Abstract: Provided herein are cell-free systems for generating carbapenems, e.g., a compound of the Formula (I): or salts thereof; wherein , R1, R2, R3, R4, R5, and R6 are defined herein. Also provided are pharmaceutical compositions comprising a compound generated by the inventive cell-free system, and use of these compounds and compositions for the treatment of bacterial infections.

Abstract: Methods are provided for controlling metabolic flux rate in a cell-free system comprising a complex set of enzymes, to produce a desired product of a pathway of interest. In the methods of the invention, measurements of metabolic performance parameters are taken by continuous monitoring or intermittent monitoring. Based on the metabolic performance parameters, the system is modified by one or more steps comprising: (i) altering enzyme levels in the cell-free system; (ii) altering feed rate of a substrate that controls redox flux or carbon flux to the cell-free system; (iii) altering O2 addition to the cell-free system; (iv) controlling efficiency of electron transport system by altering leakage across a membrane; wherein enzymes present in the pathway of interest catalyze production of a desired product.

Abstract: 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.

Abstract: The embodiments described herein pertain to cells, and methods for preparing cells, that can be used as biocatalysts by altering enzymes that compete for a substrate or product of a pathway of interest such that the targeted enzyme is sensitive to a site-specific protease, which protease is expressed but relocated in the cell to a site where it is not in contact with the targeted enzyme in the intact cell. Upon cell lysis, the protease contacts the target enzyme, which is then inactivated by protease cleavage.

Abstract: 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.

Abstract: Various aspects and embodiments herein relate to recombinant proteins with at least one protease recognition sequence that can be inactivated by a cognate protease and methods of preparing such proteins. In some embodiments, recombinant phosphoglucose isomerase (Pgi) proteins are provided. In other embodiments, recombinant phosphotransacetylase (Pta) proteins are provided. In yet other embodiments, recombinant transketolase A (TktA) proteins are provided.

Abstract: The embodiments described herein pertain to cells, and methods for preparing cells, that can be used as biocatalysts by altering enzymes that compete for a substrate or product of a pathway of interest such that the targeted enzyme is sensitive to a site-specific protease, which protease is expressed but relocated in the cell to a site where it is not in contact with the targeted enzyme in the intact cell. Upon cell lysis, the protease contacts the target enzyme, which is then inactivated by protease cleavage.

Abstract: The present disclosure relates, in some aspects, to cell-free methods and systems for large-scale conversion of methane to isobutanol, comprising combining, in a bioreactor at elevated pressure, methane, oxygen, and cell lysates containing methane monooxygenase, methanol dehydrogenase, and enzymes that catalyze the conversion of formaldehyde to isobutanol, to form a cell-free reaction mixture, and incubating under suitable conditions the cell-free reaction to convert methane to isobutanol.

Abstract: The invention pertains to a method for preparing cells that can be used as biocatalysts by inducing in them a growth-decoupled state, in which interferase inhibits the expression of genes except the ones that code for the pathway enzymes of interest. mRNAs that code for interferase-resistant products are overexpressed in the background of a metabolically-frozen cell. Enzymes that compete for a substrate or product of the pathway of interest may be altered such that the enzyme is sensitive to a site-specific protease, which protease is inducible in the host cell.