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

Research output: Contribution to journalArticle

Authors

  • Martina Pasini
  • Alfonso Jaramillo
  • Carles De Mas
  • Gloria Caminal
  • Pau Ferrer

Colleges, School and Institutes

Abstract

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.

Details

Original languageEnglish
Pages (from-to)78-90
Number of pages13
JournalNew Biotechnology
Volume33
Issue number1
Early online date31 Aug 2015
Publication statusPublished - 25 Jan 2016