous enough to modify the supplied product, as described in (“Modularity of RiPP Enzymes Enables Designed Synthesis of Decorated Peptides,” 2015), incorporated herein by reference in its entirety. This may also be done external to the cell-free reaction.
In some embodiments, non-canonical amino acids are utilized in the composition. Non-canonical amino acids can may be found naturally in the cellular-produced product, or can be artificially added to the product to produce desirable properties, such as tagging, visualization, resistance to degradation, or targeting. While implementation of non-canonical amino acids is difficult in cells, in cell-free systems implementation rates are higher due to the ability to saturate with the non-canonical amino acid. Examples, without limitation, of non-canonical amino acids, including ornithine, norleucine, homoarginine, tryptophan analogs, biphenylalanine, hydrolysine, pyrrolysine, or as described in (Blaskovich, 2016) broadly for medicinal chemistry and specifically in (Baumann et al., 2017) for natural products, are incorporated herein by reference in its entirety.
In some embodiments, the input polypeptides and/or factors are derived from environmental sequences or nucleic acids. These nucleic acids can be further derived from microbiomes, such as human gut, animal, oral, skin, vaginal, soil, ocean, rhizosome, umbilical, vaginal, conjunctival, intestinal, stomach, nasal, gastrointestinal tract, or urogenital tract. Of particular interest is the human gut microbiome, which is known to be highly overexpressed in ribosomal natural products, and the soil microbiome, from which many commercially valuable natural products (such as ‘nisin’) have been isolated. Those skilled in the art will recognize that the composition can produce the desired product using these environmental sequences and effectively emulate the activity of the host cell by doing so, thereby acting as an “artificial cell” or an alternate heterologous expression platform. In other embodiments, the input polypeptides and/or factors are derived from non-environmental (synthetic) sources. This can be to produce non-natural analogs of natural products or to speed up production of natural products (e.g., modifying flexible residues of a input lasso peptide propeptide to produce a scaffold that has the bioactivity characteristic of an antibody but the ability to enter cells, enzyme evolution to accelerate activity of limiting enzymes, enzyme evolution to allow the production of new products).
In some embodiments, the product of the reaction is a molecule that has bio-active activity. Those skilled in the art will recognize that if the input sequences are environmental, they are likely to be evolved by nature to product useful, bio-active molecules. The activity of the bio-active molecule can include antibiotic, herbicide, pesticide, insecticide, animal feed additive, signaling molecule, receptor agonist, receptor antagonist, activator, inhibitor, quorum sensing molecule, or anticancer therapeutic, toxin, or venom. In other embodiments, the bio-active molecule is derived from synthetic input sources. In these cases, many times knowledge of a known bio-activity is utilized to produce the synthetic variant, for example computationally designing a structure to bind a receptor, NMR, or crystallographiclly-defined site, and then scaffolding a polypeptide and utilize the composition and known natural product chemistry to produce a agonist/antagonist/effector.
In some embodiments, crowding agents are used in the reaction to simulate the macromolecular crowding activity in the cell and to encourage the protein-protein and protein-nucleic acids interactions necessary to drive the reaction to completion. Macromolecular crowding is an important effect in biochemical reactions, affecting, transcription, DNA replication, and protein folding. Macromolecular crowding helps to stabilize proteins in their folded state by varying excluded volume—the volume inaccessible to the proteins due to their interaction with macromolecular crowding agents. This is critical to cells; for example, E. coli cytoplasm contains 300-400 mg/mL of macromolecules. Examples, without limitation, of typical crowding agents, which are typically, e.g., above 100 Daltons, above 150 Daltons, or above 200 Daltons, include: Ficoll, polyethylene glycol, polyethylene oxide, cyclodextrin, dextran, bovine serum antigen, glucose, among others. There are assumptions that (1) crowding is not critical for some cell-free systems to function, especially those that are driven by T7 expression. This is a reasonable assumption, as the interaction of T7 RNA polymerase to the T7 operator is very strong and may not need crowding conditions to occur. Also, that (2) crowding should best emulate the cellular condition, as described in the Cytomin™ system, which uses spermidine and putrescene (cations/polyamines, not crowding agents) and particularly avoids polyethylene glycol due to negative effects. However, surprisingly, in contrast to the findings of U.S. Pat. No. 8,357,529, it has been discovered herein that: (1) crowding, while not important for the interaction of T7 RNA polymerase to the T7 operator, assists the production of natural products in cell-free lysates, and (2) that polyethylene glycol is a positive effector to crowding and crowding conditions do not need to emulate cellular conditions.
Crowding can encourage the protein-protein interactions resulting from the input polypeptide of less than 110 amino acids with either endogenous and exogenous factors. While the activity of crowding agents to impact cellular expression is well-understood, there is limited work defining the activity of crowding agents with respect to cell-free systems (e.g., (Ge, Luo, & Xu, 2011; Tan, Saurabh, Bruchez, Schwartz, & LeDuc, 2013)), and no publically available work to date (other than our example demonstration) showing activity of crowding agents in encouraging either protein-protein interactions or interactions for producing ribosomal natural products.
