Compositions, methods and uses for multiplexed trackable genomically-engineered polypeptides
AssigneeThe Regents of the University of Colorado, a body corporate
InventorsRyan T. Gill, William Grau
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Abstract
Embodiments herein concern compositions, methods, systems and uses for in vivo selection of optimum target proteins of use in designing genomically-engineered cells or organisms. Some embodiments relate to compositions and methods for generating constructs mimicking benefits of megasynthases in a non-natural organism or cell of use in systems and methods disclosed herein. Yet other embodiments relate to compositions and methods for generating agents using constructs disclosed herein of use in treating genetically-linked conditions.
Claims42
A construct, comprising:
- a non-naturally occurring polypeptide having a formula represented by (X—B)n—Z, wherein:
- X comprises at least one polypeptide encoding at least one domain of a first target protein;
- Z comprises at least one polypeptide encoding at least one domain of a second target protein;
- B comprises a polypeptide for linking X and/or Z;
- and
- n is a number from 1 to 100;
- wherein the non-naturally occurring polypeptide having (X—B)n—Z as a formula is capable of synthesizing a secondary metabolite in a cell or organism.
The construct according to any one of the preceding claims, wherein the first target protein comprises a megasynthase, a polyketide synthase, a non-ribosomal peptide synthase, and/or hybrid thereof.
The construct according to any one of the preceding claims, wherein the second target protein comprises a megasynthase, a polyketide synthase, a non-ribosomal peptide synthase, and/or hybrid thereof.
The construct according to any one of the preceding claims, wherein the construct comprises a modular megasynthase.
The construct according to any one of the preceding claims, wherein the first target protein and the second target protein are the same protein.
The construct according to any one of the preceding claims, wherein X comprises a polypeptide sequence of at least one domain selected from the group consisting of:
- Acyltransferase (AT), Acyl Carrier Protein (ACP), Keto-Synthase (KS), Ketoreductase (KR), Dehydratase (DH), Enoylreductase (ER), Methyltransferase (MT), Sulfhydrolase (SH), and Thioesterase (TE).
The construct according to any one of the preceding claims, wherein Z comprises a polypeptide sequence of at least one domain selected from the group consisting of:
- Acyltransferase (AT), Acyl Carrier Protein (ACP), Keto-Synthase (KS), Ketoreductase (KR), Dehydratase (DH), Enoylreductase (ER), Methyltransferase (MT), Sulfhydrolase (SH), and Thioesterase (TE).
The construct according to any one of the preceding claims, wherein B comprises a polypeptide sequence selected from the group consisting of:
- Acyl Carrier Protein-Condensation Domain linkers (ACP Condensation), Acyl Carrier Protein-Heterocyclization Domain linkers (ACP Heterocyclization), Acyl Carrier Protein-Ketosynthase Domain linkers (AC-PKS), Acyl Carrier Protein-Thioesterase Domain linkers (ACP-TE), Adenylation Domain-Peptide Carrier Protein linkers (A-PCP), Acyltransferase Domain-Acyl Carrier Protein linkers (AT-ACP), Acyltransferase Domain-Dehydratase Domain linkers (AT-DH), Acyltransferase Domain-Ketoreductase Domain linkers (AT-KR), Condensation Domain-Adenylation Domain linkers (Condensation A), Dehydratase Domain-Enoylreductase Domain linkers (DH-ER), Dehydratase Domain-Ketoreductase Domain linkers (DH-KR), Dual Condensation/Epimerization Domain-Adenylation Domain linkers (Dual Condensation A), Enoylreductase Domain-Ketoreductase Domain linkers (ER-KR), Heterocyclization Domain-Adenylation Domain linkers (Heterocyclization A), (Both Acyl and Peptide) Carrier Protein-Condensation Domain linkers (Joint AC-PC), Ketoreducatse Domain-Acyl Carrier Protein linkers (KR-ACP), and Ketosynthase Domain-Acyltransferase Domain linkers (KS-AT).
The construct according to any one of the preceding claims, wherein the secondary metabolite comprises:
The construct according to any one of the preceding claims, wherein the secondary metabolite is selected from the group consisting of:
- delta-hexalactone, Rapanycin, Actinorhodin, Erythromycin A, 6-Methylsalicyclic acid, Aflatoxin B1, Rifamycin S, Lovastatin, Amphotericin B, and Monensin A.
The construct according to any one of the preceding claims, wherein the first target protein or the second target protein is a prokaryotic protein or a eukaryotic protein.
The construct according to any one of the preceding claims, the construct having the formula (X—B)n—Z is a polypeptide having at least 95 percent identity to at least one polypeptide selected from the group consisting of the polypeptides represented by at least one of SEQ ID NOs:
- 33-64, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143.
The construct according to any one of the preceding claims, wherein B comprises a polypeptide sequence represented by at least one of SEQ ID NOs:
- 70-71, 73-74, 76-79, 81-82, 84-86, 88-91, 93-96, 98-99, 101-107.
A non-naturally occurring polynucleotide comprising a polynucleotide encoding the construct of any one of the preceding claims.
A method for generating a construct, comprising:
- obtaining at least one polynucleotide sequence encoding at least one domain of a first target protein and at least one polynucleotide sequence encoding at least one domain of a second target protein;
- determining a linker polynucleotide that is capable of encoding a protein linking the at least one domain of the first target protein and the at least one domain of the second target protein;
- and
- generating a polypeptide construct having the at least one polypeptide sequence encoding the first target protein and at least one polypeptide sequence encoding the second target protein and a polypeptide sequence encoding the linker polypeptide.
A method comprising:
- assigning ranks pertaining to biological effects of genetic variations of a plurality of genes or genetic loci capable of coding for a target protein;
- assigning ranks pertaining to the biological effect due to the genetic variations of the plurality of genes or genetic loci;
- obtaining and analyzing one or more rank(s) of the genetic variations of the genes or genetic loci pertaining to a predetermined selection process;
- obtaining one or more composite rank(s) based on the ranks of the biological effects as they pertain to the predetermined selection process and biological context rank;
- and
- designing a genomically-engineered process, cell or organism based on the composite rank(s).
A computer-readable medium having computer-readable instructions, which, when executed by a computer, cause the computer to carry out a method comprising:
- receiving first gene(s) or genetic segment score representing a score of a biological effect or condition due to a genetic variation of a gene or gene segment of a target protein;
- receiving at least a second gene(s) or genetic score representing a second score of another genetic variation of the target protein;
- combining the scores;
- and
- assigning a combined score related to one or more genetic variations in order to assess a value of the genetic variations related to a trait for the target protein.
A system comprising:
- a component for assessing a score of a genetic variation of genes or genetic segments pertaining to a trait of one or more target proteins;
- and
- a component for reporting the score of the genetic variation of genes or genetic segments pertaining to a trait of one or more target proteins;
- and
- a component for compiling the scores of one or more target proteins.
Dependencies are inferred from the claim text.