Reduced evolvability of <Emphasis Type="Italic">Escherichia coli</Emphasis> MDS42, an IS-less cellular chassis for molecular and synthetic biology applications

Background  

Evolvability is an intrinsic feature of all living cells. However, newly emerging, evolved features can be undesirable when genetic circuits, designed and fabricated by rational, synthetic biological approaches, are installed in the cell. Streamlined-genome E. coli MDS42 is free of mutation-generating IS elements, and can serve as a host with reduced evolutionary potential.     

The new pLAI (<Emphasis Type="Italic">lux</Emphasis> regulon based auto-inducible) expression system for recombinant protein production in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Background  

After many years of intensive research, it is generally assumed that no universal expression system can exist for high-level production of a given recombinant protein. Among the different expression systems, the inducible systems are the most popular for their tight regulation. However, induction is in many cases less favorable due to the high cost and/or toxicity of inducers, incompatibilities with industrial scale-up or detrimental growth conditions. Expression systems using autoinduction (or self-induction) prove to be extremely versatile allowing growth and induction of recombinant proteins without the need to monitor cell density or add inducer. Unfortunately, almost all the actual auto inducible expression systems need endogenous or induced metabolic changes during the growth to trigger induction, both frequently linked to detrimental condition to cell growth. In this context, we use a simple modular approach for a cell density-based genetic regulation in order to assemble an autoinducible recombinant protein expression system in E. coli.     

Extracellular overexpression of recombinant <Emphasis Type="Italic">Thermobifida fusca</Emphasis> cutinase by alpha-hemolysin secretion system in <Emphasis Type="Italic">E. coli</Emphasis> BL21(DE3)

Background  

Extracellular expression of proteins has an absolute advantage in a large-scale industrial production. In our previous study, Thermobifida fusca cutinase, an enzyme mainly utilized in textile industry, was expressed via type II secretory system in Escherichia coli BL21(DE3), and it was found that parts of the expressed protein was accumulated in the periplasmic space. Due to the fact that alpha-hemolysin secretion system can export target proteins directly from cytoplasm across both cell membrane of E. coli to the culture medium, thus in the present study we investigated the expression of cutinase using this alpha-hemolysin secretion system.     

In vivo observation of gold nanoparticles in the central nervous system of <Emphasis Type="Italic">Blaberus discoidalis</Emphasis>

Background  

Nanoparticles (NPs) are widely studied for biomedical applications. Understanding interactions between NPs and biomolecules or cells has yet to be achieved. Here we present a novel in vivo method to study interactions between NPs and the nervous system of the discoid or false dead-head roach, Blaberus discoidalis. The aims of this study were to present a new and effective method to observe NPs in vivo that opens the door to new methods of study to observe the interactions between NPs and biological systems and to present an inexpensive and easy-to-handle biological system.     

Overproduced <Emphasis Type="Italic">Brucella abortus</Emphasis> PdhS-mCherry forms soluble aggregates in <Emphasis Type="Italic">Escherichia coli</Emphasis>, partially associating with mobile foci of IbpA-YFP

Background  

When heterologous recombinant proteins are produced in Escherichia coli, they often precipitate to form insoluble aggregates of unfolded polypeptides called inclusion bodies. These structures are associated with chaperones like IbpA. However, there are reported cases of "non-classical" inclusion bodies in which proteins are soluble, folded and active.     

The Escherichia coli BarA-UvrY two-component system is a virulence determinant in the urinary tract

Background  

The Salmonella enterica BarA-SirA, the Erwinia carotovora ExpS-ExpA, the Vibrio cholerae BarA-VarA and the Pseudomonas spp GacS-GacA all belong to the same orthologous family of two-component systems as the Escherichia coli BarA-UvrY. In the first four species it has been demonstrated that disruption of this two-component system leads to a clear reduction in virulence of the bacteria. Our aim was to determine if the Escherichia coli BarA-UvrY two-component system is connected with virulence using a monkey cystitis model.     

