WO2007089382A2 - Compositions de nano-usines et procédés de fabrication et d'utilisation des nano-usines - Google Patents

Compositions de nano-usines et procédés de fabrication et d'utilisation des nano-usines Download PDF

Info

Publication number
WO2007089382A2
WO2007089382A2 PCT/US2007/000130 US2007000130W WO2007089382A2 WO 2007089382 A2 WO2007089382 A2 WO 2007089382A2 US 2007000130 W US2007000130 W US 2007000130W WO 2007089382 A2 WO2007089382 A2 WO 2007089382A2
Authority
WO
WIPO (PCT)
Prior art keywords
enzymes
pathway
pharmacophores
different
diverse population
Prior art date
Application number
PCT/US2007/000130
Other languages
English (en)
Other versions
WO2007089382A3 (fr
Inventor
Glen A. Evans
Malcolm Finlayson
Original Assignee
Pharmagic, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Pharmagic, Inc. filed Critical Pharmagic, Inc.
Publication of WO2007089382A2 publication Critical patent/WO2007089382A2/fr
Publication of WO2007089382A3 publication Critical patent/WO2007089382A3/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1086—Preparation or screening of expression libraries, e.g. reporter assays
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1079—Screening libraries by altering the phenotype or phenotypic trait of the host
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1093—General methods of preparing gene libraries, not provided for in other subgroups

