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Rapid production and characterization of antimicrobial colicins using Escherichia coli-based cell-free protein synthesis

ABSTRACT: Abstract Colicins are antimicrobial proteins produced by Escherichia coli, which, upon secretion from the host, kill non-host E. coli strains by forming pores in the inner membrane and degrading internal cellular components such as DNA and RNA. Due to their unique cell-killing activities, colicins are considered viable alternatives to conventional antibiotics. Recombinant production of colicins requires co-production of immunity proteins to protect host cells; otherwise, the colicins are lethal to the host. In this study, we used cell-free protein synthesis (CFPS) to produce active colicins without the need for protein purification and co-production of immunity proteins. Cell-free synthesized colicins were active in killing model E. coli cells with different modes of cytotoxicity. Pore-forming colicins E1 and nuclease colicin E2 killed actively growing cells in a nutrient-rich medium, but the cytotoxicity of colicin Ia was low compared to E1 and E2. Moreover, colicin E1 effectively killed cells in a nutrient-free solution, while the activity of E2 was decreased compared to nutrient-rich conditions. Both colicins E1 and E2 decreased the level of persister cells (metabolically dormant cell populations that are insensitive to antibiotics) by up to six orders of magnitude compared to that of the rifampin pretreated persister cells. This study finds that colicins can eradicate non-growing cells including persisters, and that CFPS is a promising platform for rapid production and characterization of toxic proteins.

SUBMITTER: Jin X

PROVIDER: S-EPMC7445778 | biostudies-literature | 2018 Jan

REPOSITORIES: biostudies-literature

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Project description:BackgroundHydroxycinnamoyl anthranilates, also known as avenanthramides (avns), are a group of phenolic alkaloids with anti-inflammatory, antioxidant, anti-itch, anti-irritant, and antiatherogenic activities. Some avenanthramides (avn A-H and avn K) are conjugates of hydroxycinnamic acids (HC), including p-coumaric acid, caffeic acid, and ferulic acid, and anthranilate derivatives, including anthranilate, 4-hydroxyanthranilate, and 5-hydroxyanthranilate. Avns are primarily found in oat grain, in which they were originally designated as phytoalexins. Knowledge of the avns biosynthesis pathway has now made it possible to synthesize avns through a genetic engineering strategy, which would help to further elucidate their properties and exploit their beneficial biological activities. The aim of the present study was to synthesize natural avns in Escherichia coli to serve as a valuable resource.ResultsWe synthesized nine avns in E. coli. We first synthesized avn D from glucose in E. coli harboring tyrosine ammonia lyase (TAL), 4-coumarate:coenzyme A ligase (4CL), anthranilate N-hydroxycinnamoyl/benzoyltransferase (HCBT), and anthranilate synthase (trpEG). A trpD deletion mutant was used to increase the amount of anthranilate in E. coli. After optimizing the incubation temperature and cell density, approximately 317.2 mg/L of avn D was synthesized. Avn E and avn F were then synthesized from avn D, using either E. coli harboring HpaBC and SOMT9 or E. coli harboring HapBC alone, respectively. Avn A and avn G were synthesized by feeding 5-hydroxyanthranilate or 4-hydroxyanthranilate to E. coli harboring TAL, 4CL, and HCBT. Avn B, avn C, avn H, and avn K were synthesized from avn A or avn G, using the same approach employed for the synthesis of avn E and avn F from avn D.ConclusionsUsing different HCs, nine avns were synthesized, three of which (avn D, avn E, and avn F) were synthesized from glucose in E. coli. These diverse avns provide a strategy to synthesize both natural and unnatural avns, setting a foundation for exploring the biological activities of diverse avns.

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Rapid production and characterization of antimicrobial colicins using Escherichia coli-based cell-free protein synthesis

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