Increasing the Pentose Phosphate Pathway Flux to Improve Plasmid DNA Production in Engineered <i>E. coli</i>
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 <i>E. coli</i> strain lacking the phosphotransferase system and the pyruvate kinase A...
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MDPI AG
2024-01-01
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| Series: | Microorganisms |
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| Online Access: | https://www.mdpi.com/2076-2607/12/1/150 |
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| author | Mitzi de la Cruz Flavio Kunert Hilal Taymaz-Nikerel Juan-Carlos Sigala Guillermo Gosset Jochen Büchs Alvaro R. Lara |
| author_facet | Mitzi de la Cruz Flavio Kunert Hilal Taymaz-Nikerel Juan-Carlos Sigala Guillermo Gosset Jochen Büchs Alvaro R. Lara |
| author_sort | Mitzi de la Cruz |
| collection | DOAJ |
| description | 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 <i>E. coli</i> 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. |
| first_indexed | 2024-03-08T09:49:47Z |
| format | Article |
| id | doaj.art-85225fdfb16a458787de813dd9d8e949 |
| institution | Directory Open Access Journal |
| issn | 2076-2607 |
| language | English |
| last_indexed | 2024-03-08T09:49:47Z |
| publishDate | 2024-01-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Microorganisms |
| spelling | doaj.art-85225fdfb16a458787de813dd9d8e9492024-01-29T14:06:38ZengMDPI AGMicroorganisms2076-26072024-01-0112115010.3390/microorganisms12010150Increasing the Pentose Phosphate Pathway Flux to Improve Plasmid DNA Production in Engineered <i>E. coli</i>Mitzi de la Cruz0Flavio Kunert1Hilal Taymaz-Nikerel2Juan-Carlos Sigala3Guillermo Gosset4Jochen Büchs5Alvaro R. Lara6Departamento de Procesos y Tecnología, Universidad Autónoma Metropolitana, Mexico City 05348, MexicoChair of Biochemical Engineering (AVT.BioVT), RWTH Aachen University, 52074 Aachen, GermanyDepartment of Genetics and Bioengineering, Istanbul Bilgi University, 34060 Istanbul, TurkeyDepartamento de Procesos y Tecnología, Universidad Autónoma Metropolitana, Mexico City 05348, MexicoInstituto de Biotecnología, Universidad Nacional Autónoma de México, Cuernavaca 62210, MexicoChair of Biochemical Engineering (AVT.BioVT), RWTH Aachen University, 52074 Aachen, GermanyDepartment of Biological and Chemical Engineering, Aarhus University, 8000 Aarhus, DenmarkThe 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 <i>E. coli</i> 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.https://www.mdpi.com/2076-2607/12/1/150plasmid DNA vaccinespentose phosphate pathwayglucose-glycerol co-utilization |
| spellingShingle | Mitzi de la Cruz Flavio Kunert Hilal Taymaz-Nikerel Juan-Carlos Sigala Guillermo Gosset Jochen Büchs Alvaro R. Lara Increasing the Pentose Phosphate Pathway Flux to Improve Plasmid DNA Production in Engineered <i>E. coli</i> Microorganisms plasmid DNA vaccines pentose phosphate pathway glucose-glycerol co-utilization |
| title | Increasing the Pentose Phosphate Pathway Flux to Improve Plasmid DNA Production in Engineered <i>E. coli</i> |
| title_full | Increasing the Pentose Phosphate Pathway Flux to Improve Plasmid DNA Production in Engineered <i>E. coli</i> |
| title_fullStr | Increasing the Pentose Phosphate Pathway Flux to Improve Plasmid DNA Production in Engineered <i>E. coli</i> |
| title_full_unstemmed | Increasing the Pentose Phosphate Pathway Flux to Improve Plasmid DNA Production in Engineered <i>E. coli</i> |
| title_short | Increasing the Pentose Phosphate Pathway Flux to Improve Plasmid DNA Production in Engineered <i>E. coli</i> |
| title_sort | increasing the pentose phosphate pathway flux to improve plasmid dna production in engineered i e coli i |
| topic | plasmid DNA vaccines pentose phosphate pathway glucose-glycerol co-utilization |
| url | https://www.mdpi.com/2076-2607/12/1/150 |
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