In some embodiments, the reaction comprises more than 0.1% (w/v) of crowding agent. The crowding agent used may be from a single source, or may be a mix of different sources. The crowding agent may be from varied sizes. In some embodiments, the crowding agents used limit polyethylene glycol and its derivate, polyethylene oxide or polyoxyethylene, to less than 0.2% (w/v). While polyethylene glycol and its derivatives are similar as other crowding agents in their biochemical and biophysical effect, polyethylene glycol and its derivatives can interfere with analytical methods of downstream detection of the resulting product, which can be critical for diagnosing and/or reading out the resulting reaction. To minimize this effect, polyethylene glycol and its derivatives can be limited and substituted for other crowding agents. In addition, the size used of polyethylene glycol and its derivatives can be varied.
Protection of Peptides of Less than 110 Amino Acids in Length by Utilizing Non-Proteolytically Active Lysates
In some embodiments, peptides of less than 110 amino acids in length are easily degraded in either lysates, or in cell-free systems. Ribosomal natural products are generally produced by a propeptide that is later modified by other coding sequences to produce a final product. The propeptide, therefore, is one of the rate-limiting steps of producing a ribosomal natural product. In cells, there is proteolytic activity that can degrade unfolded, misfolded, or peptides that have no secondary or tertiary structure. However, many ribosomal natural product wildtype propeptides do not have secondary or tertiary structure, leaving them open to proteolytic degradation. In our examples, we demonstrate that in cell-free systems that have active proteolysis, the propeptide degrades away without protection. This limits the yields achievable of ribosomal natural product production in cell-free systems if no protective strategies are enforced. The protective strategies are unexpected as many of them would not be able to be applied directly to cells.
In some embodiments, to protect peptides of less than 110 amino acids in length, lysates or cell-free systems are made that are highly productive at transcription and translation but also able to avoid proteolytic activity. We demonstrate in examples that proteolytic degradation is not unique to E. coli cell-free systems, but can selectively occur in different lysates from different backgrounds. While some systems, such as E. coli cell-free systems, are enriched in proteolytic components, other systems are less enriched.
In some embodiments, the lysates and/or cell-free systems produced are made from Rhodococcus jostii, Vibrio natriegens, Clostridium acetobutylicum, HeLa whole cell extract.
In some embodiments, the lysates and/or cell-free systems produced are made from organisms that are known to be devoid of proteolytic ability due to known or predicted properties of cellular biochemistry.
In some embodiments, the lysates and/or cell-free systems that are less proteolytically active are experimentally determined. In this method, a sample test, unstructured (e.g., having no secondary or higher structures) peptide under 110 amino acids in length is provided, such as MTKRTYETPVLVSAGSFARRTGSGSPKAARDPFGRRWLP (SEQ ID NO.: 21). This unstructured peptide can be produced synthetically, or produced in a system that is devoid of proteases, such as the PURExpress™ system, where the peptide is expressed with a saturating amount of T7-encoding DNA, such as that in SEQ ID NO.: 2. The unstructured peptide and/or the solution in which the peptide is made is then combined with ideally 10%, or anywhere from 1% to 100%, of a solution of either lysates or cell-free systems derived from different hosts. This solution is incubated for different time periods at ideally 29 C, or anywhere from 20 C to 80 C, for time periods from 0 min to 60 min. The amount of peptide left at the longest time periods is compared to the amount of peptide at the shortest time periods by methods such SDS-PAGE or by selective labeling. The ratio of these numbers determine proteolytic ability. Those skilled in the art will recognize that in parallel, cell-free systems formed by those that have low proteolytic ability need to be evaluated for active transcription-translation ability.
In some embodiments, it is necessary to produce a Vibrio natriegens cell-free system that is proteolytically inactive. For eVN1, Vibrio natrigens cell-free system is produced using the methods of Sun et al. (2013), but with select modifications to the protocol outlined in
In some embodiments, modifications can be made to increase the protein synthesis capacity of the Vibrio natriegens extract expression chassis to increase transcription and translation ability. Modifications to the protocol included replacing LB with 3% NaCl with Brain Heart Infusion Broth with 3% NaCl, increasing the concentration of K-glutamate in the wash buffer to 200 mM, lysing via French press rather than bead beating, conducting a 60 minute runoff incubation of the lysate at 37° C., increasing the energy substrate and amino acid concentrations 2.5×, and increasing the volume of the extract in the final reaction to 50% of the total volume. These changes are summarized in
In some embodiments, the lysates and/or cell-free systems used can be depleted of proteolytic components. The depletion can be either before or after the production the cell-free system, at either the host stage, lysate preparation stage, or post-lysate preparation stage.