Definitions

  • Polypeptides are highly complex molecules formed by direct genomic DNA coding of molecular weights 14,000 to 100,000 kD. Polypeptides are composed of structural motifs or folds that are decorated with functional groups to form different activities or functions. Polypeptide folds are highly conserved throughout evolution and only about to 1000 to 1500 basic folds appear to occur in nature.
  • Small organic molecule therapeutics generally fall into two classes: (1) simple compound therapeutics produced by traditional organic synthesis, and (2) complex small molecules best exemplified by natural products that are produced by fermentation or extraction from biological sources such as plants or microorganisms.
  • the basic structural motif termed a pharmacophore herein, corresponds to the product of a pathway composed of enzymes that produce it biologically. The diversification of a pharmacophore is performed with single enzymes that tailor the pharmacophore's structure. All pharmaceuticals developed appear to be based on no more than 200 such basics structures or pharmacophores.
  • compositions and methods of the invention overcome such inefficiencies and the limited diversity that can be achieved in pharmacophore structure.
  • the compositions and methods of the invention also allow for the identification and development of entirely new pharmacophores having useful activities. Unlike polypeptide folds whose number is limited by basic biology and evolution, there is essentially no limit to the number of new pharmacophores that can be produced as drug precursors. Therefore, the invention satisfies the additional need for producing a wide variety of novel pharmaceutical structures that can be used directly or modified for use as therapeutics.
  • the invention provides a diverse population of pharmacophores having a plurality of non-naturally occurring different structural motifs produced from the biosynthesis of unique combinations of enzymes within one or more metabolic pathways.
  • the plurality of non-naturally occurring different structural motifs can further include a plurality of different functionalizations or other decorative modifications.
  • a diverse population of nanofactories that include a diverse population of pharmacophores wherein each nanofactory within the diverse population expresses at least one different non-naturally occurring structural motifs produced from the biosynthesis of unique combinations of enzymes within one or more metabolic pathways.
  • a method of producing an isolated pharmacophore also is provided.
  • the method includes: (a) inserting into a vector a plurality of genes encoding enzymes from one or more metabolic pathways operationally linked to expression elements; (b) introducing the vector into a host cell; (c) culturing the host cell under conditions sufficient for expression of the encoding enzymes from the metabolic pathway, and (d) isolating a compound having a non-naturally occurring structural motif biosynthetically produced from the encoding enzymes from the metabolic pathway.
  • Figure 1 shows structures for baicalein and some exemplary derivatives.
  • Figure 2 shows a schematic diagram for flavenoid biosynthesis.
  • Figure 3 shows a listing of the enzymes involved in the five branches for flavenoid biosynthesis shown in Figure 2.
  • Figure 4A shows a schematic diagram for the enzymes, products and reactants involved in flavenoid biosynthesis for the flavenoid pathways dipected in Figure 2.
  • Figure 4B shows a pathway in the chalone flavenoid biosynthetic schema depicted in Figure 4A.
  • Figure 4C shows a flavenoid biosynthetic pathway from chalcone to catechin depicted in Figure 4A.
  • Figure 4D shows a flavenoid pathway for naringenin biosynthesis depicted in Figure 4A and
  • Figure 4E shows the luteolin biosynthetic pathway depicted in Figure 4A.
  • Figure 5A shows the amino acid sequence for enzyme EC 5.5.7.9 chalone isomerase from Arabadopsis.
  • Figure 5B shows significant sequence alignments for chalone isomerase as an example of evolutionary conservation among a variety of different species.
  • Figure 6A shows a schematic diagram for an exemplary lambdoid nanofactory vector and representative junctions for synthetic or enzymatic assembly such as by polymerase chain reaction (PCR).
  • Figure 6B shows a schematic diagram for the genes encoding metabolic pathway enzymes introduced into a nanofactory of the invention.
  • Figure 7A shows exemplary primer sequences for synthesis and assembly of the vector replacement modules containing genes encoding metabolic pathway enzymes for a representative nanofactory.
  • Figures 7B and 7C show a schematic diagram exemplifying the organization and order of the encoding genes as they can be placed in a vector for nanofactory expression.
  • Figure 8 shows the amino acid sequence for phenylalanine ammonia-lyase from A. thaliana.
  • Figure 9 shows the amino acid sequence for trans-cinnamate 4- monooxhygenase from P. crispum.
  • Figure 10 shows the amino acid sequence for 4-coumarate: CoA ligase from S. baicalensis.
  • Figure 11 shows the amino acid sequence for naringenin-chalcone synthase from P. sativum.
  • Figure 12 shows the amino acid sequence for chalcone isomerase from P. x hybrida.
  • Figure 13 shows the amino acid sequence for flavone synthase I from P. crispum.
  • Figure 14 shows the amino acid sequence for dihydroflavonol 4-reductase from P. communis.
  • Figure 15 shows the amino acid sequence for flavo ⁇ oid 3% 5 '-hydroxylase from E. grandiflorum.
  • Figure 16 shows the amino acid sequence for quercetin 3-O-methyltransferase
  • Figure 17 shows the amino acid sequence for naringenin, 2-oxoglutarate 3- dioxygenase (flavonone-3 -hydroxylase) from D. caryophyllus.
  • Figure 18 shows the amino acid sequence for fiavonoid 3', 5 '-hydroxylase from E. grandiflorum.
  • Figure 19 shows the amino acid sequence for fiavonoid 3'-monooxygenase from P. x hybrida.
  • Figure 20 shows the amino acid sequence for dihydrokaempferol 4-reductase from P. x hybrida.
  • Figure 21 shows the amino acid sequence for leucoanthocyanbidin reductase from Phaseolus coccineus.
  • Figure 22 shows the design of a nanofactory harboring a 13 enzyme pathway producing camptothecins and precursors.
  • Figure 23 illustrates synthetic pathway library combinatorics.
  • FIG. 24 illustrates the production of diverse synthetic pathway libraries.
  • Figure 25 shows a nanofactory harboring glycolysis pathway enzyme encoding genes.
  • the nanofactories of the invention constitute biological devices that include a host bioparticle such as a cell harboring heterologous and/or exogenous genes encoding metabolic pathways.
  • the genes can be introduced using one or more vectors where the encoding genes are operationally linked to expression and/or regulation elements. Phage, virus, phage vectors, plasmid vectors and the like are particularly useful for introduction and expression of the encoded metabolic and/or other biochemical pathways.
  • the nanofactories can be genetically engineered to express the encoding genes stably, transiently, constitutively or in a regulated fashion.
  • the encoded metabolic and/or other biochemical pathways can be derived from known pathways or from as yet undiscovered pathways.
  • An exemplary known pathway includes the flavenoid biosynthetic pathways, which is exemplified further below.
  • Exemplary undiscovered pathways include those derived from, for example, pathways from as yet uncharacterized plant, microorganisms or other species. In the latter specific example, pathways can be expressed from genomic DNA fragments and the host nanofactory can be screened for the production of a new naturally occurring pharmacophore.
  • the encoded metabolic pathways can be derived from any combination of known and uncharacterized metabolic pathway encoding genes to create large, diverse populations of novel pharmacophores.
  • genes encoding metabolic enzymes within the same pathway but from different branchs or subclasses of metabolite can be combined orderly or randomly to create new and useful pharmacophores.
  • genes encoding metabolic enzymes within different metabolic pathways also can be combined orderly or randomly to create new and useful pharmacophores. All combinations and permutations of these specific examples can be employed in the methods of the invention to generate and identify new and/or useful pharmacophores.
  • the identified pharmacophores also can be isolated, for example, and employed in a variety of therapeutic settings. Populations of pharmacophores and isolated pharmacophores can be further diversified by decorating or functionalizing the structural motif either rationally or randomaly to create large numbers of biologically useful molecules.
