Integration of (S)-2,3-oxidosqualene enables E. coli to become Iron Man E. coli with improved overall tolerance

Abstract

Background: While representing a model bacterium and one of the most used chassis in biomanufacturing, performance of Escherichia coli is often limited by severe stresses. A super-robust E. coli chassis that could efficiently tolerant multiple severe stresses is thus highly desirable. Sterols represent a featured composition that distinguishes eukaryotes from bacteria and all archaea, and play a critical role in maintaining the membrane integrity of eukaryotes. All sterols found in nature are directly synthesized from (S)-2,3-oxidosqualene. However, in E. coli, (S)-2,3-oxidosqualene is not present.

Results: In this study, we sought to introduce (S)-2,3-oxidosqualene into E. coli. By mining and recruiting heterologous enzymes and activation of endogenous pathway, the ability of E. coli to synthesize (S)-2,3-oxidosqualene was demonstrated. Further analysis revealed that this non-native chemical confers E. coli with a robust and stable cell membrane, consistent with a figurative analogy of wearing an "Iron Man's armor"-like suit. The obtained Iron Man E. coli (IME) exhibited improved tolerance to multiple severe stresses, including high temperature, low pH, high salt, high sugar and reactive oxygen species (ROS). In particular, the IME strain shifted its optimal growth temperature from 37 °C to 42-45 °C, which represents the most heat-resistant E. coli to the best of our knowledge. Intriguingly, this non-native chemical also improved E. coli tolerance to a variety of toxic feedstocks, inhibitory products, as well as elevated synthetic capacities of inhibitory chemicals (e.g., 3-hydroxypropionate and fatty acids) due to improved products tolerance. More importantly, the IME strain was effectively inhibited by the most commonly used antibiotics and showed no undesirable drug resistance.

Conclusions: Introduction of the non-native (S)-2,3-oxidosqualene membrane lipid enabled E. coli to improve tolerance to various stresses. This study demonstrated the effectiveness of introducing eukaryotes-featured compound into bacteria for enhancing overall tolerance and chemical production.

Keywords: (S)-2,3-oxidosqualene; Chemicals production; E. coli; Severe stresses; Sterols biosynthesis.

Fig. 1

(S)-2,3-oxidosqualene serves as the common precursor of synthesis of all types of sterols. Multiple arrows represent more than one reaction step; squalene hopene cyclase: SHC; tetrahymanol synthase: THS. lanosterol synthases: LSS; cyloartenol synthases: CAS; cucurbitadienol synthases: CDS; α-amyrin synthases: α-AS; β-amyrin synthases: β-AS; lupeol synthases: LUS

Fig. 2

Introduction and optimization of (S)-2,3-oxidosqualene biosynthesis pathway in E. coli and exhibits decreased membrane leakage and increased intracellular ATP level. a Full biosynthetic pathway of (S)-2,3-oxidosqualene consisting of native MEP pathway and heterologous pathway; b integration of heterologous S. cerevisiae terg9 gene, Methylococcus capsulatus smo gene with M1–93 artificial promoter into genomic DNA of E. coli MG1655 at the mgsA site and pta site, respectively, based on the two pathways genes, the native promoter of idi was replaced with artificial promoter M1–46. c GC–MS detection of (S)-2,3-oxidosqualene for the engineered strains. The engineered strain with the heterologous pathway and promoter replacement of rate-limiting enzyme idi synthesized the representative (S)-2,3-oxidosqualene, TMS derivative while control strain not. d Engineered strain had a 24% decrease in membrane leakage relative to the control strain when challenged with 5 mM H₂O₂. Membrane leakage was assessed using the SYTOX Green nucleic acid stain. e Engineered strain had a 70% increase in ATP content relative to the control strain during challenge with 5 mM H₂O₂. ATP content was assessed using an ATP assay kit (Beyotime). Error bars indicate standard deviation of at least three biological replicates. f We termed the engineered strain Iron Man E. coli (IME) with a figurative analogy of wearing “Iron Man’s armor”-like suit for E. coli cell

Fig. 3

Synthesis of (S)-2,3-oxidosqualene in E. coli strain and resistance to adverse environmental stresses. a–d IME strain had improved thermotolerance, shifted its optimal growth temperature from 37 °C to 42–45 °C. e IME strain had improved tolerance to heat shock. f–h IME strain showed acid-resistant phenotype. i, no challenge. j–l IME strain showed improved tolerance to other adverse environmental stresses such as high salt condition, high sugar condition and ROS (H₂O₂). Error bars indicate standard deviation of at least three biological replicates

Fig. 4

IME improved tolerance to inhibitors in lignocellulose-derived feedstocks. a–c IME had improved tolerance to inhibitors existing in the hydrolysate of lignocellulose (HMF, levulinic acid and vanillic acid). Growth curves were recorded in MOPS + 2% glucose medium with different inhibitors in a clear bottom 96-well plate at 37 °C, pH 7.0. d IME showed increased cell mass and glucose consumption with addition of representative inhibitors existing in the hydrolysate of lignocellulose. Both control and IME strains were cultivated in MOPS + 2% (w/v) glucose mineral salt medium containing 16 mM HMF (2 g/L), 43 mM levulinic acid (5 g/L), and 6 mM vanillic acid (0.5 g/L) in shake flasks at 37 °C and an initial pH of 7.0. Error bars indicate standard deviation of at least three biological replicates. HMF, hydroxymethylfurfural

Fig. 5

IME improved tolerance to inhibitory organic acids and production of organic acids. a IME had improved tolerance to organic acids. Growth curves were recorded in MOPS + 2% glucose medium with different inhibitors in a clear bottom 96-well plate at 37 °C with an initial pH 7.0. Error bars indicate standard deviation of at least three biological replicates. b IME had improved tolerance to 3-HP and increased 3-HP production. Both control and IME strains with pTrc99a-dhaB-aldH plasmid were cultivated in MOPS + 2% (w/v) glycerol medium in shake flasks at 37 °C, initial pH 7.0 for 48 h. c IME showed improved tolerance to octanoic acid and increased fatty acids production. Both strains with pXZ18Z plasmid harboring the genes encoding RcTE and E. coli 3-hydroxy-acyl-ACP dehydratase (FabZ) were cultivated in MOPS + 2% (w/v) glucose medium in shake flasks at 30 °C, initial pH 7.0 for 48 h. Error bars indicate standard deviation of at least three biological replicates. 3-HPA, 3-hydroxypropionaldehyde; 3-HP, 3-hydroxypropionate; DhaB, glycerol dehydratase; AldH, aldehyde dehydrogenase; RcTE, Ricinus communis thioesterase. FabZ, 3-hydroxy-acyl-ACP dehydratase

Fig. 6

Sensitivity of the IME strain to various antibiotics. a IME and control strains showed no growth differences in the absence of antibiotics. The IME strain had increased sensitivity to b chloramphenicol; c erythromycin; d spectinomycin; e tetracycline; f streptomycin; g rifamycin; h sulfadiazine; i combination of sulfamethoxazole (SMZ) and trimethoprim (TMP); j polymyxin B; k carbenicillin; l meropenem. Growth curves were recorded in MOPS + 2% glucose medium with different antibiotics in a clear bottom 96-well plate at 37 °C, initial pH 7.0. Error bars indicate standard deviation of at least three biological replicates