In some embodiments, the depletion of proteolytic components is done by adding an effector, such as protease inhibitors. Protease inhibitors inhibit the function of proteases (enzymes that aid the breakdown of proteins). Classes of protease inhibitors include aspartic protease inhibitors, cysteine protease inhibitors, metalloprotease inhibitors, serine protease inhibitors threonine protease inhibitors, trypsin inhibitors, suicide inhibitor, transition state inhibitor, serpins, chelating agents. The inhibitors can be specific, or can be general. The inhibitors can be individual chemicals or provided in cocktail form. Examples, without limitation, of protease inhibitors include SIGMAFAST™, MS-SAFE™ cOmplete™, Halt Protease™, EDTA, pepstatin A, PMSF, E-64, bestatin, aprotinin, AEB SF, Sodium phyophosphate, beta-glycerophophate, sodium orthovandate, sodium fluoride. The method of testing protease inhibitors in cell-free systems involves providing the protease inhibitor in a cell-free system and testing the transcription-translation activity of the cell-free system through the expression of a constitutively active GFP-producing DNA (e.g., Addgene 40019 or 21p) in the presence and in the absence of the protease inhibitor, as demonstrated in
In some embodiments, the depletion of proteolytic components is done by adding to the solution a dummy or competing peptide that is unstructures (e.g., without any secondary or higher structures). By including dummy peptides, any present protease will competitively degrade the dummy peptide as well as the input peptide. Due to the larger amount of dummy peptides present, the input peptide can be protected from degradation. In addition, increased turnovers of protease can result in the inactivity of the protease. This has been shown for AAA+ protein degradation enzymes (e.g., ClpXP) in previous work (Sun, Kim, Singhal, & Murray, 2015) in cell-free systems. The dummy peptides will need to not contain signaling regions or other regions that can bind, recruit, activate, or inhibit proteins or molecules essential for the cell-free system to function. Any such regions will cause the dummy peptides to interfere with transcription, translation, or essential processes. These properties can be found by comparing dummy peptides to data in publically available databases (e.g., NCBI, EMBL). The ideal source of dummy peptides is generated from random amino acid sequences. The dummy peptide can be generated by peptide synthesis or by production in another cell or cell-free reaction. In a sample reaction, random dummy peptide SFAVHGIWET YLRDQMNKCP (SEQ ID NO.: 25) (or as otherwise generated by RandSeq) is synthesized, purified of contaminating components (such as salts), and resuspended in a neutral protein buffer such as 50 mM Tris-Cl, 100 mM NaCl, 1 mM DTT, 2% DMSO, pH 7.5. The dummy peptide is added to a cell-free reaction at 0.1 mg/ml, 0.5 mg/ml, 1 mg/ml, 5 mg/ml, 10 mg/ml, and the transcription-translation of the desired peptide of less than 110 amino acids is monitored. A comparison is done of adding the dummy peptide before the reaction and incubating for 60 minutes, or adding the dummy peptide concurrently with transcription-translation of the desired peptide. In addition, the toxicity of the dummy peptides is monitored by the expression of a positive control plasmid (e.g., 21p, 40019). Parallel methods for tracking ClpXP degradation of fluorescent proteins are described in (Sun et al., 2015), incorporated herein by reference in its entirety.
In some embodiments, the depletion of proteolytic components is done by genetically engineering out proteases out of a host before or during the lysate production process to effectively produce a cell-free system deprived of protease activity. However, care must be taken to not inhibit essential growth and regulatory processes of the host species while the host species is still growing. To remove these agents, host cells will be first genetically engineered using a CRISPR-Cas9, TALEN, MAGE, or other genetically engineering approach to remove proteases that do not effect growth and regulatory processes. This can be tested by conducting a cycle of genetic engineering (e.g., by inserting a nonsense codon into a putative protease) and then conducting an OD growth curve using growth conditions for producing lysate in rich media and comparing to control growth.
