• 제목/요약/키워드: bacilysin

검색결과 6건 처리시간 0.016초

Improvement of Bacilysin Production in Bacillus subtilis by CRISPR/Cas9-Mediated Editing of the 5'-Untranslated Region of the bac Operon

  • Hadeel Waleed Abdulmalek;Ayten Yazgan-Karatas
    • Journal of Microbiology and Biotechnology
    • /
    • 제33권3호
    • /
    • pp.410-418
    • /
    • 2023
  • Bacilysin is a dipeptide antibiotic composed of L-alanine and L-anticapsin produced by certain strains of Bacillus subtilis. Bacilysin is gaining increasing attention in industrial agriculture and pharmaceutical industries due to its potent antagonistic effects on various bacterial, fungal, and algal pathogens. However, its use in industrial applications is hindered by its low production in the native producer. The biosynthesis of bacilysin is mainly based on the bacABCDEF operon. Examination of the sequence surrounding the upstream of the bac operon did not reveal a clear, strong ribosome binding site (RBS). Therefore, in this study, we aimed to investigate the impact of RBS as a potential route to improve bacilysin production. For this, the 5' untranslated region (5'UTR) of the bac operon was edited using the CRISPR/Cas9 approach by introducing a strong ribosome binding sequence carrying the canonical Shine-Dalgarno sequence (TAAGGAGG) with an 8 nt spacing from the AUG start codon. Strong RBS substitution resulted in a 2.87-fold increase in bacilysin production without affecting growth. Strong RBS substitution also improved the mRNA stability of the bac operon. All these data revealed that extensive RBS engineering is a promising key option for enhancing bacilysin production in its native producers.

Bacillus subtilis JW-1 균주가 생산하는 bacilysin의 풋마름병 억제 효과 및 특성 (Isolation and characterization of bacilysin against Ralstonia solanacearum from Bacillus subtilis JW-1)

  • 김신덕
    • 미생물학회지
    • /
    • 제54권2호
    • /
    • pp.136-139
    • /
    • 2018
  • 풋마름병 균에 대해 강한 저해활성을 갖는 활성물질 Compound S와 conversion product인 compound S'이 Bacillus subtilis JW-1 배양액에서 일련의 크로마토그래피 방법으로 분리 정제되었고, $^1H$ NMR, $^{13}C$ NMR, $^1H-^1H$ COSY와 HMBC 등의 spectra 분석에 의해 구조가 alanyl-L-${\beta}$-(2,3-epoxycyclohexyl-4-one)alanine와 alanyl-L-${\beta}$-(2,3-dihydroxycyclohexyl-4-one) alanine로 각각 동정되었다. Compound S는 $G^+$, $G^-$ bacteria, yeast와 Candida albicans 등에 대해 광범위한 antimicrobial activity를 나타내며, conversion product의 활성 상실을 통해 intact oxirane ring이 Compound S의 활성에 필수적임이 밝혀졌다.

고추장에서 분리된 Bacillus subtilis BS16045의 유전체 서열 분석 (Complete genome sequence of Bacillus subtilis BS16045 isolated from Gochujang)

  • 전새봄;허준;엄태붕
    • 미생물학회지
    • /
    • 제53권1호
    • /
    • pp.55-57
    • /
    • 2017
  • 한국 발효식품의 보존성을 높이기 위한 스타터 균주를 얻기 위하여 고추장에서 Bacillus subtilis BS16045를 분리하였다. B. subtilis BS16045에 대한 유전체 분석을 실시하였으며, G+C 비율이 43.6%인 4,165,121 bp 크기의 염기서열을 얻었다. 또한 이 유전체로부터 항진균 및 항균 활성에 연관이 있는 surfactin, kanosamine, bacillaene, plipastatin, subtilosin A, bacilysin 생산 유전자들을 확인하였다. 이러한 결과들을 통해 B. subtilis BS16045는 장류 제조시설에서 유해균의 오염문제를 해결할 수 있는 스타터로 이용될 수 있을 것으로 보인다.

