Prokaryotic cells have been widely exploited in genetic engineering in the manufacturing of useful products (e.g., for the production of food, drinks, drugs, agricultural chemicals and polymers). Prokaryotic cells such as Escherichia coli (E. coli), a well-characterized and well-studied organism, have been used as host organisms in biotechnology. Owing to its physiological simplicity and wide-array of molecular tools available, E. coli can be easily genetically manipulated for bio-production of useful and valuable compounds. Useful as it seems, E. coli however is limited by its inherent inability to export macromolecules such as therapeutic proteins (e.g. human-derived insulin [1]) and valuable bio-polymers (e.g. polylactic acid [2]) out of the cell.
As such, industrial players may have to resort to mechanical (e.g. ultrasonication), chemical (e.g. detergent) or enzymatic (e.g. lysozyme) treatment to disrupt the bacteria cells for macromolecule extraction [3]. These methods have proved to be useful, but require additional purchase of expensive reagents and equipments.
Naturally occurring lytic and temperate bacteriophages have the ability to provoke host cell lysis through the expression of specific proteins during the lytic cycle. In many phages, like the T4 phage and the lambda phage, these proteins have been identified and widely studied. In particular, holins form stable and non-specific lesions in the cytoplasmic membrane that allow the lysozymes to gain access to the peptidoglycan layer. Lysozymes are generally soluble proteins with one or more muralytic activities against the three different types of covalent bonds (glycosidic, amide, and peptide) of the peptidoglycan polymer of the cell wall. The combined work of holin and lysozyme results in the degradation of the two cell membranes of gram-negative bacteria, thus causing cell lysis. Antiholin is a third protein involved in this process as it inhibits holin and is responsible for the regulation of its activity. The described lytic mechanism can be exploited for the release of useful recombinant proteins which cannot be secreted by the engineered host strain.
In an attempt to improve the efficiency and economy of the downstream processing for product extraction, Morita and colleagues [4] had introduced the concept of programmed cell lysis by having E. coli express T4 bacteriophage lytic proteins. The lytic proteins such as holin are responsible for forming a lesion in the host cell membrane [5]. Though the use of lytic protein certainly eases and simplifies the cell disruption process, this method however, still involves supplementary chemical inducers to regulate the lytic protein expression which can be quite costly for industrial scale-up.
To address this challenge, Yun et al. [6] proposed the use of an inducible promoter that does not require additional materials for induction. This proposed promoter, a mutated P1 promoter of ptsG, the gene for major glucose PTS transporter in E. coli, was found to be up-regulated upon glucose exhaustion. By placing the lysis genes under the control of the mutant ptsG P1 promoter (ptsGPL), this approach ensures that there is sufficient cell growth before cell lysis and avoids premature lysis. Though this approach has the merit of enabling E. coli to release product macromolecules at high cell density without any additional step for cell disruption, the authors reported that this method did not lead to a huge reduction in the E. coli's viability after glucose exhaustion due to the low activity of ptsGPL promoter. Further, a closer look into the ptsG promoter suggested that this promoter can be regulated by factors other than glucose. Other factors such as oxygen concentration [7] and oxidative stress [8] are known to influence the activity ptsG promoter. Hence, the use of ptsG promoter is plagued by its low promoter activity and poor specificity, which may hamper its use in industrial settings.
An alternative approach to the glucose-regulated ptsG promoter is the use of the auto-regulatory quorum sensing based expression system, such as the lux regulon from Vibrio fischeri [9]. The quorum sensing system enables users to link recombinant gene expression to population density since the cells would produce a specific signal molecule, N-acyl-homoserine lactone (AHL) that would up-regulate the promoter once the threshold AHL concentration is reached. Such a system is auto-inducible, and it allows cells to activate recombinant gene expression at high cell density. Further, the quorum sensing system can be organized in a way that a positive feedback loop is created for amplifying protein expression. However, this auto-regulatory system is designed in such a way that the users may find it difficult to assert control over the threshold cell density at which the system is activated. To assume control over the activating cell density, users may have to perform trial and error to select the best combination of synthetic constitutive promoter and ribosome binding site for tuning the expression of AHL synthetase, the enzyme responsible for producing AHL from metabolite S-adenosylmethionine. This brute-force method is laborious and time-consuming.
Thus, there remains need in the art for programmable cell lysis systems that overcome the drawbacks of existing technologies.