Short communication: Homologous expression of recombinant and native thurincin H in an engineered natural producer

Abstract

The Bacillus bacteriocin thurincin H exhibits a wide inhibitory spectrum of activity against various foodborne pathogens, such as Listeria monocytogenes, and dairy spoilage bacteria, especially different Bacillus species commonly existing in dairy products. Previously, we constructed 3 plasmids to express native thurincin H homologously in an engineered natural producer, Bacillus thuringiensis SF361thnH(-). This host is deficient in thurincin H production because of an in-frame deletion of structural genes thnA1, thnA2, and thnA3 from the chromosome of the natural producer B. thuringiensis SF361. The previously constructed expression vectors were constructed by cloning the native thurincin H promoter, 3 (or 1) copies of structural genes, and the native (or Cry protein) terminator into an Escherichia coli-B. thuringiensis shuttle vector pHT315. In this study, 3 corresponding expression vectors (pGW134, pGW135, and pGW136) were constructed to express recombinant thurincin H-His6 in the same host, in which a 6-histidine tag was fused to the C terminus of each structural gene. The resulting low level of bacteriocin production indicated that the His tag might negatively interfere with subsequent posttranslational modification or exportation processes after the thurincin H-His6 prepeptide was translated. Additionally, in order to overexpress native thurincin H, 2 additional plasmids (pGW137 and pGW138) were constructed, consisting of the sporulation-dependent Cry protein dual promoter BtI and BtII, the thnA1 structural gene, and the thurincin H native or Cry protein terminator. However, production was low on Luria broth plates and absent on sporulation plates. It is possible that the resulting thurincin H prepeptide was not correctly modified or exported to the extracellular environment, due to the undesired biochemical and physiological changes during the sporulation phase.

Bioinformatic Mapping of Radical S-Adenosylmethionine-Dependent Ribosomally Synthesized and Post-Translationally Modified Peptides Identifies New Cα, Cβ, and Cγ-Linked Thioether-Containing Peptides

“…Reported sactipeptides typically have a narrow spectrum of activity towards Gram-positive organisms, most notably Clostridium difficile, Listeria monocytogenes, and Bacillus cereus. 16,17,[46][47][48][49] Consistent with previously characterized sactipeptides, huazacin inhibited the growth of L. monocytogenes 4b F2365 with a minimum inhibitory concentration (MIC) of 4 μM (Table S4). These results with huazacin reinforce the concept that RODEO is a useful tool to accelerate RiPP discovery.…”

Current Advancements in Sactipeptide Natural Products

“…In addition, subtilosin A also exhibited spermicidal activity (Silkin et al, 2008 ; Sutyak et al, 2008 ). Thurincin H produced by B. thuringiensis SF361 is effective against Bacillus spp., Micrococcus spp., Listeria spp, such as L. monocytogenes which are the main pathogens of human listeriosis (Lee et al, 2009 ; Sit et al, 2011b ; Wang et al, 2014c ). Simultaneously, thurincin H can be used as a food preservative for the control of food originated Gram-positive bacteria that cause the deterioration of sunflower honey (Lee et al, 2008 , 2009 ).…”

“…With the advancement of bioinformatics methods and genomic sequencing technology, more and more sactipeptides have been discovered and identified in recent years. In 2014, Hertweck et al utilized anti-SMASH (Lee et al, 2008 ; Wang et al, 2014c ; Lee and Lee, 2016 ), BAGEL3 (Wang et al, 2014b ), BLAST and other bioinformatics tools to perform RiPPs genome mining on anaerobic bacteria with known genome sequences. The results demonstrated that 59 of 211 strains (28%) contain the RiPPs gene cluster.…”