Evaluation and Genome Mining of Bacillus stercoris Isolate B.PNR1 as Potential Agent for Fusarium Wilt Control and Growth Promotion of Tomato

  • Rattana Pengproh;Thanwanit Thanyasiriwat;Kusavadee Sangdee;Juthaporn Saengprajak;Praphat Kawicha;Aphidech Sangdee
    • The Plant Pathology Journal
    • /
    • 제39권5호
    • /
    • pp.430-448
    • /
    • 2023
  • Recently, strategies for controlling Fusarium oxysporum f. sp. lycopersici (Fol), the causal agent of Fusarium wilt of tomato, focus on using effective biocontrol agents. In this study, an analysis of the biocontrol and plant growth promoting (PGP) attributes of 11 isolates of loamy soil Bacillus spp. has been conducted. Among them, the isolates B.PNR1 and B.PNR2 inhibited the mycelial growth of Fol by inducing abnormal fungal cell wall structures and cell wall collapse. Moreover, broad-spectrum activity against four other plant pathogenic fungi, F. oxysporum f. sp. cubense race 1 (Foc), Sclerotium rolfsii, Colletotrichum musae, and C. gloeosporioides were noted for these isolates. These two Bacillus isolates produced indole acetic acid, phosphate solubilization enzymes, and amylolytic and cellulolytic enzymes. In the pot experiment, the culture filtrate from B.PNR1 showed greater inhibition of the fungal pathogens and significantly promoted the growth of tomato plants more than those of the other treatments. Isolate B.PNR1, the best biocontrol and PGP, was identified as Bacillus stercoris by its 16S rRNA gene sequence and whole genome sequencing analysis (WGS). The WGS, through genome mining, confirmed that the B.PNR1 genome contained genes/gene cluster of a nonribosomal peptide synthetase/polyketide synthase, such as fengycin, surfactin, bacillaene, subtilosin A, bacilysin, and bacillibactin, which are involved in antagonistic and PGP activities. Therefore, our finding demonstrates the effectiveness of B. stercoris strain B.PNR1 as an antagonist and for plant growth promotion, highlighting the use of this microorganism as a biocontrol agent against the Fusarium wilt pathogen and PGP abilities in tomatoes.

Brief Introduction of Research Progresses in Control and Biocontrol of Clubroot Disease in China

