Design of Synthetic Quorum Sensing Achieving Induction Timing-Independent Signal Stabilization for Dynamic Metabolic Engineering of E. coli

ABSTRACT Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is responsible for outbreaks of bloody diarrhea and hemolytic-uremic syndrome in many countries. EHEC virulence mechanisms include the production of Shiga toxins (Stx) and formation of attaching and effacing (AE) lesions on intestinal epithelial cells. We recently reported that genes involved in the formation of the AE lesion were regulated by quorum sensing through autoinducer-2, which is synthesized by the product of the luxS gene. In this study we hybridized an E. coli gene array with cDNA synthesized from RNA that was extracted from EHEC strain 86-24 and its isogenicluxS mutant. We observed that 404 genes were regulated by luxS at least fivefold, which comprises approximately 10% of the array genes; 235 of these genes were up-regulated and 169 were down-regulated in the wild-type strain compared to in theluxS mutant. Down-regulated genes included several involved in cell division, as well as ribosomal and tRNA genes. Consistent with this pattern of gene expression, theluxS mutant grows faster than the wild-type strain (generation times of 37.5 and 60 min, respectively, in Dulbecco modified Eagle medium). Up-regulated genes included several involved in the expression and assembly of flagella, motility, and chemotaxis. Using operon::lacZ fusions to class I, II, and III flagellar genes, we were able to confirm this transcriptional regulation. We also observed fewer flagella by Western blotting and electron microscopy and decreased motility halos in semisolid agar in the luxS mutant. The average swimming speeds for the wild-type strain and the luxS mutant are 12.5 and 6.6 μm/s, respectively. We also observed an increase in the production of Stx due to quorum sensing. Genes encoding Stx, which are transcribed along with λ-like phage genes, are induced by an SOS response, and genes involved in the SOS response were also regulated by quorum sensing. These results indicate that quorum sensing is a global regulatory mechanism for basic physiological functions of E. coli as well as for virulence factors.

Download Full-text

Metabolic engineering of Escherichia coli for de novo production of 3-phenylpropanol via retrobiosynthesis approach

Microbial Cell Factories ◽

10.1186/s12934-021-01615-1 ◽

2021 ◽

Vol 20 (1) ◽

Author(s):

Zhenning Liu ◽

Xue Zhang ◽

Dengwei Lei ◽

Bin Qiao ◽

Guang-Rong Zhao

Keyword(s):

Escherichia Coli ◽

Metabolic Engineering ◽

De Novo ◽

Microbial Cell ◽

Cell Factory ◽

Phosphopantetheinyl Transferase ◽

Chromosome Engineering ◽

Microbial Cell Factory ◽

E Coli ◽

Artificial Pathway

Abstract Background 3-Phenylpropanol with a pleasant odor is widely used in foods, beverages and cosmetics as a fragrance ingredient. It also acts as the precursor and reactant in pharmaceutical and chemical industries. Currently, petroleum-based manufacturing processes of 3-phenypropanol is environmentally unfriendly and unsustainable. In this study, we aim to engineer Escherichia coli as microbial cell factory for de novo production of 3-phenypropanol via retrobiosynthesis approach. Results Aided by in silico retrobiosynthesis analysis, we designed a novel 3-phenylpropanol biosynthetic pathway extending from l-phenylalanine and comprising the phenylalanine ammonia lyase (PAL), enoate reductase (ER), aryl carboxylic acid reductase (CAR) and phosphopantetheinyl transferase (PPTase). We screened the enzymes from plants and microorganisms and reconstructed the artificial pathway for conversion of 3-phenylpropanol from l-phenylalanine. Then we conducted chromosome engineering to increase the supply of precursor l-phenylalanine and combined the upstream l-phenylalanine pathway and downstream 3-phenylpropanol pathway. Finally, we regulated the metabolic pathway strength and optimized fermentation conditions. As a consequence, metabolically engineered E. coli strain produced 847.97 mg/L of 3-phenypropanol at 24 h using glucose-glycerol mixture as co-carbon source. Conclusions We successfully developed an artificial 3-phenylpropanol pathway based on retrobiosynthesis approach, and highest titer of 3-phenylpropanol was achieved in E. coli via systems metabolic engineering strategies including enzyme sources variety, chromosome engineering, metabolic strength balancing and fermentation optimization. This work provides an engineered strain with industrial potential for production of 3-phenylpropanol, and the strategies applied here could be practical for bioengineers to design and reconstruct the microbial cell factory for high valuable chemicals.

Download Full-text

Escherichia coli as a platform microbial host for systems metabolic engineering

Essays in Biochemistry ◽

10.1042/ebc20200172 ◽

2021 ◽

Author(s):

Dongsoo Yang ◽

Cindy Pricilia Surya Prabowo ◽

Hyunmin Eun ◽

Seon Young Park ◽

In Jin Cho ◽

...

