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Recombinant bacteria comprising novel sucrose transporters

a technology of sucrose transporter and recombinant bacteria, which is applied in the field of microbiology and molecular biology, can solve the problems of high glucose cost, inability to operate as efficiently, and many bacteria lack the ability to utilize sucros

Inactive Publication Date: 2015-03-05
EI DU PONT DE NEMOURS & CO
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The recombinant bacteria effectively metabolize sucrose to produce glycerol and glycerol-derived products, enhancing production efficiency and reducing costs by utilizing a more readily available and cheaper carbon source.

Problems solved by technology

However, a disadvantage of the use of glucose by microorganisms developed for production of commercially desirable products is the high cost of glucose.
Therefore, when a production microorganism does not have the ability to utilize sucrose efficiently as a major carbon source, it cannot operate as efficiently.
Moreover, many bacteria lack the ability to utilize sucrose.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

examples

[0207]The present invention is further defined in the following Examples. It should be understood that these Examples, while indicating preferred embodiments of the invention, are given by way of illustration only. From the above discussion and these Examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various uses and conditions.

General Methods

[0208]Standard recombinant DNA and molecular cloning techniques described in the Examples are well known in the art and are described by Sambrook, J., Fritsch, E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, (1989) (Maniatis) and by T. J. Silhavy, M. L. Bennan, and L. W. Enquist, Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1984) and by Ausubel, F. M. et al....

examples 1-34

Recombinant E. coli Strains Containing Putative Sucrose Transporter Genes

[0213]The purpose of these Examples was to construct recombinant E. coli strains containing various putative sucrose transporter genes. First an E. coli strain, referred to herein as PDO3513, lacking a sucrose transporter but containing the sucrose invertase and fructokinase genes (i.e. a cscA+K+B− (kanR) strain) was constructed. Then, various putative sucrose transporter genes were introduced into E. coli strain PDO03513.

Construction of E. coli Strain PDO3513

[0214]E. coli strain PDO03513 was constructed from an E. coli strain (referred to herein as PDO03085) containing the wild type cscAKB gene duster from E. coli ATCC®13281, integrated at the yihP gene in E. coli strain FM5 (ATCC® No. 53911). The cscAKB gene duster (SEQ ID NO:61) was integrated at the yihP location in E. coli strain FM5 (ATCC® No. 53911) by the Lambda Red method. The cscAKB gene duster was amplified from plasmid pBHR-cscBKA (SEQ ID NO:133), w...

examples 35-52

Recombinant E. coli Strains Containing Sucrose Transporter Genes

[0223]The purpose of these Examples was to construct recombinant E. coli strains containing the sucrose transporters disclosed herein, and having the ability to produce glycerol and 1,3-propanediol (PDO) from sucrose.

[0224]The E. coli strains containing the sucrose transporter genes were constructed from PDO producing strain TTab pSYCO400 / AGRO. E. coli strain TTab pSYCO400 / AGRO, a PTS minus strain, was constructed as follows. Strain TTab was generated by deletion of the aldB gene from strain TT aldA, described in U.S. Pat. No. 7,371,558 (Example 17). Briefly, an aldB deletion was made by first replacing 1.5 kbp of the coding region of aldB in E. coli strain MG1655 with the FRT-CmR-FRT cassette of the pKD3 plasmid (Datsenko and Wanner, Proc. Natl. Acad. Sci. USA 97:6640-6645, 2000). A replacement cassette was amplified with the primer pair SEQ ID NO:174 and SEQ ID NO:175 using pKD3 as the template. The primer SEQ ID NO:1...

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Abstract

Recombinant bacteria capable of metabolizing sucrose are described. The recombinant bacteria comprise in their genome or on at least one recombinant construct, a novel nucleotide sequence encoding a polypeptide having sucrose transporter activity and a nucleotide sequence encoding a polypeptide having sucrose hydrolase activity. These nucleotide sequences are each operably linked to the same or a different promoter. Recombinant bacteria capable of metabolizing sucrose to produce glycerol and / or glycerol-derived products such as 1,3-propanediol and 3-hydroxypropionic acid are also described.

Description

FIELD OF THE INVENTION[0001]The invention relates to the fields of microbiology and molecular biology. More specifically, recombinant bacteria comprising novel sucrose transporters and methods of utilizing such recombinant bacteria to produce products such as glycerol and glycerol-derived products from sucrose are provided.BACKGROUND OF THE INVENTION[0002]Many commercially useful microorganisms use glucose as their main carbohydrate source. However, a disadvantage of the use of glucose by microorganisms developed for production of commercially desirable products is the high cost of glucose. The use of sucrose and mixed feedstocks containing sucrose and other sugars as carbohydrate sources for microbial production systems would be more commercially desirable because these materials are usually readily available at a lower cost.[0003]A production microorganism can function more efficiently when it can utilize any sucrose present in a mixed feedstock. Therefore, when a production micro...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12P7/42C12N9/10C07K14/195C12P7/20C12P7/18C12N9/26C12N9/12
CPCC12P7/42C12N9/2431C12N9/1051C12N9/1205C12P7/20C12Y207/01004C07K14/195C12Y204/01007C12Y207/01001C12Y207/01003C12P7/18C07K14/24C07K14/245C07K14/33C07K14/335C12N9/2402C12Y302/01026
Inventor RUEBLING-JASS, KRISTINTOMB, JEAN-FRANCOISVAN DYK, TINA KYOU, ZHENG
Owner EI DU PONT DE NEMOURS & CO