De novo creation of MG1655-derived E. coli strains specifically designed for plasmid DNA production

The interest in plasmid DNA (pDNA) as a biopharmaceutical has been increasing over the last several years, especially after the approval of the first DNA vaccines. New pDNA production strains have been created by rationally mutating genes selected on the basis of Escherichia coli central metabolism and plasmid properties. Nevertheless, the highly mutagenized genetic background of the strains used makes it difficult to ascertain the exact impact of those mutations. To explore the effect of strain genetic background, we investigated single and double knockouts of two genes, pykF and pykA, which were known to enhance pDNA synthesis in two different E. coli strains: MG1655 (wild-type genetic background) and DH5α (highly mutagenized genetic background). The knockouts were only effective in the wild-type strain MG1655, demonstrating the relevance of strain genetic background and the importance of designing new strains specifically for pDNA production. Based on the obtained results, we created a new pDNA production strain starting from MG1655 by knocking out the pgi gene in order to redirect carbon flux to the pentose phosphate pathway, enhance nucleotide synthesis, and, consequently, increase pDNA production. GALG20 (MG1655ΔendAΔrecAΔpgi) produced 25-fold more pDNA (19.1 mg/g dry cell weight, DCW) than its parental strain, MG1655ΔendAΔrecA (0.8 mg/g DCW), in glucose. For the first time, pgi was identified as an important target for constructing a high-yielding pDNA production strain.MIT-Portugal ProgramFundação para a Ciência e a Tecnologia (project PTDC/ EBB-EBI/113650/2009, PhD grant SFRH/BD/33786/2009

Acetate fluxes in Escherichia coli are determined by the thermodynamic control of the Pta-AckA pathway

Escherichia coli excretes acetate upon growth on fermentable sugars, but the regulation of this production remains elusive. Acetate excretion on excess glucose is thought to be an irreversible process. However, dynamic (13)C-metabolic flux analysis revealed a strong bidirectional exchange of acetate between E. coli and its environment. The Pta-AckA pathway was found to be central for both flux directions, while alternative routes (Acs or PoxB) play virtually no role in glucose consumption. Kinetic modelling of the Pta-AckA pathway predicted that its flux is thermodynamically controlled by the extracellular acetate concentration in vivo. Experimental validations confirmed that acetate production can be reduced and even reversed depending solely on its extracellular concentration. Consistently, the Pta-AckA pathway can rapidly switch from acetate production to consumption. Contrary to current knowledge, E. coli is thus able to co-consume glucose and acetate under glucose excess. These metabolic capabilities were confirmed on other glycolytic substrates which support the growth of E. coli in the gut. These findings highlight the dual role of the Pta-AckA pathway in acetate production and consumption during growth on glycolytic substrates, uncover a novel regulatory mechanism that controls its flux in vivo, and significantly expand the metabolic capabilities of E. coli

High level biosynthesis of a silk-elastin-like protein in E. coli

Silk-elastin-like proteins (SELPs) have enormous potential for use as customizable biomaterials in numerous biomedical and materials applications yet success in harnessing this potential has been limited by the lack of a commercially viable industrially relevant production process. We have developed a scalable fed-batch production approach which enables a SELP volumetric productivity of 4.3 g L-1 with E. coli BL21(DE3). This is the highest SELP productivity reported to date and is 50-fold higher than that reported by other groups. As compared to typical fed-batch processes, high pre-induction growth rates and low inducer and oxygen concentrations are allowed whereas reduced post-induction feeding rates are preferred. Limiting factors were identified and productivity was found to be strongly influenced by a trade-off between the rate of production and plasmid stability. The process developed is robust, reproducible and applicable to scale up to the industrial level and moves these biopolymers a step closer to the marketplace.Fundação para a Ciência e a Tecnologia (FCT)This work was financed by the European Commission, via the 7th Framework Programme Project EcoPlast (FP7-NMP-2009- SME-3, collaborative project number 246176), by FEDER through POFC − COMPETE and by national funds from Fundação para a Ciência e Tecnologia (FCT) through PEst- OE/BIA/U14050/2014. RM acknowledges FCT for SFRHBPD/ 86470/2012 grant. TC is thankful to FCT for its support through Programa Ciência 2008