Growth-dependent recombinant product formation kinetics can be reproduced through engineering of glucose transport and is prone to phenotypic heterogeneity

Abstract

BackgroundEscherichia coli W3110 and a group of six isogenic derivatives, each displaying distinct specific rates of glucose consumption were characterized to determine levels of GFP production and population heterogeneity. These strains have single or combinatory deletions in genes encoding phosphoenolpyruvate:sugar phosphotransferase system (PTS) permeases as PtsG and ManX, as well as common components EI, Hpr protein and EIIA, also the non-PTS Mgl galactose/glucose ABC transporter. They have been transformed for expressing GFP based on a lac-based expression vector, which is subject to bistability.ResultsThese strains displayed specific glucose consumption and growth rates ranging from 1.75 to 0.45 g/g h and 0.54 to 0.16 h−1, respectively. The rate of acetate production was strongly reduced in all mutant strains when compared with W3110/pV21. In bioreactor cultures, wild type W3110/pV21 produced 50.51 mg/L GFP, whereas strains WG/pV21 with inactive PTS IICBGlc and WGM/pV21 with the additional inactivation of PTS IIABMan showed the highest titers of GFP, corresponding to 342 and 438 mg/L, respectively. Moreover, we showed experimentally that bistable expression systems, as lac-based ones, induce strong phenotypic segregation among microbial populations.ConclusionsWe have demonstrated that reduction on glucose consumption rate in E. coli leads to an improvement of GFP production. Furthermore, from the perspective of phenotypic heterogeneity, we observed in this case that heterogeneous systems are also the ones leading to the highest performance. This observation suggests reconsidering the generally accepted proposition stating that phenotypic heterogeneity is generally unwanted in bioprocess applications.Electronic supplementary materialThe online version of this article (10.1186/s12934-019-1073-5) contains supplementary material, which is available to authorized users.

Engineered chromosome-based T7 RNA polymerase in Escherichia coli W3110 for orthogonal T7 promoter circuit as a cell factory

“…reported with high capability of withstanding different toxic chemicals [21][22]. Consequently, W3110 is a suitable strain to be equipped with the T7RNAP onto the chromosome.…”

“…Alternatively, the K-12 strains such as MG1655 and W3110, are commonly used as they express more heat shock genes, are less sensitive to certain stress and also possess higher rates of glucose consumption [21][22]. A comparative proteomics study between BL21 and W3110 manifested that W3110 maintained growth and metabolism at lower oxygen levels, thus enabling foreign protein to be gradually expressed [23].…”

The Rocky Road From Fed-Batch to Continuous Processing With E. coli

“…Decreased enzyme availability might lead to high sugar accumulation in the fermentation broth, especially when exponential feeding is applied (Slouka et al, 2018a), as already stressed cells might additionally suffer from osmotic stress, triggering cell death (Slouka et al, 2019). It is shown that high sugar uptake rates tend to shift toward acetate formation, finally leading to decreased levels of production (Fragoso-Jiménez et al, 2019). Therefore, appropriate feeding rates beneath μ max should be set to avoid overflow metabolism; however, acetate production can also be significantly reduced using engineered strains, as shown by Lara et al (2008), Valgepea et al (2010), Peebo et al (2015), and Anane et al (2017).…”

“…A reduction of cell densities, temperature, or separation of biomass growth and RPP might facilitate stability in continuous processes (Rugbjerg and Sommer, 2019). Enabling higher sugar uptake with engineered strains shows that specific productivity and specific sugar uptake rates were correlated in a bell-shaped curve; extreme sugar uptake rates lead to fermentative growth (Basan et al, 2015; Peebo et al, 2015; Basan, 2018; Fragoso-Jiménez et al, 2019). The metabolic load therefore has to be considered, and feeding strategies and model-based approaches that consider the shifts in the growth rate have to be established (Kopp et al, 2018).…”

“…Baert et al also used flow cytometry as a tool to determine variations in phenotypes (Baert et al, 2015). Cell filamentation and its correlation with high productive subpopulations are also monitored with flow cytometry in fed-batch cultivations (Fragoso-Jiménez et al, 2019). Measuring filamented populations via PI-staining showed that the filamented subpopulation had enhanced PI uptake, forming the so-called red but not dead phenotype (Shi et al, 2007; Davey and Hexley, 2011).…”