In some embodiments, depletion of proteolytic components is done by genetically engineering proteases in a host to degrade during the production of the lysate. For those proteases that affect growth and regulatory processes, host cells will be genetically modified to introduce tags that do not affect the protein but provide a residue that can be targeted during or after lysis creation. This can include non-destructive tags on the N terminus, C terminus, or the middle of the protein in between domains, including, but not limited to, polyhistidine (His6), maltose binding protein (MBP), calmodulin binding peptide (CBP), DYKDDDDK (SEQ ID NO.: 26) peptide (FLAG), glutathione S-transferase (GST), hemagglutin (HA), histidine-biotin-histidine (HBH), polypeptide tag from the cMyc gene (Myc), S-tag derived from pancreatic ribonuclease A, small ubiquitin-related modifier (SUMO), tandem affinity purification (TAP), thioredoxin (TRX), and V5 from a small epitope found on the P and V proteins of paramyxovirus of SV5. With non-destructive tags, after production of the lysate according to methods in Sun et al. (2013) JovE the tagged proteases can be removed from the system by column filtration of the processed lysate, antibody pull-down of the endonuclease and exonuclease, or other methods to deprive the system of the tagged molecule. This can also include destructive tags on the 5′, 3′, or the middle of the protein in between domains, including, but not limited to, ssrA, cln8, cln2, hsl1, UmuD, MerB. With destructive tags, the proteases must be protected from degradation during the growth of the cell, either by spatial localization (e.g., periplasm vs cytoplasm) or select control of the degradation enzyme that recognizes the degradation tag. Then, during lysis either spatial or select control of the degrading enzyme is released, and the targeted proteases degrade with an incubation step. The protease in question can also be reengineered such that the function is preserved, but a specific site is introduced such that it can be degraded after extract production. As an illustrative example, a target enzyme can be engineered to include a degradation tag exogenous to the organism that does not inhibit function and is not recognized by the host organism. However, upon creation of the lysate an enzyme that recognizes the degradation tag can be added, thereby removing the endonuclease and exonuclease in question. Methods that are described in U.S. Pat. Nos. 8,916,358, 8,956,833 are incorporated herein by reference in its entirety.
Protection of Peptides of Less than 110 Amino Acids in Length by Modifying the Peptide to Resist Proteolysis
While cell-free systems with minimal proteolytic ability can be used to protect peptides of less than 110 amino acids, there are many cases where the protection may not be sufficient. This includes (1) if the cell-free system is not as productive for catalyzing the reaction vs. a standard E. coli cell-free system (e.g., PURExpress yields are 7.5-20× lower than E. coli cell-free systems); (2) if the ribosomal natural product reaction cannot be catalyzed in the protease-limited cell-free system, due to lack of necessary co-factors, chaperones, or other additives to catalyze the reaction; (3) if the proteolytic ability of the cell-free reaction is still present; among others. One can also maintain the propeptide by modifying the peptide to resist proteolysis. In examples, we demonstrate the tagging of peptides less than 110 amino acids in length to prevent degradation in cell-free systems.
In some embodiments, the expressed protein of choice is fused to a partner protein tag, or a “stabilizing domain,” to prevent degradation, promote solubility, and aid purification. Many biologically important peptides are intrinsically disordered proteins and are thus vulnerable to proteolysis/degradation. These disordered proteins can be fused to stabilizing domains that are highly structured and soluble proteins to aid in the solubility and protection of the fusion partner. Further, some tags provide a “handle” for single-step purification. Examples of stabilizing domains that can be added to the 5′ or 3′ site, without limitation, include polyhistidine (His6), maltose binding protein (MBP), calmodulin binding peptide (CBP), DYKDDDDK (SEQ ID NO.: 26) peptide (FLAG), glutathione S-transferase (GST), hemagglutin (HA), histidine-biotin-histidine (HBH), polypeptide tag from the cMyc gene (Myc), S-tag derived from pancreatic ribonuclease A, small ubiquitin-related modifier (SUMO), tandem affinity purification (TAP), thioredoxin (TRX), and V5 from a small epitope found on the P and V proteins of paramyxovirus of SV5, N-utilizing substance A (NusA), green fluorescent protein (GFP), and ubiquitin. Novel protein tags can be chosen from extremely structured and stable proteins, or protein domains, using structure prediction analysis programs, such as PONDR (Predictor Of Natural Disordered Regions). The addition of a tag may affect the activity of the expressed protein or the ability for downstream enzymes to use the expressed peptide as a substrate. Therefore, the tag must be experimentally tested to confirm downstream utility or activity, and/or modeled to determine if downstream utility or activity is affected.
In some embodiments, the tag may be fused to the partner protein with a linker. The linker can be selected from one or more of a cleavable linker, a non-cleavable linker, a peptide linker, a flexible linker, a rigid linker, a helical linker, or a non-helical linker. In certain embodiments, the linker is a neutral linker that allows the stabilizing domain to be less likely to inhibit downstream utility or activity. The linker can be human serum albumin or an Fc domain, or a sequence comprising glycine and serine. Exemplary linkers can include, without limitation, Gly-Gly-Gly-Gly-Ser-Ser (SEQ ID NO.: 22), Gly-Gly-Ser-Gly (SEQ ID NO.: 23), Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly (SEQ ID NO.: 24), or any other combinations of Gly and Ser that is placed in between the fused domain and the protein or peptide coding sequence.
