Increasing the Pentose Phosphate Pathway Flux to Improve Plasmid DNA Production in Engineered E. coli

Author:

de la Cruz Mitzi1,Kunert Flavio2ORCID,Taymaz-Nikerel Hilal3ORCID,Sigala Juan-Carlos1,Gosset Guillermo4,Büchs Jochen2ORCID,Lara Alvaro R.5ORCID

Affiliation:

1. Departamento de Procesos y Tecnología, Universidad Autónoma Metropolitana, Mexico City 05348, Mexico

2. Chair of Biochemical Engineering (AVT.BioVT), RWTH Aachen University, 52074 Aachen, Germany

3. Department of Genetics and Bioengineering, Istanbul Bilgi University, 34060 Istanbul, Turkey

4. Instituto de Biotecnología, Universidad Nacional Autónoma de México, Cuernavaca 62210, Mexico

5. Department of Biological and Chemical Engineering, Aarhus University, 8000 Aarhus, Denmark

Abstract

The demand of plasmid DNA (pDNA) as a key element for gene therapy products, as well as mRNA and DNA vaccines, is increasing together with the need for more efficient production processes. An engineered E. coli strain lacking the phosphotransferase system and the pyruvate kinase A gene has been shown to produce more pDNA than its parental strain. With the aim of improving pDNA production in the engineered strain, several strategies to increase the flux to the pentose phosphate pathway (PPP) were evaluated. The simultaneous consumption of glucose and glycerol was a simple way to increase the growth rate, pDNA production rate, and supercoiled fraction (SCF). The overexpression of key genes from the PPP also improved pDNA production in glucose, but not in mixtures of glucose and glycerol. Particularly, the gene coding for the glucose 6-phosphate dehydrogenase (G6PDH) strongly improved the SCF, growth rate, and pDNA production rate. A linear relationship between the G6PDH activity and pDNA yield was found. A higher flux through the PPP was confirmed by flux balance analysis, which also estimates relevant differences in fluxes of the tricarboxylic acid cycle. These results are useful for developing further cell engineering strategies to increase pDNA production and quality.

Funder

CONAHCyT

MDPI

Publisher

MDPI AG

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