  • the term "pharmacophore” is intended to mean a organic chemical structural motif used as a core or basic component for at least 2 different organic compounds. Therefore, the terms “structural motif,” “basic structural motif and “pharmacophore” are used herein as synonyms unless explicitly stated otherwise. Pharmacophores are found in nature as basic structural units for a variety of metabolic and/or biochemical reactants and/or products and include, for example, polymer compounds such as isoprenoids, cyclic compounds and/or areomatic compounds and the like such as flavenoids. A variety of other pharmacophores are well known in the art and are included within the meaning of the term as it is used herein. Additionally, a large variety of other pharmacophores exist in nature but have not yet been discovered or isolated. The term
  • pharmacophore also includes non-naturally occurring core structural motifs not found in nature, such as those produced by the methods of the invention.
  • a compound produced by the methods of the invention can include one or more pharmacophores.
  • non-naturally occurring when used in reference to a basic structural motif or pharmacophore of the invention is intended to mean that the structural motif has at least one chemical constituent contained within the basic structural motif not normally found in a comparative structural motif produced by enzymatic synthesis from wild-type or naturally occurring metabolic and/or biochemical pathways.
  • a comparative structural motif can be a pharmacophore found in a compound synthesized by the same or similar metabolic pathway that contains an enzyme catalyzing a reaction within the biosynthetic pathway of the non-naturally occurring structural motif. Therefore, the term as it is used herein refers to structural motifs that differ in its chemical structure compared to pharmacophores found in nature or otherwise chemically synthesized
  • an isolated pharmacophore of the invention when used in reference to a pharmacophore is intended to mean that the structural motif is free from at least one cellular constituent as the pharmacophore is found in nature.
  • an isolated pharmacophore of the invention includes a substantially pure pharmacophore or a pharmacophore that is produced in a heterologous biological system.
  • the term "diverse,” when used in reference to a population of pharmacophores is intended to mean that the population of pharmacophores exhibits variability in the core structural motif. The variability can be small such or large and will depend on the pharmacophore biosynthetic scheme as it taught and disclosed herein.
  • diverse populations of pharmacophores can include, for examples, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 175, 200, 250, 300, 350, 400, 450, 500, 750, 1000, 10,000, 100,000, 1,000,000, 10 7 , 10 8 , 10 9 , 10 10 or more different structural motifs within the population. All numbers in between and above these exemplary diversities of different structural motifs or pharmacophores within a population also are included within the definition of the term as it is used herein.
  • the term "plurality" when used in reference to the number of different structural motifs is intended to mean at least 2 different structural motifs or pharmacophores.
  • the term can include any number of different structural motifs or pharmacophores in the range from 2 to the maximum possible diversity that can be achieved by combination of some or all possible enzymes within a metabolic pathway or among enzymes of different metabolic pathways.
  • the term can include, for example, at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 175, 200, 250, 300, 350, 400, 450, 500, 750, 1000, 10,000, 100,000, 1 ,000,000, 10 7 , 10 8 , 10 9 , 10 10 or more different structural motifs or pharmacophores. All numbers in between and above these exemplary pluralities of different structural motifs or pharmacophores also are included within the definition of the term as it is used herein.
  • the number of different structural motifs or pharmacophores also can be expressed as a portion of the total number of possible different structural motifs or pharmacophores derived from some or all combinations of enzymes within a metabolic pathway or between metabolic pathways such as at least 20%, 30%, 50%, 60%, 75%, 90%, 95% or 98% of the possible number of different structural motifs that can be achieved given the number of enzymes expressed by a referenced nanofactory of the invention.
  • all percentages above, below and in between these exemplary percentages are included within the definition of the term as it is used herein.
  • central when used in reference to a metabolic pathway, is intended to mean a metabolic pathway selected from glycolysis, the pentose phosphate pathway (PPP), the tricarboxylic acid (TCA) cycle and the electron transfer system (ETS) and associated anapleurotic reactions.
  • PPP pentose phosphate pathway
  • TCA tricarboxylic acid
  • ETS electron transfer system
  • peripheral when used in reference to a metabolic pathway, is intended to mean a metabolic pathway that includes one or more reactions that are not a part of a central metabolic pathway.
  • polypeptide folds contain the following characteristics. Their structures are dictated by evolution and gene sequence. There are only about 1500 different structures and this limited diversity will likely not be found to further increase. Polypeptide and polypeptide folds are encoded in DNA and made by DNA decoding. Functional polypeptides and drug are made by decorating the fold with different side chain to form different functions and are therefore also limited by the diversity of the existing number of different polypeptide folds. Further, decoration of individual polypeptides is determined by the individual polypeptide sequence in the genome.
  • pharmacophores are basic chemical structures generally made by organisms as natural products. There is a large number of known pharmacophores but an unlimited number possible pharmacophores. Pharmacophores are decorated by side chain and other chemical moiety modifications that further increase diversity and impart various additional functions. These structural motifs constitute the product of metabolic and/or biochemical pathways, which are sets of polypeptide enzymes that work together to produce the basic structure. Pharmacophore structures also are modified and diversified by other tailoring enzymes, that add or modify side chains and/or various chemical moieties.
  • the current available diversity of pharmacophores is limited by their discovery and by biochemical methods of isolation from naturally occurring organisms. Such an approach is unable to generate non-naturally occurring pharmacophores. Moreover, such an approach also is limited by available resources and methodology for finding, harvesting and discovering naturally occurring pharmacophores from, for example, exotic, rare and/or hard to obtain species.
  • Metabolic and other biochemical pathways are genetic systems that produce complex molecules where the enzymes encoding the biosynthetic reactions are encoded in the genome. Metabolic pathways are conserved though evolution and among different organisms. Further, the metabolic enzymes also are conserved and often the order or arrangement their encoding genes is conserved in the genomes among different organisms. However, the product or natural product produced by metabolic enzymes can be very different between different pathways or even between different branches within a pathway.
  • the creation and screening of nanofactory populations expressing combinations of metabolic or other biochemical enzymes for the production of diverse populations pharmacophores is particularly useful for the creation and/or identification new pharmacophores for pharmaceutical purposes.
  • the identified pharmacophores can be used as initially created or identified. Alternatively, the created or identified pharmacophores can be further modified, either individually or by generating diverse populations of functional modifications. This pharmacophore methodological approach is particularly useful compared to polypeptide based therapeutics because it allows for the generation of large, diverse populations and the screening of these populations for pharmacophores having essentially any desired activity.
  • the number of basic polypeptide folds, including enzymes and drug targets is limited whereas the number of pharmacophores, which are target drug ligands, is essentially unlimited. Therefore, matching a target with a ligand, an important task in all drug development, is readily accomplished by creation and screening of pharmacophore libraries for a desired activity, binding ability or structural feature.
  • a nanofactory of the invention is a biological device encoded in DNA and made by synthetic biology.