In some embodiments, the tag may include protease sites that allow the fused domain to be cleaved away from the protein or peptide. This may be necessary if the addition of the fused domain alters the conformation of the protein, interferes with the downstream applications of the proteins, or prevents the proteins from being crystallized, among others. Protease sites include, without limitation, Tobacco Etch Virus (TEV) sites, PreScission Protease sites, Thrombin Protease sites, Factor Xa protease sites, Enterokinase protease sites, among other sites. Those skilled in the art will be able to express a peptide using the tag, optionally purify out the peptide using an affinity tag or other method (e.g., size exclusion, FPLC), and then incubate the resulting solution with the protease tag followed by a size exclusion or other isolation method and an optional concentration method to have purified peptide without tag. This purified peptide can then be added in downstream cell-free reactions at high concentration.
In some embodiments, the tag may include other sites that allow the final fused protein to be detected by small molecule interactions, antibodies, affinity purification, or other reagents. These include FLASH/REASH sites, MBP, NusA, GST, His6, CBP, FLAG, HA, HBH, Myc, S-tag, SUMO, TAP, TRX, V5.
In some embodiments, the tag may be a combination of fusion proteins, fusion domains, natural linkers, proteases sites, and regions assisting detection by small-molecule reaction. This combination may occur on the 5′ end, 3′ end, or in the middle of the peptide.
Those skilled in the art will note that the tag serves to provide the input peptide less than 110 amino acids structure, thereby resisting proteolytic activity against the peptide. The addition of this tag may also assist the transcription and translation of the peptide, but any assistance is external to the main purpose of the tag, to resist proteolytic activity.
In some embodiments, the input peptide less than 110 amino acids can be modified to resist proteolysis, either through modification of side-chains on the amino acid, implementation of non-specific amino acids, or other modifications as described in Blaskovich, 2016 broadly for medicinal chemistry and specifically in Baumann et al., 2017 for natural products or D. Knappe, Henklein, Hoffmann, & Hilpert, 2010 for antimicrobial peptides, are incorporated herein by reference in its entirety. This modification can be installed synthetically, biochemically, enzymatically, or through a combination thereof. The resulting input peptide is resistant to proteolysis but can be modified by enzymes and other factors in the cell-free reaction composition, either endogenous or exogenously provided, to produce a final product.
Microcin J25 is a model peptide in the lasso peptide family, first discovered in 1992 from a fecal-isolated E. coli strain. Like most lasso peptides, its synthesis involves 4 genes: mcjA (peptide precursor), mcjB (cysteine protease), mcjC (lactam synthase), and mcjD (ABC transporter) and only mcjA, mcjB, and mcjC are necessary for its biosynthesis in E. coli. Microcin J25's mechanism of action is two-fold, targeting the E. coli RNA polymerase and interfering with membrane stability. It has activity against E. coli (MIC 0.02 μg/mL), Shigella flexneri, and Salmonella enteriditis. The peptide demonstrated 3-fold decrease in Salmonella infection in mouse models, without inducing hemolytic activity.
Microcin J25 is representative of lasso peptide family (Hegemann, Zimmermann, Xie, & Marahiel, 2015). This family is relatively new, first discovered in 1991. Lassomycin analogs are phylogenetically distributed, ranging from gram-positive Streptomyces and Rhodococcus to gram-negative E. coli and thermophilic Thermobaculum. From 1992-2007, lasso peptides were mainly isolated from functional compound-driven screens (Hegemann et al., 2015) and this led to the identification of candidate therapeutics such as anantin (atrial naturetic factor antagonist), microcin J25 (gram-negative antibiotic), and siamycin (a HIV inhibitor). Since 2008, genome mining against a lasso peptide motif has led to the discovery of additional peptides yet to be characterized. Recent advances in the field include identifying critical regions of the peptide (Pan & Link, 2011), scaffolding peptide epitopes (T. A. Knappe et al., 2011), re-engineering the peptide for stronger antimicrobial activity (Pan, Cheung, & Link, 2010) and fusion-protein stability (Zong, Maksimov, & Link, 2015), and the identification of lassomycin (Gavrish et al., 2014). Since 2017, 1,300 (35×) more lasso peptides were identified from two new independent genome mining studies of which the vast majority have not been characterized. Total chemical synthesis has been unsuccessful at making functional protein (Lear et al., 2016) and heterologous expression is difficult. The actual number is likely larger than the reported 1,300: the Tietz et al. (2017) dataset primarily consists of lasso peptide clusters from Actinobacteria, while the Skinnider et al. (2016) dataset primarily consists of clusters from Proteobacteria.
The small 4.8 kb gene cluster size of microcin J25 is extremely conducive to testing in cell-free platforms. The sequences do not carry any risk factors for cell-free expression—none require known complex co-factors and none are membrane-bound. The sequence is well-defined and expressible in E. coli cells carrying mcjD. Combination of purified mcjA, mcjB, and mcjC produces functional microcin J25 in vitro (Duquesne et al., 2007). Expressing in E. coli cell-free avoids microcin J25's toxicity by inhibition of E. coli RNA polymerase (a T7 polymerase can be introduced to native E. coli RNA polymerase) and by membrane-based toxicity (cell-free does not require membranes).
