Lisansüstü Eğitim Enstitüsü

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http://hdl.handle.net/11527/19177

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Yazar "Abdulmalek, Hadeel Waleed" ile Lisansüstü Eğitim Enstitüsü'a göz atma
  • Öge
    Improvement of Bacilysin biosynthesis in Bacillus subtilisPY79 VIA CRISPR/CAS9 technology
    (Graduate School, 2023-09-18) Abdulmalek, Hadeel Waleed ; Karataş Yazgan, Ayten ; 521172109 ; Molecular Biology-Genetics and Biotechnology
    Bacillus subtilis, a common Gram-Positive soil bacterium, has received significant attention from scientists and researchers in various fields such as agriculture, industry, medicine, and pharmacology. It is widely regarded as an excellent host model due to its highly adaptable metabolic system, which allows it to withstand nutrient scarcity by forming highly resistant endospores. Furthermore, B. subtilis is capable of producing biofilms in response to cell density. From an environmental perspective, B. subtilis is a crucial probiotic bacterium that helps plant, animal, and human health. Whole genome analysis reveals its low G+C content and over 4000 functional protein-coding genes, making it an attractive genetic tool extensively utilized in genetic engineering and biotechnology. Moreover, this rod-shaped aerobic (facultatively anaerobic) prokaryote exhibits a remarkable secretion capacity for a wide range of valuable proteins, enzymes, and significant secondary metabolites with antimicrobial properties. Bacilysin, also known as bacillin or tetaine, is a non-ribosomal synthesized dipeptide antibiotic made up of two amino acids: The N-terminal L-alanine and the C-terminal non-proteinogenic amino acid L-anticapsin. It is produced by various species of Bacillus, including Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus pumilis, and Bacillus velenzensis. Despite its simple structure, bacilysin has a wide range of antimicrobial activity against bacteria and fungi. The antimicrobial activity of bacilysin is due to its anticapsin component, which is released when it is transported into the cell and undergoes proteolysis. This anticapsin then inhibits glucosamine-6-phosphate synthase, preventing the biosynthesis of mannoprotein in fungi and peptidoglycan in bacteria. As a result, the microbial cells undergo lysis. Recently, bacilysin was found to be effective in controlling fire blight disease in orchard trees and rice diseases caused by Xanthomonas sp. It also exhibited anticyanobacterial activity against harmful algal species. Bacilysin is highly heat-stable and remains active over a wide pH. These characteristics make it a promising candidate for various industries, including pharmaceuticals, food, and agriculture. However, the low production by the native producer hinders its use in industrial applications. Therefore, in this thesis study, we aimed to improve the bacilysin biosynthesis in its native producer, B. subtilis PY79. In bacteria, translation initiation is often the rate-limiting step of translation and a major factor in determining gene expression levels. Translation initiation occurs at the ribosome binding site (RBS) within the 5′ untranslated region (5′UTR) of an mRNA. In bacteria, the RBS contains the Shine Dalgarno (SD) sequence, the start codon, and a short spacer sequence. The SD sequence plays a crucial role in translation initiation by recruiting the 30S ribosomal subunit to the start codon through base-pairing with the anti-SD sequence in the 16S rRNA. The effectiveness of the SD sequence depends on its base pairing potential and its spacing from the start codon. The canonical SD sequence for most bacteria is UAAGGAGG, which complements well with the anti-SD sequence of 16S rRNA. Studies have shown that increasing the SD-anti-SD interaction improves protein production and overall translation levels. The spacing between the SD sequence and start codon also affects translation initiation efficiency. If the spacing is too close or too far, initiation levels decrease. In bacterial genomes, the spacing of the SD sequence generally varies from 5 to 13 nt, with 8 to 10 nt being ideal for E. coli genes. Similarly, the ideal spacing between the canonical SD sequence and the AUG start codon is 7-9 nt for various B. subtilis genes. In B. subtilis 168, the bacABCDEF operon and the monocistronic gene bacG encode enzymes responsible for the synthesis of anticapsin and bacilysin. The sequence around the bacA transcriptional start site shows a strong consensus signature for the B. subtilis sigma factor σA, but there is no obvious strong SD-like sequence surrounding the translation start codon AUG. This suggests that improving the RBS region could enhance bacilysin production. Therefore, in this study, by using a CRISPR/cas9 single plasmid system (pJOE9958.1), the putative RBS region of bacA, as the first gene of the bacilysin biosynthetic operon, was edited by introducing the conanical SD sequence (TAAGGAGG) at 8 nt optimum spacing from the bacA translation start codon (AUG). To achieve this, a 932 bp homology template was initially generated through overlapped-extension PCR. This template contained a strong ribosomal binding site (5'-GCTTAAGGAGGACAAACTC-3') (bacAstrongRBS), which was then cloned into the SfiI cutting site of pJOE9958. A 20 nt spacer sequence from the upstream region was selected, specifically at positions -28 and -8 relative to the bacA start codon, by using the software CCTop. This sequence was synthesized into two complementary oligonucleotides and inserted it between the BsaI sites of pJOE9958. The resulting plasmid was named pJOE9958.1.bacAstrongRBS.sgRNA, and it was used for the transformation of B. subtilis PY79-competent cells. Transformants were selected on