A vector that enables the high-volume production of the dipeptide basilisin.

Abstract

The invention relates to a vector that enables the modification of the weak ribosome binding region (5'UTR) sequence of the Bacillus subtilis PY79 strain using CRISPR / Cas9. The vector modifies the 5'UTR sequence with SEK ID NO:2 to SEK ID NO:1 in wild-type Bacillus subtilis PY79. Transformation of the Bacillus subtilis PY79 strain with this vector increases the biosynthesis of the bacteria by 2.86-fold compared to the wild-type strain. As a result of this genetic modification in the Bacillus subtilis PY79 strain, these bacteria can be used both as probiotics and against pathogens in agricultural activities. Furthermore, the ability to continuously produce high levels of Bacillus during cultivation eliminates the need to separate the growth and production phases, significantly reducing costs in industrial production.

Description

1 TARIFF A method that enables the high-level production of dipeptidian bacilli. VECTOR Technical Area The invention describes a weakly characteristic ribosome binding region (5'UTR) of Bacillus subtilis PY79 strain. It relates to a vector that enables the replacement of the sequence using CRISPR / Cas9. The invention concerns 5. obtained as a result of the transformation of the vector into Bacillus subtilis PY79 The organism's biosynthesis of basilisin is 2.86 times higher compared to the wild-type strain. This is happening and thanks to the Bacillus antibiotic produced, it acts as both a probiotic. It is also used in agricultural activities against pathogens, as well as in cultivation. growth through the acquisition of consistently high levels of bacillus production capability during the process and 10 Cost reduction in industrial production is achieved by eliminating the need to separate the production phase. is significantly reduced. State of the Art Bacillus spp. are aerobic or facultative anaerobic, Gram-positive bacteria, numbering from two dozen to 15 the potential to produce a large number of bioactive compounds, the majority of which are low molecular weight, bioactive Peptides have been known for over 50 years [1]. The simplest known peptide The antibiotic basilisin was developed in 1946 to inhibit the growth of Staphylococcus aureus cultures. synthesized by a B. subtilis strain that causes partial disintegration It was discovered as an antibiotic [2]. Also, Bacillus amyloliquefaciens, Bacillus 20 obtained from Bacillus velezensis, Bacillus licheniformis, Bacillus pumilus and B. subtilis In silico genome analysis of the strains revealed that the basilisin gene cluster was present in all strains except B. licheniformis. It has been shown that it is present in species [3], but in different strains, for example B. Even between B. subtilis PY79 and B. subtilis 168, the level of basilic acid biosynthesis differs. The biosynthesis of basilis occurs primarily through the conversion of prephenate to basilis. It is regulated by the bac operon (bacABCDEF), which plays a key role, and the basilisin, Active against a wide range of bacteria, including some fungi such as Candida albicans. and its antibacterial effect is primarily due to its transport to host cells, the cell intracellular peptidases enable the activation of the bacillus within the cell. hydrolyzing of anticapsin and 30, a compound necessary for cell wall biosynthesis 2 It is based on the inhibition of glucosamine 6-phosphate (GlcN6P) synthase [4]. In addition to its antibacterial effect, the dipeptide antibiotic is synthesized by Bacillus. Bacillus spp. bacteria are used directly as probiotics. Bacillus probiotics, It can have a direct effect on pathogenic bacteria, which makes them prevention, inhibition or 5 of its growth and colonization of the intestine By being able to stop the spread of infection, they strengthen the intestinal barrier. It helps alleviate inflammatory responses in the gut and potentially ongoing... It exhibits properties that can help prevent chronic inflammation [5]. This direct effect on pathogens is characteristic of antimicrobial peptides, in particular. It results from the production of basilisin and its metabolites [6]. In addition, basilisin 10 Bacillus spp., which are produced by this species, reduce the use of synthetic pesticides in agricultural activities. and an environmentally friendly approach to reduce the use of harmful chemicals against pathogens It is also used as a biocontrol agent with this approach [7]. However, both antibiotics or as a probiotic, or in agricultural activities against microbial pathogens. Its use as a biocontrol agent involves mRNA 15 of Bacillus spp. in the current technique. their stability is low and consequently their mRNA degradation times are short and due to the low level of bacillus biosynthesis, it is not sufficiently effective. It is not. Bacillus species are diverse and can be easily scaled up for large-scale industrial production. 