Produced microcin J25 can be rapidly screened against known sensitive wildtype E. coli and non-pathogenic Pseudomonas spp., as well as known insensitive gram-positive Rhodococcus spp. and Streptomyces spp. 10 μg is sufficient for conducting 24 MIC assays at 200 μL scale. With cell-free expression yields of 0.75 mg/mL and ability to run 10 mL reactions, theoretical yields are ˜750× above required.
We demonstrate that microcin J25 and produced is active in our cell-free systems. This is a surprising outcome, as lasso peptides, of which microcin J25 is an example, have not been able to be synthesized using synthetic chemistry techniques, as shown by reference to (Lear et al., 2016). SEQ ID NO.: 1, SEQ ID NO.: 3, and SEQ ID NO.: 4 show the promoter, utr, coding sequence, and terminator sequences of the mcjA (726), mcjB (727), and mcJC (728) expressed under sigma70 promoter, respectively in a E. coli cell-free system. It is understood that these can be tested as linear DNA as written, or can be tested as plasmids when cloned on a backbone (e.g., colE1, ampR). A control provided is SEQ ID NO.: 5, caulA (729).
When expressing combinations of 726, 727, 728, 729, and the plasmid pBEST-OR2-OR1-Pr-UTR1-deGFP-T500 (40019, Addgene), it can be seen in
It is seen in
Another way to visualize the killing effect of microcin J25 produced in cell-free systems is through a high-throughput experiment shown in
We were able to expand our functional assay as a screen for lasso peptides that are able to inhibit native RNA polymerase. We incorporated a panel of known lassos including those capable of inhibiting RNA polymerase and not, as well as a few predicted lasso clusters, as shown in
In the first round, the known lasso peptides microcinJ25, capistruin, burhizin and caulosegnin we obtained plasmids with the native coding sequences and cloned them into our expression system as parts. The number of individual coding sequences for each lasso cluster vary, but generally have a core of A, B (or B1) C and E (or B2). E is often fused to another gene. Our expression system incorporates these coding sequences individually with a sigma70 promoter, UTR and terminator. For the remaining known lassos that we screened; klebsidin, lariatin, and acinetodin, as well as a few predicted lassos, we synthesized DNA coding sequences to recapitulate the native peptides and assembled these synthetic coding sequences into our system. A GFP construct (40019 in linear) was assembled in the same way. Linear DNAs were generated either by PCR amplification from plasmid or the DNA assemblies themselves. We used 3.5 μM GamS to prevent template degradation in E. coli lysate. Reactions were run at 10 μl scale in a 384-well format. The reaction is done in E. coli extract “eZS6” at 25% lysate, 75% buffer concentration, prepared as listed in (Sun et al., 2013). Timecourses of GFP intensity were taken, and 12 hr endpoints were used to generate the heatmap. GFP intensity of lasso-cluster-containing reaction was normalized to their paired negative controls.
We expressed our GFP construct in the same reactions as our lasso constructs. GFP (4009 in linear) was expressed at 4 nM and the lasso cluster genes at 0.6 nM. For the negative controls we substituted the lasso A genes that code for the propetide substrates used to make functional lassos with an A gene from the caulosegnin cluster, which has been shown not to have RNA polymerase inhibitory activity. By comparing the GFP intensity of negative controls to reactions expressing the complete cluster, we were able to screen for RNA polymerase inhibition and our screen indicated that our klebsidin constructs assembled functional lasso. The specific sequences of the klebsidin constructs are 938 sigma70-klebsidinB (SEQ ID NO.: 6), 939 sigma70-klebsidinC (SEQ ID NO.: 7), 940 sigma70-klebsidinA (SEQ ID NO.: 8), and of the actinodein constructs are 908 sigma70-acinetodinC (SEQ ID NO.: 9), 909 acinetodinB (SEQ ID NO.: 10), 910 acinetoinA (SEQ ID NO.: 11).
From our functional screens, we demonstrate that microcin J25 and klebsidin are produced and active in our cell-free systems. Again, this is a surprising result given that these are characteristic lasso peptides that cannot be synthesized using synthetic chemistry techniques. We go on to show one can also detect lasso peptide production in cell-free systems even if there is no functional screen.
Shown in
Shown in
Lactazoles are a novel family of thiopeptides, which are representative ribosomal natural products, isolated in 2014 from the genome mining of Streptomyces lactacystinaeus OM-6519. The resulting thiopeptides produced are macrocylic rings of 11 amino acids, with up to 56% post-translationally modified serine/threonine/cysteine residues. The lactazole biosynthetic gene cluster is a demonstration of the cell-free platform disclosed herein as a formerly cryptic cluster with minimal published data. The gene cluster is also short, spanning 9.8 Kb in size and composed of six genes.
