Biomanufacturing Potential of Streamlined Cells

Abstract

A series of Escherichia coli streamlined strains was developed by removing the expression of genes encoding extracellular structures and unessential enzymes. The streamlined strains exhibited improved metabolic performance, including lower overflow metabolism and ATP maintenance coefficient, as well as a higher growth rate, compared to their parental strain. The intracellular levels of ATP were monitored using a genetic sensor, showing the improved resource stewardship of the streamlined cells. The streamlined strains were tested as cell factories to produce plasmid DNA (pDNA) in batch cultures, exhibiting a 23% increase in the specific pDNA production rate, compared to the parental strain. Recombinant protein expression was evaluated in microbioreactors in batch and fed-batch modes. In batch mode, recombinant protein yield from biomass was up to 82% higher in the streamlined strains than in the parental strain. Furthermore, in fed-batch mode, the recombinant protein yield was 79% greater in the streamlined cells compared to the parental strain. Our results show the benefits of reducing cellular complexity on the biomanufacturing of pDNA and recombinant proteins in culture schemes typical of industrial settings.

Keywords: ATP maintenance; microbial engineering; minimal cells; proteome reduction.

Figure 1

Schemes of the genetic circuits used. (a) ATP sensor. (b) Constitutive protein generator.

Figure 2

Specific rates and yields of the wild type (WT) and streamlined strains (PR01‐PR06) in batch cultures. (a) specific growth rate; (b) biomass yield on glucose; (c) acetate yield on glucose; (d) specific glucose uptake rate; (e) specific acetate production rate; (f) specific oxygen consumption rate. All values were calculated during the exponential growth phase. Error bars indicate the standard deviation between replicates (n = 3). * Indicates significant difference (p < 0.05), compared to the WT strain under the same conditions.

Figure 3

Estimated specific ATP consumption rates for cellular maintenance (m _ATP ) for the different strains. Values were calculated via FBA as ATP maintenance requirement in the metabolic model (Taymaz‐Nikerel et al. 2010).

Figure 4

(a) Culture profile of strains bearing the ATP sensor. Biomass was monitored by scattered light intensity (ScL). Shaded bands indicate the standard deviation between replicates. (b) Specific GFP fluorescence calculated over the exponential growth phase. Vertical lines indicate the standard deviation between replicates. The specific fluorescence of all the streamlined strains was significantly different from that of the WT (p < 0.05).

Figure 5

Performance of the different strains producing pDNA in batch cultures. (a) Specific growth rate; (b) pDNA yield from biomass; (c) specific pDNA production rate; (d) pDNA supercoiled fraction. The values were determined during the exponential growth phase. * Indicates significant difference (p < 0.05), compared to the WT strain under the same conditions.

Figure 6

(a) Batch culture profile of strains expressing GFP constitutively. Biomass was monitored by scattered light intensity (ScL). Shaded bands indicate the standard deviation between replicates. (b) Specific GFP fluorescence calculated over the exponential growth phase. (c) Specific GFP fluorescence emission rate calculated over the exponential growth phase. Vertical lines indicate the standard deviation between replicates. The values in (b) and (c) of all the streamlined strains were significantly different from those of the WT (p < 0.05).

Figure 7

(a) Fed‐batch culture profiles of strains expressing GFP constitutively. Biomass was monitored by scattered light intensity (ScL). Shaded bands indicate the standard deviation between replicates. (b) Specific GFP fluorescence calculated over 40 to 48 h of culture. The values in (b) of all the streamlined strains were significantly different from those of the WT (p < 0.05).