Efficient production of soluble recombinant single chain Fv fragments by a <Emphasis Type="Italic">Pseudomonas putida</Emphasis> strain KT2440 cell factory

Background  

Recombinant antibody fragments have a wide range of applications in research, diagnostics and therapy. For many of these, small fragments like single chain fragment variables (scFv) function well and can be produced inexpensively in bacterial expression systems. Although Escherichia coli K-12 production systems are convenient, yields of different fragments, even those produced from codon-optimized expression systems, vary significantly. Where yields are inadequate, alternative production systems are needed. Pseudomonas putida strain KT2440 is a versatile biosafety strain known for good expression of heterologous genes, so we have explored its utility as a cell factory for production of scFvs.     

Genomic transcriptional response to loss of chromosomal supercoiling in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Background  

The chromosome of Escherichia coli is maintained in a negatively supercoiled state, and supercoiling levels are affected by growth phase and a variety of environmental stimuli. In turn, supercoiling influences local DNA structure and can affect gene expression. We used microarrays representing nearly the entire genome of Escherichia coli MG1655 to examine the dynamics of chromosome structure.     

Encapsulated <Emphasis Type="Italic">Escherichia coli</Emphasis> in alginate beads capable of secreting a heterologous pectin lyase

Background  

Production of heterologous proteins in the E. coli periplasm, or into the extracellular fluid has many advantages; therefore naturally occurring signal peptides are selected for proteins translocation. The aim of this study was the production in high yields of a recombinant pectin lyase that is efficiently secreted and the encapsulation of transformed E. coli cells for pectin degradation in a biotechnological process.     

Evaluation of Petrifilm™ Select <Emphasis Type="Italic">E. coli</Emphasis> Count Plate medium to discriminate antimicrobial resistant <Emphasis Type="Italic">Escherichia coli</Emphasis>

Background  

Screening and enumeration of antimicrobial resistant Escherichia coli directly from samples is needed to identify emerging resistant clones and obtain quantitative data for risk assessment. Aim of this study was to evaluate the performance of 3M™ Petrifilm™ Select E. coli Count Plate (SEC plate) supplemented with antimicrobials to discriminate antimicrobial-resistant and non-resistant E. coli.     

A fed‐batch based cultivation mode in Escherichia coli results in improved specific activity of a novel chimeric‐truncated form of tissue plasminogen activator

Aims

A novel chimeric‐truncated form of tissue‐type plasminogen activator (t‐PA) with improved fibrin affinity and resistance to PAI was successfully produced in CHO expression system during our previous studies. Considering advantages of prokaryotic expression systems, the aim in this study was to produce the novel protein in Escherichia coli (BL21) strain and compare the protein potency in batch and fed‐batch processes.

Methods and Results

The expression cassette for the novel t‐PA was prepared in pET‐28a(+). The E. coli expression procedure was compared in traditional batch and newly developed fed batch, EnBase^® Flo system. The protein was purified in soluble format, and potency results were identified using Chromolize t‐PA Assay Kit. The fed‐batch fermentation mode, coupled with a Ni‐NTA affinity purification procedure under native condition, resulted in higher amounts of soluble protein, and about a 30% of improvement in the specific activity of the resulted recombinant protein (46·66 IU mg^?1) compared to traditional batch mode (35·8 IU mg^?1).

Conclusions

Considering the undeniable advantages of expression in the prokaryotic expression systems such as E. coli for recombinant protein production, applying alternative methods of cultivation is a promising approach. In this study, fed‐batch cultivation methods showed the potential to replace miss‐folded formats of protein with proper folded, soluble form with improved potency.

Significance and Impact of the Study

Escherichia coli expression of recombinant proteins still counts for nearly 40% of marketed biopharmaceuticals. The major drawback of this system is the lack of appropriate post‐translational modifications, which may cause potency loss/decline. Therefore, applying alternative methods of cultivation as investigated here is a promising approach to overcome potency decrease problem in this protein production system.