Each coding sequence (lazA, lazB, lazC, lazD, lazE, lazE) has been synthesized and assembled onto sigma70 constitutive promoters using the methods outlined in (Sun et al., 2014). Set concentrations of these coding sequences, and tagged and untagged variants, and additives such as DMSO will be varied, each in a range of 1 nM to 16 nM, and expressed in cell-free systems as a reaction in a size range of 10 uL to 1 mL. The reaction will have low amounts of amounts of polyethylene glycol (0.1%-0.2% w/v) and will have another crowding agent (4% Ficoll 400). We will detect expression using both a qTOF LC-MS as well as MALDI and search for the three possible lactazole analogs using ion extraction of m/z 1401.3975 [M+H]+ for lactazole A, m/z 1,529.4586 [M+H]+ for lactazole B and m/z 1,176.2830 [M+H]+ for lactazole C30.
Those skilled in the art will recognize that each coding sequence will need to be properly expressed in a cell-free system for the reaction to take place. To get each coding sequence to express, tags or additives may need to be added to the systems to ensure proper transcription and translation. In one example, we have expressed different variants of lazA, the propetide, under either a untagged version but with a T7 promoter replacing a sigma70 promoter (890/992, SEQ ID NO.: 12) or a tagged version with 5′ CAT (1071, SEQ ID NO.: 13). The results of expressing these constructs in PURExpress™, a system by NEB, that does not have proteolysis, are presented in
LazC (773, SEQ ID NO.: 14) is experimentally found to express best at with 4% DMSO addition and no tag. LazD is experimentally found to express best as cat-lazD (897, SEQ ID NO.: 15), after testing multiple sequence variants of lazD (no tag 891, BCD2 tag from (Mutalik et al., 2013) 896, CAT tag 897, CAT tag plus ‘GGSG’ (SEQ ID NO.: 23) protein linker 898, CAT tag plus FLASH tag plus ‘GGSG’ (SEQ ID NO.: 23) protein linker 899, CAT tag plus his6 tag plus ‘GGSG’ (SEQ ID NO.: 23) protein linker 900, FLASH tag plus ‘GGSG’ (SEQ ID NO.: 23) protein linker 901) that can be assembled by those skilled in the art. This shows the need to test different variants of genes with different tags and different conditions in order to experimentally determine conditions that cause expression with little loss of activity. Expression of lazB (812, SEQ ID NO.: 16), of lazE (892, SEQ ID NO.: 17) and lazF (893, SEQ ID NO.: 18) is detectable without modification in an E. coli cell-free system.
Expression of the variants will result in E. coli cell-free reactions that will have detectable amounts of lactazole and/or lactazole intermediates. If detectable amounts are not made, but each gene is expressed and can be verified as active, the problem of expression may be due to the lack of cofactors in E. coli cell-free systems that are required for the production and activity of lactazole. If detectable amounts are not made, we will first utilize alternate cell-free systems, broadly made by gram-positive organisms, more specially actinomycetes, more specifically Streptomyces spp., more specifically Streptomyces lactacystinaeus OM-6519, in an attempt to supply the missing cofactors. This will involve utilizing cell-free systems that are non E. coli but are adept at transcription and translation, an example which is given later for Vibrio natrigens cell-free systems. If alternate cell-free systems fail, we will then utilize mixing 1%-50% of lysates of gram-positive organisms with our E. coli or other cell-free systems, more specially actinomycetes, more specifically Streptomyces spp., more specifically Streptomyces lactacystinaeus OM-6519, in an attempt to supply the missing cofactors. We will also purify specific cofactors that are known to affect lactazole production and add to the open cell-free systems, as isolated in (Hayashi et al., 2014), hereby incorporated herein by reference in its entirety.
We demonstrate that novel ribosomal natural products can be produced in cell-free systems. The workflow for doing this is outlined in
In a sample bioinformatics throughput, the current largest collection of automatically mined gene clusters is the “Atlas of Biosynthetic Gene Clusters”, a component of the “Integrated Microbial Genomes” Platform of the Joint Genome Institute (JGI IMG-ABC). IMG-ABC has annotations of 960,000 putative gene clusters from JGI's genome and metagenome datasets that are sorted by phylum and gene count. 217,395 clusters are from the phyla Actinobacteria. Of these, 33,364 clusters have the probability score of 1.0 and 18,202 are 1-20 genes in length. Only 311 of these were verified experimentally. Its gene cluster family network, comprising 11,422 gene clusters grouped into the main natural product gene cluster family of NRPS, type I and type II PKS, NISs, RiPPs, and TOMMs was validated in hundreds of strains by correlating confident mass spectrometric detection of known small molecules with the presence or absence of their established biosynthetic gene clusters.