LB agar plates with Km and 0.2% mannose at 30oC to induce cas9 expression. Within two days, 21 colonies appeared, and they were streaked again on LB plates with Km and incubated at 30oC. Before the plasmid curing step, colonies resistant to Km were tested for their ability to produce bacilysin, eliminating any mutants that were unable to do so. Only three colonies exhibited antibacterial activity against S. aureus, indicating that the efficiency of homology-directed repair (HDR) was insufficient to repair Cas9-induced double-strand breaks (DSBs), and most of the colonies in our study likely arose from the NHEJ pathway. Subsequently, only colonies resistant to Km and capable of producing bacilysin were plated on LB plates without Km and incubated at 50oC overnight, which prevents plasmid replication. They were then restreaked on LB plates at 42oC to generate plasmid-cured cells, and approximately 42% of the examined colonies were found to be plasmid-cured. In the final step, 11 randomly selected Km-sensitive (plasmid-cured) transformants were screened with colony PCR to identify mutants with the substitution of the bacAstrongRBS sequence with the bacAnativeRBS sequence. Out of the 11 transformants, 10 displayed the expected PCR fragment of approximately 440 bp. DNA sequencing of one randomly chosen PCR-positive colony confirmed the successful replacement of the bacAnativeRBS sequence with the bacAnativeRBS. To assess the impact of the strong RBS insertion, eight mutants, including the strong RBS mutant confirmed by sequence analysis and the seven randomly selected mutants (from the PCR-positive colonies), were cultured in PA media for 16-18 hours. The level of bacilysin in their culture fluids was then determined using a paper disk-diffusion assay. Compared to the PY79 strain, each of the examined mutants produced more bacilysin, and there were no significant differences in their increased bacilysin levels. These results demonstrated that the strong RBS substitution led to enhanced bacilysin production. Finally, one of the mutants with the bacAstrongRBS sequence was chosen to represent the strong RBS-carrying mutants and designated as B. subtilis HWA for further investigation. To further investigate the impact of the strong RBS substitution on bacilysin overproduction, HWA and the parental strain PY79 were grown in PA medium at 37°C with shaking for 24 hours, and their growth and bacilysin production levels were monitored at regular intervals. no significant differences in the growth patterns or the accumulation of bacilysin in HWA compared to PY79. In both strains, the highest bacilysin activity was detected during the early stage of the stationary phase, which occurred around 16-18 hours of cultivation. However, the most notable finding was that HWA consistently exhibited overproduction of bacilysin at all growth stages, with the peak occurring at 18 hours of incubation. The bacilysin titer in HWA was 2.87 times higher than that of the wild-type strain. This significant increase in bacilysin activity was further confirmed by UPLC-MS analysis, which revealed a higher relative abundance of the peak m/z of 271 (M + H)+, corresponding to the mass of bacilysin, in HWA compared to PY79 during the 16-18 hours culture period. In bacteria, the processes of transcription and translation are closely linked. Modifying the nucleotides in the 5' untranslated region (5'UTR) can have an impact on both transcription and translation. Therefore, to demonstrate the effect of modifying the 5'UTR through a strong ribosome binding site (RBS) substitution on intracellular mRNA levels, the relative amounts of transcripts from the bac operon were examined in two bacterial strains, HWA and PY79, by using the RT-qPCR technique. These measurements were performed during the early and late stationary phases of growth, which correspond to 18 hours and 24 hours of growth, respectively. In HWA, the mRNA levels of the bac operon showed a significant change. Specifically, during the early stationary phase, the transcript levels were three times higher compared to PY79. Additionally, the transcript level in HWA was detected at 25 quantitative cycles (Cq) during the early stationary phase (18 hours) and 21 Cq during the late stationary phase (24 hours). In contrast, PY79 exhibited a Cq value of 29 at 24 hours. These findings indicated that the high transcript level of the bac operon in HWA is maintained throughout the stationary phase. In conclusion, these results provide evidence that the strong RBS substitution also improves the stability of the bac operon mRNA. In summary, in this study, a CRISPR/Cas9 single-plasmid system was used to edit the putative RBS region of the bac operon. The canonical SD sequence (TAAGGAGG) at an optimal spacing of 8 nt from the bacA start codon (AUG) was introduced. Because the upstream region of the bac operon did not have an ideal core SD sequence with a spacing of 7-9 nt. Therefore, editing the bacA-RBS most likely has a positive impact on bacilysin production in its native producer. As expected, modifying the 5'UTR through strong RBS substitution led to a significant increase in bacilysin production. Additionally, the transcript level of the bac operon was significantly increased, especially in the late stationary phase. This suggests that a strong RBS substitution in the bac operon can improve translation initiation efficiency and mRNA stability. Overall, these findings indicate that extensive RBS engineering could be a promising approach to enhancing bacilysin production in native producers. This study is the first to employ RBS editing to improve bacilysin production in B. subtilis PY79, and our results will contribute to further enhancing bacilysin production in native strains.