Keyword(s):

Escherichia Coli ◽

Natural Products ◽

Metabolic Engineering ◽

Food Ingredients ◽

E Coli ◽

Renewable Biomass ◽

Microbial Cell Factories ◽

Cell Factories ◽

Systems Metabolic Engineering ◽

Microbial Host

Abstract Bio-based production of industrially important chemicals and materials from non-edible and renewable biomass has become increasingly important to resolve the urgent worldwide issues including climate change. Also, bio-based production, instead of chemical synthesis, of food ingredients and natural products has gained ever increasing interest for health benefits. Systems metabolic engineering allows more efficient development of microbial cell factories capable of sustainable, green, and human-friendly production of diverse chemicals and materials. Escherichia coli is unarguably the most widely employed host strain for the bio-based production of chemicals and materials. In the present paper, we review the tools and strategies employed for systems metabolic engineering of E. coli. Next, representative examples and strategies for the production of chemicals including biofuels, bulk and specialty chemicals, and natural products are discussed, followed by discussion on materials including polyhydroxyalkanoates (PHAs), proteins, and nanomaterials. Lastly, future perspectives and challenges remaining for systems metabolic engineering of E. coli are discussed.

Download Full-text

In vitro and in situ abrogation of biofilm formation in E. coli by vitamin C through ROS generation, disruption of quorum sensing and exopolysaccharide production

Food Chemistry ◽

10.1016/j.foodchem.2020.128171 ◽

2021 ◽

Vol 341 ◽

pp. 128171

Author(s):

DP Shivaprasad ◽

Neetu Kumra Taneja ◽

Anupama Lakra ◽

Divya Sachdev

Keyword(s):

Quorum Sensing ◽

Vitamin C ◽

Biofilm Formation ◽

Ros Generation ◽

Exopolysaccharide Production ◽

E Coli

Download Full-text

Metabolic engineering for glycoglycerolipids production in E. coli: Tuning phosphatidic acid and UDP-glucose pathways

Metabolic Engineering ◽

10.1016/j.ymben.2020.05.010 ◽

2020 ◽

Vol 61 ◽

pp. 106-119

Author(s):

Nuria Orive-Milla ◽

Tom Delmulle ◽

Marjan de Mey ◽

Magda Faijes ◽

Antoni Planas

Keyword(s):

Metabolic Engineering ◽

Phosphatidic Acid ◽

E Coli

Download Full-text

Metabolic engineering of the E. coli l-phenylalanine pathway for the production of d-phenylglycine (d-Phg)

Metabolic Engineering ◽

10.1016/j.ymben.2005.12.001 ◽

2006 ◽

Vol 8 (3) ◽

pp. 196-208 ◽

Cited By ~ 44

Author(s):

Ulrike Müller ◽

Friso van Assema ◽

Michele Gunsior ◽

Sonja Orf ◽

Susanne Kremer ◽

...

Keyword(s):

Metabolic Engineering ◽

E Coli

Download Full-text

Novel Quorum-Sensing Peptides Mediating Interspecies Bacterial Cell Death

mBio ◽

10.1128/mbio.00314-13 ◽

2013 ◽

Vol 4 (3) ◽

Cited By ~ 35

Author(s):

Sathish Kumar ◽

Ilana Kolodkin-Gal ◽

Hanna Engelberg-Kulka

Keyword(s):

Pseudomonas Aeruginosa ◽

Cell Death ◽

Bacillus Subtilis ◽

Quorum Sensing ◽

Bacterial Cell ◽

Content Type ◽

E Coli ◽

New Class ◽

Bacterial Cell Death ◽

Induced Module

ABSTRACTEscherichia colimazEFis a toxin-antitoxin stress-induced module mediating cell death. It requires the quorum-sensing signal (QS) “extracellular death factor” (EDF), the penta-peptide NNWNN (EcEDF), enhancing the endoribonucleolytic activity ofE. colitoxin MazF. Here we discovered thatE. coli mazEF-mediated cell death could be triggered by QS peptides from the supernatants (SN) of the Gram-positive bacteriumBacillus subtilisand the Gram-negative bacteriumPseudomonas aeruginosa. In the SN ofB. subtilis, we found one EDF, the hexapeptide RGQQNE, calledBsEDF. In the SN ofP. aeruginosa, we found three EDFs: the nonapeptide INEQTVVTK, calledPaEDF-1, and two hexadecapeptides, VEVSDDGSGGNTSLSQ, calledPaEDF-2, and APKLSDGAAAGYVTKA, calledPaEDF-3. When added to a dilutedE. colicultures, each of these peptides acted as an interspecies EDF that triggeredmazEF-mediated death. Furthermore, though their sequences are very different, each of these EDFs amplified the endoribonucleolytic activity ofE. coliMazF, probably by interacting with different sites onE. coliMazF. Finally, we suggest that EDFs may become the basis for a new class of antibiotics that trigger death from outside the bacterial cells.IMPORTANCEBacteria communicate with one another via quorum-sensing signal (QS) molecules. QS provides a mechanism for bacteria to monitor each other’s presence and to modulate gene expression in response to population density. Previously, we addedE. coliEDF (EcEDF), the peptide NNWNN, to this list of QS molecules. Here we extended the group of QS peptides to several additional different peptides. The new EDFs are produced by two other bacteria,Bacillus subtilisandPseudomonas aeruginosa. Thus, in this study we established a “new family of EDFs.” This family provides the first example of quorum-sensing molecules participating in interspecies bacterial cell death. Furthermore, each of these peptides provides the basis of a new class of antibiotics triggering death by acting from outside the cell.

Download Full-text