Plasmid DNA Production in Proteome-Reduced Escherichia coli

Cruz, Mitzi de la; Ramírez, Elisa A.; Sigala, Juan-Carlos; Utrilla, José; Lara, Alvaro R.

doi:10.3390/microorganisms8091444

Cited by 10 publications

(7 citation statements)

References 30 publications

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“…In this work, we provide a straightforward path to pursue when designing a strain with a higher potential to divert resources to a function of interest whilst performing fewer genetic interventions. Those resource‐minimized strains should perform better as microbial production hosts than their non‐improved counterparts (de la Cruz et al, 2020 ; Mizoguchi et al, 2008 ; Park et al, 2014 ).…”

Section: Discussionmentioning

confidence: 99%

Engineering resource allocation in artificially minimized cells: Is genome reduction the best strategy?

Marquez‐Zavala

Utrilla

2023

Microbial Biotechnology

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The elimination of the expression of cellular functions that are not needed in a certain well-defined artificial environment, such as those used in industrial production facilities, has been the goal of many cellular minimization projects. The generation of a minimal cell with reduced burden and less host-function interactions has been pursued as a tool to improve microbial production strains. In this work, we analysed two cellular complexity reduction strategies: genome and proteome reduction. With the aid of an absolute proteomics data set and a genome-scale model of metabolism and protein expression (MEmodel), we quantitatively assessed the difference of reducing genome to the correspondence of reducing proteome. We compare the approaches in terms of energy consumption, defined in ATP equivalents. We aim to show what is the best strategy for improving resource allocation in minimized cells. Our results show that genome reduction by length is not proportional to reducing resource use. When we normalize calculated energy savings, we show that strains with the larger calculated proteome reduction show the largest resource use reduction. Furthermore, we propose that reducing highly expressed proteins should be the target as the translation of a gene uses most of the energy. The strategies proposed here should guide cell design when the aim of a project is to reduce the maximum amount or cellular resources.

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Section: Discussionmentioning

confidence: 99%

Engineering resource allocation in artificially minimized cells: Is genome reduction the best strategy?

Marquez‐Zavala

Utrilla

2023

Microbial Biotechnology

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“…Strain VH34 is a Δ ptsI , ptsH , crr :: km , ΔP galP ::P trc , pykA :: cat derivative of W3110 [ 14 ]. The recA gene was further deleted in each strain as described elsewhere [ 16 ]. The strain VH34recA − is named here as VH36.…”

Section: Methodsmentioning

confidence: 99%

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

de la Cruz,

Kunert,

Taymaz-Nikerel

et al. 2024

Microorganisms

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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.

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“…Proteome reduction to develop minimal cells has been reported for E. coli [37]. Proteome-reduced E. coli strains have a superior performance for plasmid DNA vaccines production in batch and fed-batch mode [38]. However, overflow metabolism in proteome-reduced cells has not yet been thoroughly tested.…”

Section: Proteome Allocationmentioning

confidence: 99%

Vitreoscilla Haemoglobin: A Tool to Reduce Overflow Metabolism

Taymaz-Nikerel

Lara

2021

Microorganisms

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Overflow metabolism is a phenomenon extended in nature, ranging from microbial to cancer cells. Accumulation of overflow metabolites pose a challenge for large-scale bioprocesses. Yet, the causes of overflow metabolism are not fully clarified. In this work, the underlying mechanisms, reasons and consequences of overflow metabolism in different organisms have been summarized. The reported effect of aerobic expression of Vitreoscilla haemoglobin (VHb) in different organisms are revised. The use of VHb to reduce overflow metabolism is proposed and studied through flux balance analysis in E. coli at a fixed maximum substrate and oxygen uptake rates. Simulations showed that the presence of VHb increases the growth rate, while decreasing acetate production, in line with the experimental measurements. Therefore, aerobic VHb expression is considered a potential tool to reduce overflow metabolism in cells.

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Plasmid DNA Production in Proteome-Reduced Escherichia coli

Cited by 10 publications

References 30 publications

Engineering resource allocation in artificially minimized cells: Is genome reduction the best strategy?

Engineering resource allocation in artificially minimized cells: Is genome reduction the best strategy?

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

Vitreoscilla Haemoglobin: A Tool to Reduce Overflow Metabolism

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