  • the term "nanofactory” refers to a microorganism that is host-dependent. Therefore, a nanofactory acts like a cellular parasite, deriving much of its necessary biology from the host.
  • Specific examples of nanofactories include bacteriophage, filamentous bacteriophage, DNA viruses and RNA viruses. Other bioparticles such as these are well known in the art and also can be employed as a nanofactory of the invention given the teachings and guidance provided herein.
  • a nanomachine or ASC (artificial synthetic cell/artificial stem cell) refers to a free-living device that is host independent whereas a CFN or cell- free nanodevice, refers to a true biological machine and his host- and cell-independent.
  • ASC artificial synthetic cell/artificial stem cell
  • the nanofactories and method of the invention are exemplified herein by reference to a lambdoid-based nanofactory utilizing a lamda phage vector for purposes of illustration.
  • the lambdoid nanofactory exemplified herein is based on the biology of bacteriophage lambda. Use of this device can be compared to cloning in phage lambda except there is no bacteriophage and, optionally, no cloning.
  • packaging in the nanofactory requires an obligate 20 kb insert, which allows "forcing" enzyme encoding gene assortments and generating pathway diversity.
  • the exemplified methods and nanofactories of the invention can utilize any of a variety of vectors and microorganism or cellular system well known in the art.
  • the nanofactories also can employ filamentous phage based nanofactories.
  • the construction of enzyme encoding gene assortments into, for example, Ml 3, will additionally allow for the generation of diverse populations of surface expressed pharmacophores and screening by, for example, panning for a pharmacophore having a desired property.
  • the invention provides a diverse population of pharmacophores having a plurality of non-naturally occurring different structural motifs produced from the biosynthesis of unique combinations of enzymes within one or more metabolic pathways.
  • the plurality of non-naturally occurring different structural motifs can further include a plurality of different functionalizations or other decorative modifications.
  • the diverse population of pharmacophores having a plurality of non-naturally occurring different structural motifs can include 10 or more different structural motifs, particularly, 25 or more different structural motifs, and more particularly, 50 or more different structural motifs.
  • the enzymes within one or more metabolic pathways can be derived, for example, from the flavenoid biosynthetic pathway as described further below and in Example I.
  • the enzymes within one or more metabolic pathways also can be further derived, for example, from a metabolic pathway selected from a central metabolic pathway, from a peripheral metabolic pathway or from any other metabolic pathway.
  • the enzymes within one or more metabolic pathways encoded within a nonofactory of the invention also can be derived, for example, from the same or from different metabolic pathways.
  • a diverse population of nanofactories that include a diverse population of pharmacophores wherein each nanofactory within the diverse population expresses at least one different non-naturally occurring structural motifs produced from the biosynthesis of unique combinations of enzymes within one or more metabolic pathways.
  • a method of producing an isolated pharmacophore also is provided.
  • the method includes: (a) inserting into a vector a plurality of genes encoding enzymes from one or more metabolic pathways operationally linked to expression elements; (b) introducing the vector into a host cell; (c) culturing the host cell under conditions sufficient for expression of the encoding enzymes from the metabolic pathway, and (d) isolating a compound having a non-naturally occurring structural motif biosynthetically produced from the encoding enzymes from the metabolic pathway.
  • One or more genes encoding enzymes from other, non-metabolic biochemical pathways also can be inserted into a vector and operationally linked to expression elements.
  • the methods of the invention artificially generate pharmacophore diversity through the use of rationally designed and/or random combinations genes encoding metabolic or other biochemical pathway enzymes.
  • the methods of the invention generate nanofactories that express undiscovered naturally occurring pharmacophores produced by reconstruction of a naturally occurring pathways in a host cell or microorganism.
  • the methods of the invention also generate nanofactories that express non-naturally occurring pharmacophores produced by the creation of novel combinations of metabolic or other biochemical enzymes.
  • the methods of the invention can be used to reconstruct naturally occurring pathways by, for example, random or ordered cloning of genomic DNA populations into a nanofactory vector of the invention, expression the cloned DNA populations and screening populations of nanofactories expressing these combinations for the production of one or more pharmacophores. Screening can be accomplished by, for example, affinity, activity or by specific pharmacophore structural characteristics.
  • the methods of the invention can be used to create non-naturally occurring metabolic pathways for the generation of new and useful pharmacophores by, for example, random or ordered cloning of novel combinations of genes encoding metabolic enzymes and screening populations of nanofactories expressing these combinations for the production of one or more pharmacophores.
  • the populations can express a large or a small diversity of metabolic enzymes and the encoding genes can be from the same metabolic pathway, the same branch of a metabolic pathway or from different branches or pathways. Therefore, the methods of the invention can employ some or all possible combinations and/or permutations of metabolic enzyme repertoires.
  • screening for new and useful pharmacophores also can be accomplished by affinity, activity or by specific pharmacophore structural characteristics using any method well known in the art. Identification of positive hits in the screening methods indicates the presence of a non-naturally occurring pharmacophore, which can be further isolated by well known methods in the art.
  • Pathway extraction is one approach that can be utilized to generate nanofactories expressing a plurality of metabolic or other biochemical enzyme encoding genes. As described previously, the number of basic polypeptide folds is limited by evolution and is known — those folds encoded in the genome of man and other organisms. Since PCR allows the amplification of sequences that are related but diverse from a variety of genomes, this evolutionary conservation can be used to generate PCR primers to amplify some or all the members of a pathway, including a metabolic pathway.
  • pathway extraction refers to the approach where a pathway from a simple or complex organism is selected.
  • Each enzyme or polypeptide in the pathway will be sufficiently conserved so to allow its sequence to be amplified by PCR.
  • primers for each member within a pathway are generated and mixed together. PCR is applied to these mixtures to amplify some or the entire pathway from a target organism.
  • pathway extraction when used to generate coding regions sequences for known or suspected metabolic pathways.
  • the components of the pathway can be cloned in a way to associate and diversify the pathway, generating novel products as described previously and further below.
  • Baicalein is described herein as a representative example for production of a nanofactory expressing a known metabolic pathway or for the production of a nonofactory expressing ordered, semi-ordered, random or a combination thereof of genes encoding enzymes of known a metabolic pathway.
  • a nanofactory expressing a known metabolic pathway or for the production of a nonofactory expressing ordered, semi-ordered, random or a combination thereof of genes encoding enzymes of known a metabolic pathway.
  • the traditional Chinese herbal medicine "Sho-saiko-to" contains nine active ingredients including baicalein. The active compounds fall into two major groups: sapopnins and flavnoids.
  • Saponins are glycyrrhizin, saikosaponins a, c and d and ginsenosides RbI and RgI .
  • Favonoids are baicalin, baicalein and wogonin.
  • Baicalein and derivatives are potential drugs for the inhibition of ⁇ -synuclein aggregation and Lewy body formation, exemplary structures of which are shown in Figure 1. It is subject of investigation as a treatment for Parkinson's disease and other neurodegenerative diseases as well as a scaffold for the further decoration of functional side chains by tailoring enzymes.
  • Figure 2 shows a schematic diagram for flavenoid biosynthesis, which includes Baicalein.
