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Methods and molecules for yield improvement involving metabolic engineering

a metabolic engineering and yield improvement technology, applied in the field of cell metabolism engineering, can solve the problems of the ‘feedstock’ cost, the cost of the feedstock itself, etc., and achieve the effects of promoting degradation of protein, enhancing degradation of protein, and modifying cell metabolism

Active Publication Date: 2019-08-20
GINKGO BIOWORKS INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0005]The invention generally provides improved cells, molecules, and methods for synthesis of products by metabolic engineering. In a general embodiment, the invention provides an engineered cell that synthesizes a product more cost-effectively than current methods by making use of a cell with the following characteristics. The cell contains one or more proteins that include an enzymatic function with an engineered connection to a sequence that can promote degradation of the protein. The cell also includes a regulatory system such that upon addition or withdrawal of a regulatory factor, which may be a chemical, a protein, photons, temperature, or any other factor, the degradation of the protein is enhanced. As a result, the metabolism of the cell is altered so that the synthesis and / or secretion of a desired product is enhanced. In a further embodiment, the desired product is obtained from the cell or the medium. The enzymatic function may promote growth of the cell during an expansion phase or may allow the culturing and expansion of the cell with less or none of an expensive feedstock component.

Problems solved by technology

One major cost in metabolic engineering is the ‘feedstock’—the mixture of nutrients used in the medium in which the microbe grows.
One significant problem in metabolic engineering is that even under conditions of product production, much of the feedstock is channeled into other metabolic pathways that contribute to growth of the organism and production of its biomass.
A second problem is the cost of the feedstock itself, especially when the feedstock includes, in addition to a carbohydrate, molecules that fulfill auxotrophic requirements.

Method used

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  • Methods and molecules for yield improvement involving metabolic engineering
  • Methods and molecules for yield improvement involving metabolic engineering
  • Methods and molecules for yield improvement involving metabolic engineering

Examples

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Effect test

example 1

of Shikimic Acid from a Microbe Containing an Engineered Shikimate Kinase Gene

[0091]An E. coli strain capable of being grown in the absence of aromatic amino acids and producing shikimic acid was engineered as follows. The strain was engineered to express a shikimate kinase isoform, the product of the aroK gene, from a plasmid, while the chromosomal genes encoding shikimate kinase were non-functional. The plasmid-borne shikimate kinase isoform was engineered to have a degradation tag at its C-terminus. In this case and throughout the invention, it was and is useful to inspect the three-dimensional structure of a protein to verify that a chosen terminus is compatible with addition of a degradation tag. The solved structure of the aroK product, PDB file 1KAG, was inspected and the steric availability of the C-terminus was verified.

[0092]Plasmid vectors were generated which allow for conditional expression of E. coli shikimate kinase I, aroK. Using standard plasmid construction techniq...

example 2

n of Shikimic Acid from a Microbial Strain in Which Shikimate Kinase is Fused to a Degradation Tag and Expressed from an Episome with Conditional Replication

[0102]In an alternative method of the invention, an E. coli strain that could be grown in the absence of aromatic amino acids and produce shikimic acid was engineered as follows. Four variants were constructed from a plasmid derivative of the low-copy vector pSC101, in which the origin of the plasmid was temperature-sensitive for replication. The plasmid encoded the E. coli aroK gene expressed from its endogenous promoter. The four plasmid variant coding sequences for the degradation tags AANDENYALAA (SEQ ID NO: 1), AANDENYALVA (SEQ ID NO: 8), AANDENYADAS (SEQ ID NO: 2) and the non-degrading control variant AANDENYALDD (SEQ ID NO: 13) were fused to the 3′ end of the aroK coding sequence. These vectors also encoded a chloramphenicol-resistance marker. Expression of shikimate kinase from the E. coli chromosome was defective.

[0103]...

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Abstract

The invention features methods and compositions relating to cells that have been engineered to reduce or eliminate proteins having enzymatic activity that interfere with the expression of a metabolic product.

Description

RELATED APPLICATIONS[0001]This application is a National Phase application of International Application No. PCT / US2010 / 036902 filed Jun. 1, 2010, which claims priority to and the benefit of U.S. Provisional Application No. 61 / 182,839, filed Jun. 1, 2009. The entire teachings of the above applications are incorporated herein by reference.BACKGROUND OF THE INVENTION[0002]In general, the invention relates to metabolic engineering of cells for the enhanced production of a cellular product.[0003]Metabolic engineering involves the industrial production of chemicals from biological sources. Typically, a microbe such as a bacterium or a single-celled eukaryote is engineered to produce a compound in large amounts that is normally produced in small amounts or not at all. Examples of compounds produced by metabolic engineering include ethanol, butanol, lactic acid, various vitamins and amino acids, and artemisinin. Metabolic engineering generally involves genetic modification of a host organis...

Claims

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Application Information

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Patent Type & Authority Patents(United States)
IPC IPC(8): C12N1/00C12P13/04C12P13/00C12N5/00C12P7/42C12N15/67C12N9/12C12N15/10
CPCC12P7/42C12N9/1205C12N15/67C12N15/10C07K2319/95
Inventor WAY, JEFFREY C.DAVIS, JOSEPH H.
Owner GINKGO BIOWORKS INC
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