Aug 2, 2016

Cell-Free Mixing of Escherichia coli Crude Extracts to Prototype and Rationally Engineer High-Titer Mevalonate Synthesis

ACS Synthetic Biology
Quentin M DudleyMichael C Jewett

Abstract

Cell-free metabolic engineering (CFME) is advancing a powerful paradigm for accelerating the design and synthesis of biosynthetic pathways. However, as most cell-free biomolecule synthesis systems to date use purified enzymes, energy and cofactor balance can be limiting. To address this challenge, we report a new CFME framework for building biosynthetic pathways by mixing multiple crude lysates, or extracts. In our modular approach, cell-free lysates, each selectively enriched with an overexpressed enzyme, are generated in parallel and then combinatorically mixed to construct a full biosynthetic pathway. Endogenous enzymes in the cell-free extract fuel high-level energy and cofactor regeneration. As a model, we apply our framework to synthesize mevalonate, an intermediate in isoprenoid synthesis. We use our approach to rapidly screen enzyme variants, optimize enzyme ratios, and explore cofactor landscapes for improving pathway performance. Further, we show that genomic deletions in the source strain redirect metabolic flux in resultant lysates. In an optimized system, mevalonate was synthesized at 17.6 g·L(-1) (119 mM) over 20 h, resulting in a volumetric productivity of 0.88 g·L(-1)·hr(-1). We also demonstrate that this system...Continue Reading

  • References73
  • Citations8

References

Mentioned in this Paper

Metabolic Process, Cellular
Biochemical Pathway
Solocyte
Genetically Engineered Mouse
Alkalescens-Dispar Group
AT 17
Genome
Enzymes, antithrombotic
Isoprenoid Biosynthetic Process
Calcium ion

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