  • Figure 3 shows a listing of the enzymes involved in the five branches for the flavenoid biosynthesis shown in Figure 2. Any or all of the genes encoding these enzymes can be incorporated in an ordered or random alignment for the expression of a variety of different and diverse new and useful pharmacophores based on flavenoid pathway biosynthetic enzymes.
  • Figure 4A shows a schematic diagram for the enzymes, products and reactants involved in flavenoid biosynthesis for the flavenoid pathways dipected in
  • Figure 4B shows a pathway in the chalone flavenoid biosynthetic schema depicted in Figure 4A.
  • Figure 4C shows a flavenoid biosynthetic pathway from chalcone to catechin depicted in Figure 4A.
  • Figure 4D shows a flavenoid pathway for naringenin biosynthesis depicted in Figure 4A and
  • Figure 4E shows the luteolin biosynthetic pathway depicted in Figure 4A.
  • a nanofactory of the invention can utilize this part of the flavone synthesis pathway for nanofactory pathway extraction.
  • the enzymes are:
  • Figure 5A shows the amino acid sequence for enzyme EC 5.5.7.9 chalone isomerase from Arabadopsis.
  • Figure 5B shows significant sequence alignments for chalone isomerase as an example of evolutionary conservation among a variety of different species.
  • Figure 5 illustrates the usefulness of evolutionary conservation to generate a nanofactory having desired pathways or combinations of pathway enzymes compiled from one or many different species.
  • Figures 8-21 provide the amino acid sequences for enzymes catalyzing one flavenoid branch shown in Figure 2 compiled from several different species.
  • the nanofactories are exemplified herein for illustration by reference to a lambdoid-based nanofactory construction.
  • the lambdoid nanofactory can be constructed where roughly 20 kb of the 50kb final construct is vector and used for construction and assembly. The remaining about 30 kb is used for insert genes and there is an obligate about 20 kb insert required. Therefore, if the average size of the gene sequences are 2 kb, the nanofactory can hold on average 20 to 30 genes sequences.
  • the gene sequences can be incorporated in a predetermined order or associated at random in the vector.
  • An exemplary construction of such an exemplary lambdoid-based nanofactory is shown in Figures 6A and 6B, where the replacement modules are utilized to harbor the genes encoding pathway enzymes.
  • each primer can have, for example, co-adhesive terminal enzyme sites but different primer sequence.
  • the primer sequences can be, for example, at the extreme ends of the gene so as to amplify an expressible fragment making the entire polypeptide.
  • each enzyme is a member of a multigene family. Therefore, primers such as those described herein and exemplified in Figure 7A can be employed for amplification of entire families of related enzymes. This approach is particularly useful to generate groups of assorted sequences.
  • primers also can be organized with cohesive sites that are at the ends so as to make tandem and allow directional assembly (e.g., orientation in the same direction during assembly).
  • Directional assembly is particularly useful, especially where all enzymes are to be expressed as a single operon.
  • the assembly and organization within a nanofactory so as to place or operationally link a promoter and terminator at each end of the construct is exemplified in Figures 7B and 7C.
  • each nanofactory plaque can contain in the construct 10 or more enzymes, in order if directed assembly is used, for example,, that are homologous to enzymes in the flavone synthesis pathway.
  • the transformation efficiency can be 10 6 or more and each plaque in the library will contain different combination of enzymes depending, for example, on (1) primers used; (2) target DNA used; (3) plant or other organism source, and (4) random chance assortment.
  • the nanofactory library can be assayed for content or for component pharmacophores produced by one or several methods as exemplified below:
  • Miniature preparations ie, "minipreps" of each individual clone can be prepared and screened by gas chromatography (GS), mass spectrophotometry (MS)and/or combined GC/MS.
  • Lift-type assays e.g., lysis and blotting to nitrocellulose, nylon or other solid support
  • Lift-type assays can be used to screen for an antibody reactive compound or for a specific target by another affinity binding molecule and/or by activity.
  • Pooled screens can be performed where multiple clones are pooled and screened together. Those pools having positive hits can then be divided and rescreened until a single positive clone is obtained.
  • labeled ⁇ - synuclein can be employed in a plaque lift assay using affinity binding.
  • the library can be plaqued out at 1000 colonies/ filter and transferred to a filter or other solid support (e.g., similar to library screening by plaque hybridization).
  • the filter can be incubated with biotinylated a-syn, washed and then incubated with HR peroxidase labeled avidin followed by development.
  • HR peroxidase labeled avidin followed by development.
  • the HR peroxidase detection reagent will turn the transferred plaque yellow.
  • the plaque can be picked, grown up further and analyzed for chemical content by, for example, GC/MS, NMR and/or other methods well known in the art.
  • the above exemplified methods and nanofactories produced therefrom of the invention beneficially allow selection of new pharmacophores with a particular target polypeptide in mind.
  • a further embodiment of the invention is the use of primers made, for example, to cover all of the desired enzymes classes of interest in PCR as a pool. This approach can beneficially be used to rescue a pathway knowing nothing about it other than it must be composed of enzymes that are related to known ones.
  • the methods and nanofactories of the invention provide an assortment of enzymes that will produce structures allowing: (1) rescue of unknown pathways; (2) diversification of known pharmacophores into new compounds, and (3) generation of new pharmacophores for assortment of parts of different pathways.
  • Baicalien Baicalien, Baicalien derivatives and other flavones.
  • the NFvI nanofactory is designed to support synthesis of Baicalien and derivatives, including other flavones.
  • the derived pathway is from phenylalanine through naringenin branching to apigenin, apiforol, luteolin and others. All the enzymes to Baicalein synthesis are known except apigenin, which is likely a flavone 6 hydroxylase and a flavone 4' dehydroxylase activities. These genes can be obtained by pathway extraction from Skutellaria. Branches A and B of the pathway are needed for pharmacophore synthesis while branched C through E are for generating additional diversity. The extra branches are optional, but can be useful for additional pathway diversity and to demonstrate pathway extraction and novel pharmacophore generation.
  • the pathway schematic is shown in the figures.
  • NFvI will contain the enzymes from phenylalanine to apigenin, which will provide the basics for the production and diversification of Baicalien.
  • Nanofactory 2, or NFv2 will contain the above pathway as well as additional branches to produce other flavenoids. Branhces C, D and E in order to generate more diversity among the flavone products.
  • These exemplary nanofactories serve as models for generating new pharmacophores.
  • new pharmacophores can be produced using a collection of PCR products representing a large repertoire or all known enzymes of one or more desired pathways and cloned into the nanofactory so that each nanofactory will contain from about 6 to 16 or more genes. This number provides a very large number of new synthetic pathways that can be screened for activity.
  • Use of PCR generated fragments in lieu of synthesized genes can be useful as a model for pathway extraction.
  • the nanofactories of the invention also can be used for pharmaceutical production, pathway extraction, pharamcophore discovery.
  • a nanofactory is described herein using a synthetic lambda-like phage.
  • the insert size can be made much larger in order to get more pathway encoding DNA into the nanofactory.
  • a cosmid can allow packaging of 35 to 45 kb.
  • Sets of PCR primers are made that will amplify synthetic enzymes based on known pathways but with a lot of degeneracy.
  • PCR can be performed from some normal and some exotic organisms, like plants that make exotic products, and all the enzymes of a pathway (and some or a lot of other enzymes as well) can be amplified and cloned in the nanofactory phage. Since these enzymes generate about 1-2 kb and about 16 kb is needed to package, it will force the cloning of an end to end array of about 10 or more enzyme encoding genes into an artificial pathway.