20 long-established plants used in the production of enzymes, antibiotics, and other metabolites They are industrial microorganisms and are used in the biocontrol of plant pathogens and in plants. They produce various compounds that play a role in promoting growth, which makes them the most This makes it a potential candidate for agricultural and biotechnological applications [8]. Furthermore, when compared in terms of genetic engineering applications, Bacillus Thanks to the well-characterized genome of spp., we can introduce these genes and 25 by deleting, controlling gene expression and engineering metabolic pathways, especially This includes the ability to optimize the production of specific metabolites, such as basilisin. In addition, CRISPR-Cas based genome editing and synthetic biology approaches a number of genetic engineering strategies for Bacillus spp. developed, therefore any genetic engineering application specific to these species is 30 Due to the existence of many tools and techniques for genetic engineering applications They stand out as suitable candidates and, due to their aforementioned advantages, Bacillus spp. are a more practical and efficient choice for basil production in industry. 3 A study conducted by Wu and colleagues, which is part of the current technique, shows that Bacillus amyloliquefaciensv, or in the new classification B. velezensis FZB42. [9] strain is related to the enhancement of basilisin biosynthesis. Cre / lox area specific The promoter region of the biosynthetic operon of basilisin was formed into two different regions using recombination techniques. Modified with PrepB and Pspac promoters and increased transcription level to 5 Bacillus biosynthesis was increased by 170.4% and 315.6% compared to the wild type. However, the mRNA stability of the recombinant strain obtained in the study in question and No information regarding mRNA degradation time or translational efficiency is included. It does not contain bacA. However, bacA is found in the genome of Bacillus amyloliquefaciens FZB42. The gene and 5' UTR region, and the B. subtilis PY79 bacA and 5' UTR region show a 10-degree difference between the two strains. Many base changes even in the 5'UTR regions and in the coding region of the bacA gene. It is located. Due to this differentiation in the 5'UTR region, any action to be taken... Since genetic modification can produce different effects in the two strains, the target product must be carefully considered. The improvement rate will vary. Therefore, the genetic modifications to be made will be different for each strain. It is unique in itself, and it is not possible to compare the therapeutic effects obtained with each other. 15 This is not the case. Furthermore, the production levels of basil differ in both natural strains. This shows that the improvement rates obtained are consistent with the properties of both strains. It makes sense within that context. Furthermore, although the biosynthesis of basilisin is increased, due to the absence of a statement regarding the growth phase and in the invention in question Since the increase in biosynthesis occurs through transcriptional modification (because the high 20 Gene expression requires much more than just transcription), translational In contrast to modification, the amount of basilisin only increases from the end of the logarithmic phase. It becomes measurable. Therefore, growth and production phase in industrial production Separation will occur, and consequently, the problem of high production costs will arise. The limitations and inadequacies of the solutions in the current technology, in the known state of the technology, 25 The mRNA stability of naturally occurring Bacillus spp. strains is low, and the mRNA degradation time is long. due to its short length and the low level of basil biosynthesis The inadequacy of the use of probiotics as both a probiotic and a biocontrol agent in agriculture, The amount of the bacterial antibiotic can only be measured at the end of the logarithmic phase. Therefore, in the industrial production stage, the growth and production phases must be separated. 30 reasons such as the necessity of the product and consequently the high production costs Therefore, an improvement is needed to increase the biosynthesis of basil. It has been made. 4 Brief Description and Objectives of the Invention The invention involved a weak ribosome binding site of Bacillus subtilis PY79 strain. A vector that enables the replacement of the (5'UTR) sequence using CRISPR / Cas9. It is explained that the vector in question is transformed into a Bacillus subtilis PY79. The resulting organism had a 5% higher biosynthesis rate of basilisin compared to the wild-type strain. It occurs 2.86 times more frequently, and thanks to the produced basilisin antibiotic, both It is used both as a probiotic and against pathogens in agricultural activities, by gaining the ability to produce consistently high levels of bacilli during the cultivation process industrial Production costs are significantly reduced. 