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Using promoter libraries to reduce metabolic burden due to plasmid-encoded proteins in recombinant Escherichia coli

Paper ID Volume ID Publish Year Pages File Format Full-Text
33039 44954 2016 13 PDF Available
Title
Using promoter libraries to reduce metabolic burden due to plasmid-encoded proteins in recombinant Escherichia coli
Abstract

•Minimization of the metabolic burden.•Develop of an antibiotic-free expression system, devoid of resistance markers.•Improvement of the recombinant FucA production.

The over-expression of proteins in recombinant host cells often requires a significant amount of resources causing an increase in the metabolic load for the host. This results in a variety of physiological responses leading to altered growth parameters, including growth inhibition or activation of secondary metabolism pathways. Moreover, the expression of other plasmid-encoded genes such as antibiotic resistance genes or repressor proteins may also alter growth kinetics.In this work, we have developed a second-generation system suitable for Escherichia coli expression with an antibiotic-free plasmid maintenance mechanism based on a glycine auxotrophic marker (glyA). Metabolic burden related to plasmid maintenance and heterologous protein expression was minimized by tuning the expression levels of the repressor protein (LacI) and glyA using a library of promoters and applying synthetic biology tools that allow the rapid construction of vectors. The engineered antibiotic-free expression system was applied to the l-fuculose phosphate aldolase (FucA) over-production, showing an increase in production up to 3.8-fold in terms of FucA yield (mg g−1DCW) and 4.5-fold in terms of FucA activity (AU g−1DCW) compared to previous expression. Moreover, acetic acid production was reduced to 50%, expressed as gAc gDCW−1.Our results showed that the aforementioned approaches are of paramount importance in order to increment the protein production in terms of mass and activity.

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Using promoter libraries to reduce metabolic burden due to plasmid-encoded proteins in recombinant Escherichia coli
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Publisher
Database: Elsevier - ScienceDirect
Journal: New Biotechnology - Volume 33, Issue 1, 25 January 2016, Pages 78–90
Authors
, , , , , ,
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
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