  • the resulting library will be a diverse collection of randomly associated synthetic enzymes.
  • a minimum library set of about 10 6 or more different phage can be produced (or cosmids) and screened for the desired activity. New pharmacophore substances also can be screened for and identified among such a diverse population. Use of plaque lifts is particularly useful because it allows direct screening directly for activities such as receptor binding.
  • a first generation nanofactory will employ from about 6 to 16 kb (or about 6 to 16 enzymes). Modification of the phage nanofactory to a cosmid-based nanofactorywill allow up to about 45 kb of encoding DNA (or about 45 genes). Encoding inserts larger than about 45 kb also is possible using, for example, PACs or BACs.
  • a BAC nanomachine can harbor an assortment of up to 120 kb or 120 genes. Also, in such cases where the pathway assortment is very large (e.g., 120 genes), a small or modest error rate or base degeneracy can be beneficial. Modifications can change or enhance activities. Using phage-based nanofactories is particularly useful because non- viable combinations of pathway encoding genes do not show up in the screen.
  • nanofactories developed or being developed using the methods of the invention include a nanofactory carrying a seven enzyme pathway producing the flavonoid narigenin; a second nanofactory producing components of taxane precursors, both as exemplified above; NFv3 carrying a 15 enzyme pathway metabolizing phenylalanine to various flavenoids and NFNv5, a 13 enzyme pathway producing camptothecins and precursors, which is exemplified further in Figure 22. 000130
  • This Example shows the design of a nonofactory for the generation of cellulosic ethanol.
  • Cellulosic ethanol promises to be a cost effective and environmentally responsible alternative fuel which is currently produced by hydrolysis and conventional fermentation.
  • the substantial interest in biofuels is the result of several factors: the relative lack of worldwide resources sufficient to meet current U.S. consumer demand; the high price of oil-based fuels; and the lack of adequate expansion capacity to meet predicted future needs.
  • Biofuels offer an attractive alternative to petroleum fuels for transportation purposes but are prohibitively expensive.
  • Ethanol is an effective and efficient biofuel which could be utilized by existing engines, with minor modifications and could reduce gasoline use by 70- 80%. Ethanol can be made through fermentation from "green" waste material or agricultural products such as corn.
  • One of the additional benefits of cellulosic ethanol production is that it reduces greenhouse gas emissions by 85% over conventional fuels. However ethanol is still too expensive to be used as a replacement for gasoline (e.g.,,doegenomestolife.org/pubs/2006abstracts/index.shtml).
  • Synthetic biology is a field of science where biological "parts,” generally ⁇ polypeptides, are invented or designed by computer, encoded into synthetic DNA, and produced or “decoded” in a host cell (see, for example, Chang and Keasling, Nat Chem Biol 15:674, 2006).
  • the purpose of synthetic biology is to allow scientists to be able to build biological nanoscale machines, including designer cells, and apply engineering principles to create living things.
  • a nanofactory is designed to carry a synthetic metabolic pathway. It is designed, the desired pathway encoding genes are chemically synthesized, and introduced into a microbial host for activation and expression.
  • a nanofactory based on the phage lambda model of 44,000 base pairs is produced which will encode a synthetic 15 enzyme pathway to metabolize sugars to ethanol. It also carries a variety of cellulases in a "diversity cassette" region of the nanofactory which allows for the introduction of mutations, motif substitutions and alternate genes for the assembly of large libraries of synthetic metabolic pathways. These libraries will then be screened for improved or altered metabolic function. Using this approach, high efficiency microbial production of cellulosic ethanol for commercial development can be obtained.
  • Synthetic biology is dependent on the high accuracy synthesis of entire genes composed of thousands of base pairs. Oligonucleotide synthesis of up to 200 bases is carried routinely by a large number of commercial suppliers and in virtually every laboratory. Gene synthesis, on the other hand, is generally carried out by assembling component oligonucleotides into whole synthetic genes.
  • Figure 22 is a diagram of nanofactory NFv5, a 13 enzyme pathway producing camptothecins and precursors.
  • bacteriophages such as lambda
  • phage-based nanofactories carry out bacterial infection, injection of DNA, replication and formation of phage heads and host lysis/lysogeny.
  • the genome is engineered to carry genes encoding enzymes of a metabolic pathway and introduces this pathway genome functionally into the host organism.
  • the lambda packaging ability of this exemplified nanofactory provides for the creation of synthetic pathway libraries where randomly amplified enzyme sequences can be combinatorically assorted into libraries of high complexity.
  • lambda packaging has a absolute size requirement for packaging of 35-45kb (e.g., Evans.G., Cosmids in Birren et al Genome Analysis Vol. 3 pg. 87 CSH 1999).
  • a 29 kb nanofactory construct (leaving 6-16kb) has a carrying capacity for up to around 20 metabolic genes.
  • Figure 23 exemplifies this procedure of synthetic pathway library combinatorics by randomly cloning a library of pathway encoding genes to produce a library of synthetic pathways. While a number of the pathway combinations result in nonsense or non-productive pathways, the efficiency of cloning is more than sufficient (up to or more than 10 10 / ⁇ g) that the novel functions of resultant pharmacophores can be detected by plaque lift screening. This method of generating highly diverse libraries for the identification of novel pharmacophores is further exemplified in Figure 24.
  • Pathway optimization libraries can have the following features:
  • a "library” is a one pool of nanofactories each containing a mixture of assorted synthetic enzymes, ii. Degenerate PCR primers can be used amplify collections of enzymes from natural sources based on conserved structures.
  • Nanofactory packaging (e.g., lambda) mechanism assures an array of about 4 to 16 enzymes in order for expression and production of resultant pharmacophores, iv. DNA injection mechanism introduces the nanofactory library into a compatible host strain and desired pharmacophores are identifiable with a suitable assay by, for example, plaque lifts. v. The nanofactory survives for additional screening.
  • This exemplarly procedure is applicable to very large collections >10 6 . vii.
  • Nanofactories can be produced and used equivalently to phage display methods for metabolic pathways - allowing for the generation, replication, surface expression and screening of large (e.g., aboutlO 10 ) libraries of synthetic pathways.
  • Combinatoric libraries are "biologically" relevant since they are produced by enzymes that are a result of a evolutionary process, ix.
  • Pathway enzymes can be from known family classes but undiscovered polypeptide sequences, which will generate novel compounds within a class and novel classes of structures.
  • Nanofactories inherently exhibit production and manufacturing capability because of their biological vector/host component.
  • Exemplified in this Example is a synthetic lambda phage-based nanofactory carrying the yeast glycolytic fermentation pathway and a variety of cellulases. Briefly, the nanofactory is produced and screened for the ability to direct an E. coli host to produce ethanol from cellulose.
  • a combinatoric library of nanofactories is synthesized where each member contains a different collection of randomly amplified cellulose enzymes and which are combinatorically assorted. This library of >10 6 synthetic pathways is screened in situ through plaque lifts to identify more efficient ethanol producers.
  • the more efficient ethanol producers also are optimized for high capacity conversion of cellulosic materials into ethanol for possible fuel production.
  • the production and optimization of ethanol for possible fuel production is indicative of nanofactory use for pharmaceutical pathway optimization - to produce and optimize ethanol biosynthesis from cellulose.
  • this approach will be used to "match" cellulases with fermentation to maximize and optimize the process, which is an approach applicable to pharmaceutical pathway generation and optimization.
