Turning the screw: engineering extreme pH resistance inEscherichia colithrough combinatorial synthetic operons

Abstract

Adoption of microorganisms as platforms for sustainable biobased production requires host cells to be able to withstand harsh industrial conditions, which are usually far from the ones where these organisms are naturally adapted to thrive. However, novel survival mechanisms unearthed by the study of microbiomes from extreme habitats may be exploited to enhance microbial robustness under the strict conditions needed for different applications. In this work, synthetic biology approaches were used to engineer enhanced acidic tolerance in Escherichia coli under extreme conditions through the characterization of a library of twenty-seven unique operons composed of combinatorial assemblies of three novel genes from an extreme environment and three synthetic ribosome binding sites. The results here presented illustrate the efficacy of combining different metagenomic genes for tolerance in truly synthetic genetic operons, as expression of these gene clusters increased hundred-fold the survival percentage of cells exposed to an acidic shock in minimal media at pH 1.9 under aerobic conditions.

Synthetic reconstruction of extreme high hydrostatic pressure resistance in Escherichia coli

“…Next to metabolic engineering, the building of stress-resistant microbial chassis for applications in bioprocessing under inhospitable conditions or environments is gaining increased attention. As such, the genetic basis for engineering acid-, osmotic-, heat-, and solvent-robust microbial chassis has already been established (Appukuttan et al, 2015;de Siqueira et al, 2020;Jia et al, 2016;Lennen and Herrgård, 2014;Mukhopadhyay, 2015;Swings et al, 2017). However, despite the biotechnological potential of HHP (Aertsen et al, 2009;Speranza, 2020), only little progress has been made on engineering HHP-robust microbial chassis because of the still cryptic multitarget impact of HHP on microbial physiology.…”

Strategies to improve the stress resistance of Escherichia coli in industrial biotechnology

“…21 Synthetic biological approaches can also be combined with other new emerging strategies to enhance the acidic resistance through the design of several unique operons composed of diverse combinations of three novel genes from an extreme environment and three synthetic ribosome binding sites. 22 Heat shock proteins (GroESL) could also be overexpressed to increase the cell growth and viability against alcohols including ethanol, 2-butanol, n-butanol, and 1,2,4-butanetriol. In particular, a 12-fold increase of microbial growth under 4% (v/v) ethanol occurred, which could be explained by that misfolded proteins damaged by toxic products were repaired with the help of GroESL.…”

Synthetic acid stress-tolerance modules improve growth robustness and lysine productivity of industrial Escherichia coli in fermentation at low pH

“…For example, in a study on yeast SyBE005, stress sensing promoters were used to regulate the superoxide dismutase gene SOD1, the glutamate-cysteine ligase gene GSH1, the glutathione-disulfide reductase gene GLR1 of the antioxidant system, the glucose-6-phosphate dehydrogenase gene ZWF1 of the glycolysis pathway, and the acetyl-CoA synthase gene ACS1, which is involved in the acetyl-CoA synthesis, leading to an improved ethanol titer by 49.5% at the shake flask scale [23]. In another study on Escherichia coli DH10B, the DNA binding protein gene hu, which is involved in DNA protection, the RNA binding protein gene rbp, which is involved in RNA protection, and the ATP-dependent serine protease gene clpP, which is involved in misfolded protein degradation, were overexpressed, leading to an increase in survival rate of over 600-fold for the best strain upon acid shock at pH 1.9 [24]. It is noteworthy that many studies on acid tolerance have focused on cell survival under extreme pH conditions, however efficient cell growth and productivity under moderate acidic conditions are more valuable for industrial applications [15,25].…”