Abstract
Productivity of bacterial cell factories is frequently compromised by stresses imposed by recombinant protein synthesis and carbon-to-product conversion, but little is known about these bioprocesses at a systems level. Production of the unnatural metabolite citramalate in Escherichia coli requires the expression of a single gene coding for citramalate synthase. Multiomic analyses of a fermentation producing 25 g liter-1 citramalate were undertaken to uncover the reasons for its productivity. Metabolite, transcript, protein, and lipid profiles of high-cell-density, fed-batch fermentations of E. coli expressing either citramalate synthase or an inactivated enzyme were similar. Both fermentations showed downregulation of flagellar genes and upregulation of chaperones IbpA and IbpB, indicating that these responses were due to recombinant protein synthesis and not citramalate production. Citramalate production did not perturb metabolite pools, except for an increased intracellular pyruvate pool. Gene expression changes in response to citramalate were limited; none of the general stress response regulons were activated. Modeling of transcription factor activities suggested that citramalate invoked a GadW-mediated acid response, and changes in GadY and RprA regulatory small RNA (sRNA) expression supported this. Although changes in membrane lipid composition were observed, none were unique to citramalate production. This systems analysis of the citramalate fermentation shows that E. coli has capacity to readily adjust to the redirection of resources toward recombinant protein and citramalate production, suggesting that it is an excellent chassis choice for manufacturing organic acids.IMPORTANCE Citramalate is an attractive biotechnology target because it is a precursor of methylmethacrylate, which is used to manufacture Perspex and other high-value products. Engineered E. coli strains are able to produce high titers of citramalate, despite having to express a foreign enzyme and tolerate the presence of a nonnative biochemical. A systems analysis of the citramalate fermentation was undertaken to uncover the reasons underpinning its productivity. This showed that E. coli readily adjusts to the redirection of metabolic resources toward recombinant protein and citramalate production and suggests that E. coli is an excellent chassis for manufacturing similar small, polar, foreign molecules.
Highlights
IMPORTANCE Citramalate is an attractive biotechnology target because it is a precursor of methylmethacrylate, which is used to manufacture Perspex and other highvalue products
After initial batch growth for 18.5 h, the bioprocess continued under a fed-batch regimen, and synthesis of inactive or active CimA3.7 was induced with L-arabinose at an optical density at 600 nm (OD600) of ϳ50
Characterization of the stresses experienced by bacterial cell factories during synthesis of recombinant proteins and chemical products is essential for improving process efficiency and designing new bacterial chassis
Summary
IMPORTANCE Citramalate is an attractive biotechnology target because it is a precursor of methylmethacrylate, which is used to manufacture Perspex and other highvalue products. Further work to eliminate their formation using chassis gene deletions (ackA-pta, aceB, glcB, gltA, leuCD, and poxB) resulted in much lower yields of citramalate [6,7,8] and increased process costs [5]. This prior work has demonstrated the potential to develop bio-based production of citramalate in a simple fed-batch process using a minimally engineered E. coli strain as a precursor for manufacturing MMA. Citramalate production had relatively few effects on the host transcriptome, proteome, lipidome, or metabolome, providing a simple explanation for the exceptional product titers and yields
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