  • the methods of the invention described previously are used, for example, to: (1) design a sugar to ethanol metabolic pathway suitable for manipulation in a nanofactory of the invention; (2) select one or more cellulases suitable to generate sugars from cellulosic materials for incorporation in the nanofactory; (3) synthesize the nanofactory from component oligonucleotides, assemble and introduce into bacterial host as described previously; (4) quantify production of ethanol and function of the resultant nanofactory; (5) employ screening and/or selection methods well known in the art for identification of ethanol production, and (6) prepare and screen an optimization library of about 10 6 or more synthetic pathways, initially through combinatorial assortment of cellulase enzymes PCR amplified from Trichoderma reesei. Exemplary Design of an Ethanol-Producing Nanofactory.
  • the design and generation of the ethanol-producing nanofactory construct is performed using components of NFv5, which carries a 13 enzyme pathway designed to produce the anti-cancer drug camptothecin.
  • the right and left "phage arms" of NFv5 are loosely based on the EMBL-3 phage vector widely used for bacteriophage cloning.
  • the right arm encodes 22 genes in 20,125 base pairs of DNA which comprise the lambdoid capsid, tail components packaging and assembly machinery, and outer host membrane polypeptide in the same relative position as wild type lambda.
  • the "right arm” encodes excision, DNA replication and cell lysis functions, regulatory function and other desired features and which are generally transposed from their position in wild type lambda.
  • this right arm encodes 20 genes in 9,025 base pairs of DNA.
  • Several polypeptides are encoded as GFP (green fluorescent protein) fusions to allow color identification of phage plaques and capsid polypeptides can be 6X HIS-tagged to aid in purification.
  • the central "stuffer region" of NFv5 encodes a synthetic pathway of 13 genes in 14,454 base pairs comprising the MEP and isoprenoid pathways of plant secondary metabolism and enzymes for camptothecin biosynthesis.
  • the polypeptide sequence of a plant, fungal or microbial enzyme in the pathway was obtained from a public access database such as the KEGG pathway database (see, for example, the URL genome.jp/kegg ) or NCBI/GenBank (see, for example, the URL ncbi.nlm.nih.gov/) and a synthetic gene is designed to make it use E. coli codon preference for enhanced expression.
  • the promoter and regulatory sequence from the expression plasmid ptrcHIS were adapted for expression of these pathway polypeptides as one long polycistronic message.
  • the entire NFv5 construct contains the following components: (1) protein coat (lambda); (2) tail and injector (lambda); (3) DNA replication; (4) cell lysis; (5) DNA/head packaging machinery; (6) enzymes for a synthetic metabolic pathway, and other features such as: (a) genetic identification (copyright notice); (b) biological identification (color); (c) temperature inducible regulation; (d) periplasmic expression sequence, and (e) modified cell recognition protein.
  • the nanofactory, termed NFv8 and illustrated in Figure 25, contains essentially identical right and left arms as with NFv5, but also included are two stuffer region cassettes.
  • One stuffer region encodes the 8 polypeptides of yeast glycolysis for the breakdown of glucose to pyruvate, plus two fermentation enzymes for the conversion of pyruvate to ethanol.
  • synthetic genes are designed using bacterial codon preference, high output bacterial expression promoter and single polycistronic message.
  • the second stuffer region cassette contains synthetic genes encoding a variety of cellulase enzymes as outlined below.
  • the strategy for maximizing the ethanol production from glycolytic fermentation is to increase the availability of free sugars from cellulase. While there are may sources of cellulases, the filamentous fungus Trichoderma reesei produces large quantities of diverse cellulose degrading enzymes (Foresman et al JBC 278: 31988, 2003) and contains a least 30 genes for structurally similar cellobiohydrolases and endoglucanases as well a myriad of other cellulose processing enzymes (Carle-Urioste et al JBC 272: 10169, 1997). Production of the initial ethanol-producing nanofactory uses seven primary cellulases of T.
  • Libraries are produced by deriving degenerate PCR primers from these genes and PCR amplifying suitable natural sources (microorganisms, plants, soil) to generate diversity in the cellulase sequences.
  • Nanofactory NF v ⁇ is synthesized using methods similar to described in, for example, U.S. Patent Nos: 6,670,127 and 6,521,427 and in U.S. Patent Publications 20030165946, 20050053997, 20050118628, 20050191648, 20050221340, 20050244841 and 20060134628. Briefly, 44,000 base pairs is synthesized as 880 component 50-mers for the (+) strand and 880 50-mers for the (-) strand in 96-well plate arrays. This synthesis requires 1760 50-mer oligonucleotides or 19 96-well plates. These right and left arms have already been synthesized in the construction of NFv5 described above.
  • Synthesis was performed using a commercial synthesis company such as Blue Heron or IDT or by Egea Biosciences LLC. Following synthesis, the component oligonucleotides are assembled. Under robotic control, each ligation center consisting of three oligonucleotides (two + strand and one overlapping - strand) are subjected to a brief ligation. Following a predetermined assembly program subsequent ligation steps of 6-mers, 12-mers, etc are automatically carried out until the entire assembly is complete. The entire assembly takes about 5 hours. This method has been used to assemble single DNA molecules of > 20kb.
  • viable constructs are recovered by lambda packaging.
  • the three components: right arm, left arm and insert stuffer are mixed, allowed to briefly anneal and mixed with phage lambda packaging extracts.
  • the mixture is added to a suspension o ⁇ E.coli host strain to initiate infection.
  • the mixture is plated on LB agar in YT soft top agar.
  • the viable nanofactories i.e., synthetic phage
  • the accuracy of total gene synthesis using this exemplified methodology exhibits error rates approaching one error in 10,000 synthesized bases. In a 44,000 bp construct rate corresponds to about 4-5 incorrect base per unit. This error rate is tolerable to the nanofactories of the invention. Since the packaging efficiency of lambda is extraordinarily high (up to 10 10 or more plaques/ ⁇ g) base synthesis errors are not likely to create a problem. Most errors in non-coding regions, changes which are silent or do not affect polypeptide function will not affect the viability of the phage. Therefore, this method selects for viable particles not 100% accurate DNA synthesis.
  • nanofactory viable plaques are selected and amplified, phage stocks prepared, and large scale cultures of infectious particles, DNA and cellular polypeptides prepared.
  • the integrity and authenticity of the construct is determined using agarose DNA gels.
  • the expression, quantification and size of each polypeptide in the synthetic pathway is determined by SDS PAGE.
  • the cells carrying the selected biodevices are analyzed to determine their ability to ferment ethanol from glucose, and then from cellulose using HPLC to quantify ethanol production.
  • Tagged substrates can be used to determine fidelity of the synthetic pathway.
  • One useful characteristic of the methods and nanofactories of the invention lies in the ability to create pathway diversity libraries and screen them in situ by plaque lift techniques, similar to techniques used in original cloning procedures of molecular biology during the 1970's, 1980's and early 1990's.
  • an in situ semi-quantitative ethanol assay can be employed which is suitable for use on plaque lift-type screens.
  • a modified synthetic alcohol dehydrogenase enzyme with color indicator substrate can be applied to the plaque lawn and the intensity of blue color over the plaque will indicate the presence and rough quantification of ethanol production.
  • An optimization library also is produced which will place a vast number of different cellulases and other cellulose processing enzymes in association with the glycolytic fermentation pathway.
  • This first generation optimization library is used to increase production efficiency by identifying combination of cellulases which maximize sugar availability for fermentation.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • General Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biomedical Technology (AREA)
  • Organic Chemistry (AREA)
  • Biotechnology (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Plant Pathology (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Bioinformatics & Computational Biology (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biophysics (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Enzymes And Modification Thereof (AREA)

