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High throughput transposon mutagenesis

A transposon and gene mutation technology, applied in DNA preparation, recombinant DNA technology, etc., can solve the problems of slowness, low efficiency, and time-consuming efficiency of industrial microbial strains

Pending Publication Date: 2020-03-06
ZYMERGEN INC
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0009] However, identification of improved industrial microbial strains by traditional mutagenesis methods is time-consuming and inefficient
The method is by its nature haphazard, inefficient and slow

Method used

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Examples

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example

[0603] The following examples are provided to illustrate various embodiments of the present disclosure and are not intended to limit the present disclosure in any way. Those skilled in the art will recognize that changes therein and other uses are encompassed within the spirit of the disclosure as defined by the scope of the claims.

[0604] A brief table of contents is provided below to assist the reader only. This list is not intended to limit the scope of the examples or disclosure of this application.

[0605] Table 4 - Table of Contents for Example Chapters

[0606]

example 1

[0607] Example 1 - HTP Genome Engineering - Construction of a Transposon Mutagenesis Library to Improve Saccharopolyspora Strain Performance

[0608] This example describes a method for in vivo transposon-induced mutagenesis of S. spinosa to generate a library of strains. The resulting library can be screened to identify strains with improved phenotypes, such as potency of a particular compound (eg, spinosyn).

[0609] Strains can be further used in multiple rounds of cycle engineering or to decipher genotypes contributing to strain performance. Strains in the library can also be used in combination with other strains with different gene perturbations in order to generate improved strains with increased production of one or more desired compounds.

[0610] Thus, the present disclosure describes a method for creating a library of transposon mutagenized microbial strains using the EZ-Tn5 transposome system of S. spinosa (Epicenter Bio). The transposase is first complexed with ...

example 2

[0619] Example 2-HTP genome engineering-construction of transposon mutagenesis library to improve the strain performance of Escherichia coli

[0620] Transposon mutagenesis can be performed to generate a library of random strains of E. coli to improve strains. Libraries of these strains can be screened for desired phenotypes (eg, tryptophan production) to identify mutants with improved performance.

[0621] E. coli mutant libraries can be generated using the EZ-Tn5 transposon system. Briefly, the EZ-Tn5 transposase was incubated with payload DNA flanked by chimeric element sequences to allow the EZ-Tn5 transposase to complex with the DNA to form transposomes. The DNA / protein transposome complex was then transformed in E. coli by electroporation, and the EZ-Tn5 transposase catalyzed random integration of the payload DNA into the E. coli genome, generating a random library of strain variants.

[0622] The specific sequence of the payload DNA can be further altered to favor los...

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Abstract

The present disclosure is directed to a method of high-throughput (HTP) microbial genomic engineering, which utilizes in vivo transposon mutagenesis to develop strain libraries for the perturbation ofmicrobial phenotypes.

Description

[0001] Cross References to Related Applications [0002] This application claims priority to U.S. Provisional Application No. 62 / 515,965, filed June 6, 2017, the contents of which are hereby incorporated by reference in their entirety. technical field [0003] The present disclosure relates to a high-throughput (HTP) microbial genome engineering method, which uses in vivo transposon mutagenesis to establish a strain library for disturbance of microbial phenotypes. [0004] Statement about the sequence listing [0005] The Sequence Listing associated with this application is provided in text format in lieu of a paper copy and is incorporated herein by reference. The name of the text file containing the sequence listing is ZYMR_014_01WO_SeqList_ST25.txt. The text file is 14KB, was created on June 6, 2018, and was submitted electronically via EFS.com. Background technique [0006] Humans' ability to exploit microbial cell biosynthetic pathways to produce products of inter...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
CPCC12N15/1058C12N15/1079C12N15/102
Inventor P·凯利P·埃涅尔特
Owner ZYMERGEN INC
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