10 The aim of the invention is to increase the biosynthesis of basilisin at the translational level. This is achieved by demonstrating the weak ribosome binding characteristic of the Bacillus subtilis PY79 strain. The 5'UTR array, which is the region, was processed using the CRISPR / Cas9 system and the vector that is the subject of the invention. By regulating the translational level, the biosynthesis of basilisin was increased 2.86-fold. This is ensured. The fact that this effect is at the translational level is regulated by Article 15. the sequence is made in the ribosome binding region, that is, in the sequence where translation begins Thanks to this, mRNA stability is high and mRNA degradation is reduced. This is possible due to its long duration. Another aim of the invention is to produce it at low cost, both as a probiotic and... Bacillus spp. 20 as a biocontrol agent against pathogens in agricultural activities The goal is to provide a vector for use with high efficiency. This vector... The transformation of Bacillus subtilis into PY79 results in the bacterium's basilisin biosynthesis. Thanks to its 2.86-fold increase compared to the wild-type strain, it is suitable for use as a probiotic. Furthermore, high efficacy is achieved in the use of biocontrol agents. In this invention, weak The vector that is the subject of the invention contains a ribosome binding region (5'UTR) sequence with a specific character. is being altered and the amount of basilisin produced by Bacillus species representing this vector is being changed. It starts from the middle of the logarithmic phase, unlike the wild-type strain, in the stationary phase. It reaches its maximum level in the early stages and remains significantly stable until the late stationary phase. ...continues, meaning high basilicin production throughout the entire growth phase. Thanks to its realization, industrial production has reached the production and growth phases of 30. Production is low-cost because there is no need for separation. It can be accomplished. 5 Explanation of the Figures Figure 1. Growth and basilisin of B. subtilis PY79 and recombinant B. subtilis PY79 strain. Activity profiles (REC PY79: Recombinant PY79 strain) Figure 2. RT-qPCR method applied to PY79 and recombinant PY79 strains. Transcriptional analysis graph. (* and ** p-values, < 0.05 and < 0.01, respectively) 5 Figure 3. RT-qPCR analysis of PY79 and recombinant PY9 strains at 18 and 24 hours. Quantitative cycle (Cq) values ​​measured for the bacB gene in clock samples. Detailed Description of the Invention The invention describes the binding of a weakly characteristic ribosome binding site 10 to the Bacillus subtilis PY79 strain. It is related to a vector that enables the replacement of the (5'UTR) sequence with CRISPR / Cas9. The vector in question corresponds to the nucleotide sequence SEK ID NO:2 in wild-type Bacillus spp. It modifies the 5'UTR sequence to the nucleotide sequence with SEC ID NO:1. This invention is the subject of the invention. Biosynthesis of basilis by transformation of the vector into Bacillus subtilis PY79 strain 2.86 The ratio is increased compared to the wild-type strain. The vector in question is Bacillus subtilis 15 As a result of this genetic modification performed on the PY79 strain, the word These bacteria are used both as probiotics and against pathogens in agricultural activities. Although used, it does not produce consistently high levels of bacilli during the cultivation process. With the acquisition of capability, there is no need to separate the growth and production phase. Thanks to this, costs in industrial production are significantly reduced. 20 In the invention, the basilisk-producing strain is B. subtilis PY79 (wild type, B. subtilis 168). (prototrophic derivative) is used, also E. coli DH5a as a host for cloning. It is used to enable the transformation of B. subtilis with the vector that is the subject of the invention. The plasmid DNA used was amplified in recA-active E. coli BL21(DE3) pLysS. Appendix As such, S. aureus ATCC 9144 was used as a bioassay organism for the detection of basilisin 25. It is used. In the invention, homology for the alteration of the strong Shine-Dalgarno (5'-TAAGGAGG-3') sequence For the purpose of creating the template, pJOE9958.1 was used as the CRISPR / Cas9 plasmid. It is used. The homology template, B. 9, is used as a template with the conjugate extension PCR strategy. Use of subtilis PY79 and DNA polymerase 30 with 3´→ 5´ exonuclease activity 6 It is created using the following method: the upstream region of