Abstract
The design of optimal cell factories requires engineering resource allocation for maximizing product synthesis. A recently developed method to maximize the saving in cell resources released 0.5% of the proteome of Escherichia coli by deleting only three transcription factors. We assessed the capacity for plasmid DNA (pDNA) production in the proteome-reduced strain in a mineral medium, lysogeny, and terrific broths. In all three cases, the pDNA yield from biomass was between 33 and 53% higher in the proteome-reduced than in its wild type strain. When cultured in fed-batch mode in shake-flask, the proteome-reduced strain produced 74.8 mg L−1 pDNA, which was four times greater than its wild-type strain. Nevertheless, the pDNA supercoiled fraction was less than 60% in all cases. Deletion of recA increased the pDNA yields in the wild type, but not in the proteome-reduced strain. Furthermore, recA mutants produced a higher fraction of supercoiled pDNA, compared to their parents. These results show that the novel proteome reduction approach is a promising starting point for the design of improved pDNA production hosts.
Highlights
The availability of production strains optimized for industrial bioprocesses is critical for successful commercialization of biotechnologies
There are, to the best of our knowledge, no reports on the use of proteome-reduced strains for this purpose. pUC57kan production in the proteome-reduced strain was studied in a mineral medium and two complex media widely used in small-scale cultures: lysogeny broth (LB) and terrific broth (TB)
YpDNA/X for strain PFC was 33, 54, and 40% higher in mineral media, LB and TB, respectively, than for the wild type strain
Summary
The availability of production strains optimized for industrial bioprocesses is critical for successful commercialization of biotechnologies. An interesting approach to develop minimal cells is the elimination of genome sections considered dispensable under bioprocess conditions [2]. Such so-called genome-reduction approach has been employed in Escherichia coli by several groups [3]. A different approach consists in the identification of proteins not essential under process conditions and removing the burden of their expression [4] This would generate an optimal resource allocation that maximizes the designed cell function [5]. Lastiri and co-workers [6] developed a method to engineer the resource allocation of Escherichia coli This method identifies the minimum combinatorial set of genetic interventions that maximizes resource savings, which they applied by removing transcription factors that activate the expression of unused functions with the greater proteomic load. Fed-batch cultures in shake flasks were carried out in order to test the strain under conditions closer to industrial scales
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