| 摘要 |
Genetically manipulated cells, lysates of such cells, systems, and methods of use thereof are provided, where one or more enzymes in a pathway of interest are genetically modified to incorporate a peptide sequence that provides for relocation of the protein, e.g., to the periplasm, so as to sequester the enzyme, and where the enzyme controls flux in the pathway of interest. |
| 主权项 |
1. A method of producing a product of a biosynthetic pathway of interest, the method comprising:
(a) culturing bacterial cells that express enzymes of a biosynthetic pathway for production of a product, wherein at least one of the enzymes:
(i) is an enzyme that controls metabolic flux in the biosynthetic pathway; and(ii) is an enzyme modified by genetic means to attach a periplasmic-targeting sequence, thereby producing cultured bacterial cells that contain at least one of the modified enzymes sequestered into the periplasmic space, wherein the modification by genetic means and the periplasmic sequestering do not result in a deleterious increase in metabolic flux in the biosynthetic pathway; (b) lysing the cultured bacterial cells, thereby producing a cell lysate containing the enzymes of the biosynthetic pathway and the at least one periplasmic-targeting sequence-modified enzyme; (c) combining the cell lysate with one or more substance selected from the group consisting of substrates, enzymes, nutrients, co-factors, buffers, reducing agents, ATP generating systems; and (d) incubating the cell lysate and the one or more substance for a period of time and under conditions sufficient to enzymatically produce the product, wherein the product is selected from the group consisting of antibiotics, biosurfactants, biological fuels, amino acids, organic acids, fatty acids, alcohols, polyols, flavors, fragrances, nucleotides, vitamins, pigments, sugars, polysaccharides, biopolymers, plastics, isoprenoids, terpenes, and cell metabolites. |