the bacA start codon. (313 bp), forward primer with SEC ID NO:3 nucleotide sequence and Sfi1 exclusion site. and SEK ID NO:4 nucleotide sequence and strong ribosome binding of bacA operon. amplified first PCR reaction via back primer with region is done. Subsequently, the open read pattern (ORF) of bacA is 615 pairs of base (bp) SEK ID 5 Forward primer with nucleotide sequence NO:5 and SEC ID NO:6 nucleotide sequence It is amplified via a second PCR reaction with the reverse primer. Then, the first and the second PCR products, forward primer with SEK ID NO:7 nucleotide sequence and SEK ID Amplified by a third PCR reaction with a back primer having the nucleotide sequence NO:8. The homology template, pJOE9958.1.10, containing 932 base pairs, was prepared after the process. It is cleaved by the Sfi1 enzyme to form bacA (strong ribosome binding site), and Sfi1 It is connected via the decomposed pJOE9958.1 vector. The resulting pJOE9958.1. Vectors containing bacA (strong ribosome binding site) are cut with the BsaI enzyme and at their 5' ends TACG and AAAC single-chain extension, complementary double-chain SEK ID NO:10 It binds to a guide RNA sequence with a specific nucleotide sequence. B. subtilis PY79 has 15 nucleotide sequences. Before transformation, pJOE9958.1. bacA (strong ribosome binding site). guide RNA recA-active E. coli BL21(DE3)pLysS was used to prepare the plasmid in multimeric form. It is multiplied within it. Competent B. subtilis PY79 cells, The invention concerns pJOE9958.1. bacA (strong ribosome binding region) transformed with guide RNA vector. It is being processed. The transformed nucleotide with 5'UTR sequence SEK ID NO:2 is 20. Candidate cells whose nucleotide sequence was changed from the sequence with SEK ID NO:1 (transformers) Kanamycin and 0.2% mannose-containing LB (Luria) for Cas9 induction. (Liquid culture medium) is selected on agar. 21 colonies appear within two days, They are seeded onto LB plates containing kanamycin and incubated overnight at 30°C. For the elimination of plasmids, isolates are placed individually on antibiotic-free LB agar (25°C). the day after planting to obtain colonies and incubating at 50°C They are seeded onto LB plates at 42°C to obtain single colonies, however Before implementing the plasmid elimination steps mentioned above, potential Screening for basilisin phenopythione to eliminate non-bacillisin-producing organisms. This is because those that do not produce basilisin are 30% produced via non-homologous end joining. (NHEJ) is likely to arise from repaired cells. Therefore, kanamycin obtained by seeding into LB plates containing and incubating overnight at 30°C Colonies were bioassayed directly with sterile toothpicks containing S. aureus ATCC9144. by placing them on plates and incubating them overnight at 37°C, multi-rapid basilisin 7 Screening is being carried out. According to the results, only 3 colonies were found to contain S. aureus. It exhibits significant antibacterial activity against (Bac+), which is homology-directed. The repair efficiency (HDR) for repairing Cas9-mediated double helix fractures is very low. This suggests that the colonies that remained and the vast majority of the colonies in the discovery are homologous. It shows that it is derived via non-end joining. Thus, the 3 selected Bac+ 5 In KmR colonies, plasmids are eliminated as described above. Plating of single colonies onto antibiotic-free LB agar and incubation at 50°C. The day after incubation, the colonies will be separated into single colonies (LB). They are seeded onto plates and incubated overnight at 42°C. The resulting colonies, Grown overnight at 42°C on LB plates containing kanamycin, 10 Colonies that do not grow in the presence of hematuria (KmS) are selected. Bacillus activities... For analysis, B. subtilis strains grown on LB agar at 37°C were mixed with 10 mL of liquid Perry agar. and Abraham (PA) prepared a one-night culture in the culture medium [9]. This prepared For overnight culture, use 100 ml of fresh PA (Perry and Abraham medium) as a starting solution with a 0.1 ratio. Used for inoculation at OD600, shaken at 37°C (200 rpm) for 24 hours. It is cultivated. During this process, samples are taken at certain intervals to monitor growth (OD600). Absorbance measurement and basilar activity are monitored in culture fluids. Bacillus activity, Staphylococcus aureus ATCC 9144 as the assay organism. As shown in Figure 1, using paper disk-agar diffusion assay. It is determined. In addition, early and late 20 weeks of growth are used for total RNA extraction. cells in their stationary phases (corresponding to 18 hours and 24 hours of growth, respectively) Samples are collected and treated with an RNA extraction kit. See Figure 1. According to this, basil accumulation depends on the growth and cell density of the recombinant strain. a significant positive