Abstract

L'invention concerne une population diversifiée de pharmacophores possédant une pluralité de motifs structurels différents non naturels produits par la biosynthèse de combinaisons enzymatiques uniques dans une ou plusieurs voies métaboliques. La pluralité de motifs structurels non naturels différents peuvent en outre comprendre une pluralité de fonctions ou autres modifications décoratives différentes. L'invention se rapporte également à une population diversifiée de nano-usines comprenant une population diversifiée de pharmacophores, chaque nano-usine de la population diversifiée exprimant au moins un motif structurel non naturel différent produit par la biosynthèse de combinaisons enzymatiques uniques dans une ou plusieurs voies métaboliques. L'invention porte aussi sur un procédé permettant de produire un pharmacophore isolé, lequel consiste à: (a) insérer dans un vecteur une pluralité de gènes codant des enzymes d'une ou plusieurs voies métaboliques, fonctionnellement liés à des éléments d'expression; (b) introduire le vecteur dans une cellule hôte; (c) mettre en culture la cellule hôte dans des conditions suffisantes pour permettre l'expression des enzymes de codage de la voie métabolique; et (d) isoler un composé possédant un motif structurel non naturel produit par biosynthèse à partir des enzymes de codage de la voie métabolique.
PCT/US2007/000130 2006-01-03 2007-01-03 Compositions de nano-usines et procédés de fabrication et d'utilisation des nano-usines WO2007089382A2 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US75575806P 2006-01-03 2006-01-03
US60/755,758 2006-01-03
US78734306P 2006-03-29 2006-03-29
US60/787,343 2006-03-29

Publications (2)

Publication Number Publication Date
WO2007089382A2 true WO2007089382A2 (fr) 2007-08-09
WO2007089382A3 WO2007089382A3 (fr) 2008-05-15

Family

ID=38327845

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2007/000130 WO2007089382A2 (fr) 2006-01-03 2007-01-03 Compositions de nano-usines et procédés de fabrication et d'utilisation des nano-usines

Country Status (1)

Country Link
WO (1) WO2007089382A2 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10774050B2 (en) 2011-09-02 2020-09-15 Purdue Pharma, L.P. Pyrimidines as sodium channel blockers

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6242211B1 (en) * 1996-04-24 2001-06-05 Terragen Discovery, Inc. Methods for generating and screening novel metabolic pathways

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6242211B1 (en) * 1996-04-24 2001-06-05 Terragen Discovery, Inc. Methods for generating and screening novel metabolic pathways

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10774050B2 (en) 2011-09-02 2020-09-15 Purdue Pharma, L.P. Pyrimidines as sodium channel blockers

Also Published As

Publication number Publication date
WO2007089382A3 (fr) 2008-05-15

Similar Documents

Publication Publication Date Title
Shao et al. Refactoring the silent spectinabilin gene cluster using a plug-and-play scaffold
Gong et al. Genome shuffling: progress and applications for phenotype improvement
US7018793B1 (en) Combinatorial screening of mixed populations of organisms
EP0839185B1 (fr) Procedes de criblage pour des enzymes et des kits d'enzyme
Jenke-Kodama et al. Evolutionary mechanisms underlying secondary metabolite diversity
Hopwood et al. Genes for polyketide secondary metabolic pathways in microorganisms and plants
Wang et al. CYP76B74 catalyzes the 3′′-hydroxylation of geranylhydroquinone in shikonin biosynthesis
Kaul et al. Strategies for discovery and improvement of enzyme function: state of the art and opportunities
Khan et al. Fungi as chemical industries and genetic engineering for the production of biologically active secondary metabolites
Pettit Soil DNA libraries for anticancer drug discovery
Hou et al. Biosynthesis gene cluster and oxazole ring formation enzyme for inthomycins in Streptomyces sp. strain SYP-A7193
Akhgari et al. Single cell mutant selection for metabolic engineering of actinomycetes
Zhang et al. Developing GDi-CRISPR system for multi-copy integration in Saccharomyces cerevisiae
Li et al. Pathway elucidation of bioactive rhamnosylated ginsenosides in Panax ginseng and their de novo high-level production by engineered Saccharomyces cerevisiae
Cobb et al. Drug discovery and development via synthetic biology
CN106191155A (zh) 一种提高大肠杆菌异源合成聚酮类化合物的方法和用途
Shen et al. Macrotetrolide biosynthesis: a novel type II polyketide synthase
Liu Generate a bioactive natural product library by mining bacterial cytochrome P450 patterns
WO2007089382A2 (fr) Compositions de nano-usines et procédés de fabrication et d'utilisation des nano-usines
Zhou et al. Chromosome-scale genome assembly of Rhododendron molle provides insights into its evolution and terpenoid biosynthesis
Bapat et al. Medicinal phytometabolites synthesis using yeast bioengineering platform
US10119154B2 (en) Anthocyanins with specific properties
CN101182525A (zh) 桔霉素生物合成基因簇
CN107849586A (zh) 芳香化合物的生产
Iacovelli et al. Genome sequencing and molecular networking analysis of the wild fungus Anthostomella pinea reveal its ability to produce a diverse range of secondary metabolites

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application
NENP Non-entry into the national phase in:

Ref country code: DE

32PN Ep: public notification in the ep bulletin as address of the adressee cannot be established

Free format text: NOTING LOSS OF RIGHTS PURSUANT TO RULE 112(1)EPC,EPO FORM 1205A DATED 11/09/08

122 Ep: pct application non-entry in european phase

Ref document number: 07762925

Country of ref document: EP

Kind code of ref document: A2