JPWO2019236761A5

JPWO2019236761A5 -

Info

Publication number: JPWO2019236761A5
Application number: JP2020567524A
Authority: JP
Publication date: 2022-06-03

Claims

A method of genetically manipulating antibacterial peptides,
(A) The candidate nucleic acid encoding the candidate antibacterial peptide is translated in vitro in a translation solution, whereby the translation solution contains the candidate antibacterial peptide;
(B) Combining the candidate antibacterial peptide and microbial organisms in a solution environment;
(C) Culturing the microorganism and the candidate antibacterial peptide in the solution environment under selective culture conditions;
(D) To detect the inhibition or lack of growth and / or reproduction of the microorganism in the solution environment;
(E) Selection of the candidate nucleic acid upon detection of inhibition of growth and / or reproduction of the microorganism in the solution environment under the selective culture conditions;
(F) Producing a variant nucleic acid of the candidate nucleic acid of choice, wherein the variant nucleic acid encodes a variant of the candidate antibacterial peptide; and the microorganism in the solution environment under the selective culture conditions. Repeat (a)-(f) with one or more continuous variant nucleic acids as the candidate nucleic acid until a predetermined level of inhibition of body growth and / or reproduction is achieved, thereby the antibacterial peptide. However, methods involving genetic engineering.

Further comprising indexing the sequence of said candidate nucleic acid; and indexing the sequence to said detected inhibition of growth and / or reproduction of said microorganism in said solution environment of (d).
The method of claim 1, wherein (f) produces the variant nucleic acid based on the indexed sequence of the candidate nucleic acid.

Obtain a sequence of another candidate nucleic acid in which a lack of inhibition of growth and / or reproduction of the microorganism in another solution environment under the selective culture conditions is detected; and the sequence of the other candidate nucleic acid. Further comprising indexing for the lack of inhibition of growth and / or reproduction of the microorganism.
The method according to claim 1 or 2, wherein (f) produces the variant nucleic acid of the selection candidate nucleic acid based on the indexed sequence of the other candidate nucleic acid.

If the other candidate nucleic acid produces a lower level of inhibition of the growth and / or reproduction of the microorganism in the solution environment under the selective culture conditions, the said another candidate nucleic acid. The method of claim 3, wherein the sequence is obtained.

(I) Production of the variant nucleic acid sequence based on indexed sequence information involves machine learning such as automated machine learning.
(Ii) (e) further comprises confirming the inhibition of the growth and / or reproduction of the microorganism in the solution environment by the candidate antibacterial peptide, said confirmation being the candidate nucleic acid or its. Containing the repetition of (a)-(d) for copying, in the repetition of (d), the amount of the microorganism in the solution environment is the growth of the microorganism in the solution environment under the selective culture conditions and If / or indicates inhibition of reproduction, confirm the inhibition of growth and / or reproduction.
(Iii) The detection of inhibition of growth and / or reproduction of the microorganism or its lack thereof comprises quantifying the microorganism in the solution environment and of the microorganism in the solution environment over time. Decreased amounts indicate inhibition of growth and / or reproduction of said microorganisms.
(Iv) The inhibition of the predetermined level of growth and / or reproduction of the microorganism in the solution environment under the selective culture conditions is greater than that of the reference naturally occurring or genetically engineered antibacterial peptide. be,
(V) The genetically engineered antibacterial peptide and the candidate antibacterial peptide, respectively, of earlier repetition of the method have efficacy, and the efficacy of the genetically engineered antibacterial peptide is greater than that of the candidate antibacterial peptide. , And / or
(Vi) The genetically engineered antibacterial peptide has greater potency than the candidate antibacterial peptide over various culture conditions and / or against various microbial strains and / or species.
The method according to any one of claims 1 to 4.

Any of claims 1 to 5 , wherein the repetition of (b) of (a)-(f) comprises conjugating the translation solution with a microorganism of a strain or species different from that of the pre-repetition of (b). The method described in paragraph 1.

Any one of claims 1 to 5 , wherein the repetition of (b) of (a)-(f) comprises conjugating the translation solution with a microorganism of the same species or strain as the pre-repetition of (b). The method described in the section.

The invention according to any one of claims 1 to 7 , wherein the repetition of (c) of (a) to (f) comprises culturing the microorganism in a culture environment different from that of the prior repetition of (c). Method.

The invention according to any one of claims 1 to 7 , wherein the repetition of (c) of (a) to (f) comprises culturing the microorganism in the same culture environment as the prior repetition of (c). the method of.