change in their profiles compared to the wild-type strain This does not happen. However, in the recombinant strain, the basilisin titer reaches its maximum level at 25. According to the comparison made after 18 hours, the recombinant strain's basilisin its activity increased from 27.9 ± 4.5 U / ml (in the parent strain) to 80.3 ± 9.1 U / ml (in the recombinant strain) (in the strain) an increase of 2.86 times is observed. Most importantly, the production of basilisin... When the levels were compared, the amount of basilisin in the wild strain (B. subtilis PY79) was only While reaching a measurable level from the end of the logarithmic phase, the recombinant strain is 30 Starting from the middle of the logarithmic phase and reaching its maximum level at the beginning of the stationary phase. It reaches, with a slight decrease, a significant amount of basilisin until the late stationary phase. Production is ongoing. In addition, compared to the natural strain, the recombinant strain exhibits overall growth. It exhibits high basilicin production throughout its phases. Therefore, industrial 8 By eliminating the need to separate the production and growth phases in production, The production cost of the recombinant strain has been significantly reduced compared to the wild type. is happening. The 5'UTR modification introduced in this invention affects intracellular bac operon-mRNA. To highlight its effect at the level of relative transcript volumes of the bac operon, 5 via RT-qPCR in the early and late stationary phases of growth (corresponding). Detected in recombinant and wild-type PY79 for 18 and 24 hours, respectively. Accordingly, recombinant strains with a strong ribosome binding site are used. It shows three-fold higher bac operon mRNA levels in the early stationary phase. During the early (18-hour) and late stationary (24-hour) phases of growth, the bac operon appears in the second 10-hour phase. Relative mRNA concentration levels calculated by tracking the bacB gene. This is indicated in Figure 3. Asterisks represent parental responses according to the paired “student t-test”. The type PY79 was statistically significantly different (p-values ​​of * and ** respectively, < 0.05 and < 0.01). They show differences. In addition, the transcript level in the recombinant strain is early 25 quantitative cycles (Cq) in the stationary phase (18 hours) and 21 15 in the late stationary phase (24 hours) quantitative cycle (Cq), in contrast to the 29 quantitative cycles (Cq) in the wild-type strain, It is detected that the bac operon in the recombinant strain has a high transcript level. It is preserved throughout the stationary phase. Therefore, the strong ribosome binding region is protected. It appears that the bac operon significantly improves mRNA stability. As a result of the 5'-UTR modification introduced, ribosomes can bind much faster. with, both an increase at the translational level and exo- and bac-mRNA mRNA stability is achieved by preventing their degradation by endonucleases. The absolute level of bac operon mRNA was increased due to the increase in this factor. As a result of these effects, basil biosynthesis increases 2.86 times compared to the normal strain. In the invention, the nucleotide sequence is SEK ID NO: 9, bacA open reading region (ORF), SEK ID NO: 1 25 nucleotide sequence (the sequence obtained as a result of the modification that is the subject of the invention) and bacA upstream It includes the upstream region. The method of generating the vector that is the subject of the invention, i. It has 932 base pairs that will allow the insertion of the pJOE9958.1 vector. Creation of the homology template, 30 ii. cutting of the homology template with the Sfi1 enzyme, 9 iii. Homology obtained in (i) to the pJOE9958.1 vector cut with the Sfi1 enzyme The template binds to pJOE9958.1.bacA (strong ribosome binding site). obtaining the vector, iv. The obtained pJOE9958.1.bacA (strong ribosome binding site) was mixed with the BsaI enzyme. cutting, 5 v. 5' ends with TACG and AAAC single-chain extensions, complementary pairs. by binding the chain guide RNA to the vector obtained in step (iv) of the process Generation of the pJOE9958.1.bacA (strong ribosome binding site) guide RNA vector. It includes the steps involved in the process. The vector subject to the invention contains a guide RNA with SEK ID NO:10 nucleotide sequence and SEK ID 10 It contains a strong ribosome-binding region with the NO:1 nucleotide sequence and is said to The subject is the strong ribosome binding site of the canonical Shine-Dalgarno (5'-TAAGGAGG-3') It includes the following arrangement. 15 20 25 10 REFERENCES [1] İ;, Ö. G.Ö. (n.d.). Biochemistry, genetics and regulation of bacillysin biosynthesis and its significance more than an antibiotic. New biotechnology. 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