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Reduction of acetate accumulation in Escherichia coli cultures for increased recombinant protein production

Paper ID Volume ID Publish Year Pages File Format Full-Text
31907 44851 2008 12 PDF Available
Title
Reduction of acetate accumulation in Escherichia coli cultures for increased recombinant protein production
Abstract

The culture of Escherichia coli for the commercial production of recombinant proteins has increased significantly in recent years. The production of acetate as a byproduct retards cell growth, inhibits protein formation, and diverts carbon from biomass to protein product. Our approach to reducing acetate accumulation was to disable the phosphoenolpyruvate:sugar phosphotransferase system (PEP–PTS) by deleting the ptsHI operon in the wild-type E. coli strain GJT001. The mutation caused a severe reduction in growth rate and glucose uptake rate in glucose-supplemented M9 minimal medium, which confirmed the mutation, and eliminated acetate accumulation. The mutant strain (TC110) apparently metabolized glucose by a non-PTS mechanism that we are currently investigating, followed by phosphorylation by glucokinase. In complex medium such as 2×LB broth with 2% glucose, TC110 was able to grow quickly and still retained the phenotype of significantly reduced acetate accumulation (9.1±6.6 vs. 90.4±1.6 mM in GJT001, P<0.05). The reduced acetate accumulation resulted in a significant improvement in final OD (23.5±0.7 in TC110 vs. 8.0±0.1 in GJT001, P<0.05). We tested the strains for the production of model recombinant proteins such as green fluorescent protein (GFP) and β-galactosidase. TC110 had a 385-fold improvement in final volumetric productivity of GFP over GJT001 in shake flasks with 2×LB broth with 2% glucose. The distribution of GFP fluorescence in the cell population, as determined by flow cytometry, was much broader in GJT001 (coefficient of variation=466±35%) than in TC110 (coefficient of variation=55±1%). In corn steep liquor medium with 2% glucose, we observed a 28.5-fold improvement in final volumetric production of GFP in TC110 over GJT001. TC110 had a 7.5-fold improvement in final volumetric productivity of β-galactosidase over GJT001 in 2×LB broth with 2% glucose medium. When tested in a batch bioreactor cultures with 2×LB broth with 2% glucose medium, the volumetric production of GFP by TC110 was 25-fold higher than that of GJT001. In summary, the ptsHI mutant of GJT001 resulted in reduced acetate accumulation, which led to significant improvements in recombinant protein production in batch bioreactors.

Keywords
Reduce acetate; E. coli; GFP; β-galactosidase; ptsHI deletion
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Reduction of acetate accumulation in Escherichia coli cultures for increased recombinant protein production
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Publisher
Database: Elsevier - ScienceDirect
Journal: Metabolic Engineering - Volume 10, Issue 2, March 2008, Pages 97–108
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us