For expressing natural products, we use lasso peptides as an exemplary example. From databases such as JGI-IGI, RODEO (Tietz et al., 2017), NCBI, PRISM, EMBL, ClusterFinder, antiSMASH, one can identify predictive lasso peptides by propetide sequence and other associated genes. We identified a set of 22 predictive lasso peptides using this approach, and using the workflow outlined for
We establish a method for determining what cell-free systems degrade propeptides by generalizing a screening approach, the results of which are shown in
With the Vibrio natrigens cell-free systems produced, we test the systems using our PURExpress incubation assay. A PURExpress™ system is set up according to manufactuer's instructions with a saturating amount of T7-ARVW01000001.1A_est DNA (981). 2 μL of the PURExpress reaction is combined with 2 μL reaction buffer, 0.4 μL V. natriegens or E. coli extract, and 2.27 μL water to roughly simulate reaction proportions. The resulting mixture is incubated at 29° C. for 1 hour and loaded onto a protein gel to visualize degradation of the peptide band. E. coli extract in which the peptide is rapidly degraded or absence of extract served as controls. As seen in
We demonstrate that by utilizing the produced Vibrio natrigens cell-free systems we are able to show increased processed pro-peptide present for both mccj25 and klebsidin in
We show that the propeptide can be tagged to prevent degradation in lysates or cell-free systems. In an example, mcjA (726) is known to degrade when expressed in E. coli cell-free systems. To stabilize mcjA, the peptide can be tagged either on the N terminus or the C terminus as a fusion protein that provides a stable domain that prevents proteolysis of mcjA. A linker and/or targeting region can be added to remove the tag. In an example, we tag mcjA with a maltose binding protein (MBP), generating a construct 1065 (SEQ ID NO.: 19) and compare the expression of 1065 to a wildtype MBP, 1066 (SEQ ID NO.: 20). In a SDS-PAGE gel expressing both constructs in
We would then test the ability of enzymes mcjB and mcjC to process the product of MBP-mcjA (1065), therefore producing the final microcin J25 lasso peptide by either detection on LC/MS QTOF or by activity assay. If mcjB and mcjC are not able to process MBP-mcjA, we would then switch the tag with other tag types (e.g., SUMO, GFP) and/or add neutral linkers to avoid interference with mcjB. We note that for the lasso peptide class, the “B” enzyme typically acts on the N terminus of the lasso peptide, thereby allowing tags to not remain on the final lasso peptide product. However, the propeptide can also be tagged on the C terminus, in which case the tag would remain on the final product (and may impede activity).
In another embodiment, the propeptide can be physically modified to prevent degradation. In particular, those in the art in lasso peptides recognize that while there are restrictions known on the donor residues and the acceptor residues for lasso peptides, other residues are open to modification. Therefore, on an open residue non-canonical amino acids can be implemented into the propeptide to protect it from degradation. For example, for capistruin, it is known that while T27 and G29 of the propeptide are critical for activity, as described (T. A. Knappe, Linne, Robbel, & Marahiel, 2009)11 and incorporated herein by reference in its entirety, other residues can be modified, e.g., with non-canonical amino acids, to prevent proteolysis. We would first determine if residues are critical or not critical for propeptide processing by downstream enzymes. Then, through synthetic peptide synthesis variants to reduce degradation can be generated and tested in the cell-free system for catalyzing downstream reactions.
Crowding agents have been shown to be important in cells to assist protein-nucleic acid interactions and protein-protein interactions. To assist in protein-nucleic acid interactions and protein-protein interactions for catalyzing ribosomal natural products, one can supplement cell-free systems, that are not as crowded as cells, with crowding agents such as Ficoll, polyethylene glycol, polyethylene oxide, cyclodextrin, dextran, bovine serum antigen, glucose, among others. We show that in
The present disclosure provides among other things cell-free systems and use thereof. While specific embodiments of the subject disclosure have been discussed, the above specification is illustrative and not restrictive. Many variations of the disclosure will become apparent to those skilled in the art upon review of this specification. The full scope of the disclosure should be determined by reference to the claims, along with their full scope of equivalents, and the specification, along with such variations.
All publications, patents and sequence database entries mentioned herein are hereby incorporated by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference.
This application claims priority to and the benefit of U.S. Provisional Application Nos. 62/467,548 filed Mar. 6, 2017, 62/482,856 filed Apr. 7, 2017 and 62/620,310 filed Jan. 22, 2018, the entire disclosure of all of which is hereby incorporated by reference.
This invention was made with government support under contract number W911NF17C0008 awarded by the U.S. Defense Advanced Research Projects Agency (DARPA), and grant number 1R43AT00952201 awarded by the U.S. National Institutes of Health (NIH). The government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 62620310 | Jan 2018 | US | |
| 62482856 | Apr 2017 | US | |
| 62467548 | Mar 2017 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 16499233 | Sep 2019 | US |
| Child | 18311555 | US |
