Kluyveromyces lactis as an alternative host for the expression of phage-encoded endolysin genes
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Date
2020-08-18
Authors
Wang, Klara
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Abstract
Antibiotic resistance continues to be a long-standing issue worldwide, especially in the context of inappropriate administration of antibiotics in both humans and animals. In response to the threat posed by antibiotic resistant bacteria and the restricted use of medically important antibiotics in the animal industry, more research has been invested in the use of synthetic phage to prevent and/or treat bacterial infections. Unfortunately, one of the major caveats of synthetic phage design is selecting the appropriate genes to carry out infection and lysis of the host bacterium. This includes the selection of endolysin(s), which are phage-encoded proteins that are responsible for destabilizing the cell wall of the host. In order to efficiently screen endolysins for their antimicrobial proprieties, a universal expression system must first be developed to allow for stable expression of muralytic and non-muralytic endolysins. Though some endolysins can be recombinantly expressed in bacteria without the need for host cell manipulations, this is not the case for all endolysins. To address this concern, we report a potential yeast-based gene expression system to allow for stable expression of both muralytic and non-muralytic endolysins. Kluyveromyces lactis GG799 was used to intracellularly express genes for three different muralytic endolysins (Lys3, Lys4, Lys6) and one non-muralytic endolysin (protein E). Western blots were used to detect the 10X-His tag engineered onto the C-terminal end of each endolysin. The results from the Western blots confirmed that all four endolysins could be expressed in K. lactis. In addition to stable expression, we wanted to see if K. lactis was capable of expressing functional endolysins. To test this theory, one muralytic endolysin (Lys6) was chosen. In a high-throughput assay that was developed for this study, ethylenediaminetetraacetic acid treated Escherichia coli C was exposed to two different concentrations of Lys6 (100 and 160 μg / mL), and the growth curve of E. coli C was determined. The results revealed a delay in growth when E. coli C was treated with Lys6, thereby proving that K. lactis is capable of expressing functional endolysins.
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Keywords
Bacteriophage, Endolysin, Recominant protein expression, bacteria, yeast, Kluyveromyces lactis
Citation
Chicago