(I) The candidate nucleic acid comprises DNA and further comprises transcribing the candidate nucleic acid.
(Ii) The translation solution further comprises a transcription solution, whereby the translation solution is configured for transcription and translation of the candidate nucleic acid.
(Iii) The translation solution contains a translation reagent such as a ribosome.
(Iv) The translation solution comprises one or more post-translational modification enzymes.
(V) The translation solution contains only one candidate nucleic acid sequence encoding a candidate antibacterial peptide.
(Vi) The candidate nucleic acid encodes two or more different candidate antibacterial peptides such that the solution environment comprises two or more candidate antibacterial peptides and the variant nucleic acid is the two or more candidates. Encoding at least one variant of an antibacterial peptide, whereby two or more antibacterial peptides are co-genetically engineered to inhibit the growth and / or reproduction of the microorganism under said selective culture conditions.
(Vii) The candidate antibacterial peptide comprises a chimeric protein.
(Viii) The selective culture conditions include conditions of an industrial process, a pharmaceutical manufacturing process, or a mammalian microbial flora.
(Ix) The solution environment comprises two or more species of microorganisms and / or
(X) The translation solution further contains a substrate, and the candidate nucleic acid is immobilized on the substrate.
The method according to any one of claims 1 to 9 .

(I) The production of the variant nucleic acid amplifies the selection candidate nucleic acid by a degenerate polymerase; amplifies the selection candidate nucleic acid in the presence of a degenerate primer; the selection candidate in the presence of a degenerate nucleotide. Amplifying nucleic acid; and comprising one or more mutagenetic reactions to said candidate nucleic acid.
(Ii) Screening for a library of candidate nucleic acids,
(Iii) Production of the variant nucleic acid comprises producing a library of variant nucleic acids, further comprising performing (a)-(e) on the library of variant nucleic acids.
(Iv) Perform in a microfluidic system and / or
(V) The translation solution and / or the solution environment is on a microliter scale.
The method according to any one of claims 1 to 10 .

A microfluidic system for genetically manipulating antibacterial peptides,
A transcription station configured to perform in vitro transcription, comprising a transcription reagent;
A translation station fluidly coupled to the transfer station, configured to perform in vitro translation and comprising a translation reagent:
Fluidly linked to the translation station, the microorganism is configured to be cultured under selective culture conditions in a solution environment containing the microorganism, a candidate nucleic acid encoding a candidate antibacterial peptide, and the candidate antibacterial peptide. Culture station;
A detector that is fluid-coupled to the culture station and configured to detect inhibition of growth and / or reproduction of the microorganism in the solution environment, or lack thereof; and encodes the candidate antibacterial peptide. Contains a variant station configured to produce a variant nucleic acid of said nucleic acid.
A microfluidic system in which the variant station is fluidly connected to the transfer station.

Including more processors
The variant station further comprises a sequencing module configured to obtain sequence information from the candidate nucleic acid.
The processor is configured to index the sequence information for said detection of growth and / or reproduction inhibition or lack thereof for the microorganism.
The microfluidic system according to claim 12 .

13. The microfluidic system of claim 13 , wherein the processor is configured to select the sequence of the variant nucleic acid based on the indexing information by machine learning such as automated machine learning.

(I) The variant station produces the variant nucleic acid only if the detector detects inhibition of growth and / or reproduction of the microorganism in the solution environment.
(Ii) The variant station comprises a degenerate polymerase.
(Iii) Two or more of the transcription station, the translation station, the culture station, and / or the variant station are contained in separate chambers separated from each other and / or.
(Iv) The transcription station and the translation station are the same station or overlap with each other.
The microfluidic system according to any one of claims 12 to 14 .

The microfluidic system according to any one of claims 12 to 15 , wherein two or more of the transcription station, the translation station, the culture station, and the variant station are contained in a single chamber.

16. The microfluidic system of claim 16 , wherein the single chamber is configured to release a first set of reagents and subsequently receive a second set of reagents different from the first set. ..

(I) The selective culture conditions of the culture station include conditions of an industrial process, a pharmaceutical manufacturing process, or a mammalian microbial flora.
(Ii) The translation station is fluid-coupled to a substrate containing the immobilized candidate nucleic acid.
(Iii) The translation station comprises a chamber that is microliter scale and / or the solution environment is microliter scale.
(Iv) The translation station comprises a mixture of different candidate antibacterial peptides.
(V) The microorganism comprises and / or has a plurality of species.
(Vi) The translation station comprises one or more post-translational modification enzymes.
The microfluidic system according to any one of claims 12 to 17 .

A kit for genetically manipulating antibacterial peptides,
Candidate nucleic acid encoding a candidate antibacterial peptide;
A kit comprising the microfluidic system according to any one of claims 12 to 18 .

19. The kit of claim 19 , further comprising a library of candidate nucleic acids and / or microorganisms .

The method according to any one of claims 1 to 11 , wherein the microorganism comprises a bacterium.

The method according to any one of claims 1 to 11 or 21 , wherein the antibacterial peptide contains or is a bacteriocin, and the microfluidic system according to any one of claims 12 to 18 . , Or the kit of claim 19 or 20 .