  • He, Yueqiu;Wu, Yixin;He, Pengfei;Li, Xinyu
    • 한국균학회소식:학술대회논문집
    • /
    • 한국균학회 2015년도 춘계학술대회 및 임시총회
    • /
    • pp.45-46
    • /
    • 2015
  • Clubroot disease of crucifers has occurred since 1957. It has spread to the whole China, especially in the southwest and nourtheast where it causes 30-80% loss in some fields. The disease has being expanded in the recent years as seeds are imported and the floating seedling system practices. For its effective control, the Ministry of Agriculture of China set up a program in 2010 and a research team led by Dr. Yueqiu HE, Yunnan Agricultural University. The team includes 20 main reseachers of 11 universities and 5 institutions. After 5 years, the team has made a lot of progresses in disease occurrence regulation, resources collection, resistance identification and breeding, biological agent exploration, formulation, chemicals evaluation, and control strategy. About 1200 collections of local and commercial crucifers were identified in the field and by artificiall inoculation in the laboratories, 10 resistant cultivars were breeded including 7 Chinese cabbages and 3 cabbages. More than 800 antagostic strains were isolated including bacteria, stretomyces and fungi. Around 100 chemicals were evaluated in the field and greenhouse based on its control effect, among them, 6 showed high control effect, especially fluazinam and cyazofamid could control about 80% the disease. However, fluzinam has negative effect on soil microbes. Clubroot disease could not be controlled by bioagents and chemicals once when the pathogen Plasmodiophora brassicae infected its hosts and set up the parasitic relationship. We found the earlier the pathogent infected its host, the severer the disease was. Therefore, early control was the most effective. For Chinese cabbage, all controlling measures should be taken in the early 30 days because the new infection could not cause severe symptom after 30 days of seeding. For example, a biocontrol agent, Bacillus subtilis Strain XF-1 could control the disease 70%-85% averagely when it mixed with seedling substrate and was drenching 3 times after transplanting, i.e. immediately, 7 days, 14 days. XF-1 has been deeply researched in control mechanisms, its genome, and development and application of biocontrol formulate. It could produce antagonistic protein, enzyme, antibiotics and IAA, which promoted rhizogenesis and growth. Its The genome was sequenced by Illumina/Solexa Genome Analyzer to assembled into 20 scaffolds then the gaps between scaffolds were filled by long fragment PCR amplification to obtain complet genmone with 4,061,186 bp in size. The whole genome was found to have 43.8% GC, 108 tandem repeats with an average of 2.65 copies and 84 transposons. The CDSs were predicted as 3,853 in which 112 CDSs were predicted to secondary metabolite biosynthesis, transport and catabolism. Among those, five NRPS/PKS giant gene clusters being responsible for the biosynthesis of polyketide (pksABCDEFHJLMNRS in size 72.9 kb), surfactin(srfABCD, 26.148 kb, bacilysin(bacABCDE 5.903 kb), bacillibactin(dhbABCEF, 11.774 kb) and fengycin(ppsABCDE, 37.799 kb) have high homolgous to fuction confirmed biosynthesis gene in other strain. Moreover, there are many of key regulatory genes for secondary metabolites from XF-1, such as comABPQKX Z, degQ, sfp, yczE, degU, ycxABCD and ywfG. were also predicted. Therefore, XF-1 has potential of biosynthesis for secondary metabolites surfactin, fengycin, bacillibactin, bacilysin and Bacillaene. Thirty two compounds were detected from cell extracts of XF-1 by MALDI-TOF-MS, including one Macrolactin (m/z 441.06), two fusaricidin (m/z 850.493 and 968.515), one circulocin (m/z 852.509), nine surfactin (m/z 1044.656~1102.652), five iturin (m/z 1096.631~1150.57) and forty fengycin (m/z 1449.79~1543.805). The top three compositions types (contening 56.67% of total extract) are surfactin, iturin and fengycin, in which the most abundant is the surfactin type composition 30.37% of total extract and in second place is the fengycin with 23.28% content with rich diversity of chemical structure, and the smallest one is the iturin with 3.02% content. Moreover, the same main compositions were detected in Bacillus sp.355 which is also a good effects biocontol bacterial for controlling the clubroot of crucifer. Wherefore those compounds surfactin, iturin and fengycin maybe the main active compositions of XF-1 against P. brassicae. Twenty one fengycin type compounds were evaluate by LC-ESI-MS/MS with antifungal activities, including fengycin A $C_{16{\sim}C19}$, fengycin B $C_{14{\sim}C17}$, fengycin C $C_{15{\sim}C18}$, fengycin D $C_{15{\sim}C18}$ and fengycin S $C_{15{\sim}C18}$. Furthermore, one novel compound was identified as Dehydroxyfengycin $C_{17}$ according its MS, 1D and 2D NMR spectral data, which molecular weight is 1488.8480 Da and formula $C_{75}H_{116}N_{12}O_{19}$. The fengycin type compounds (FTCPs $250{\mu}g/mL$) were used to treat the resting spores of P. brassicae ($10^7/mL$) by detecting leakage of the cytoplasm components and cell destruction. After 12 h treatment, the absorbencies at 260 nm (A260) and at 280 nm (A280) increased gradually to approaching the maximum of absorbance, accompanying the collapse of P. brassicae resting spores, and nearly no complete cells were observed at 24 h treatment. The results suggested that the cells could be lyzed by the FTCPs of XF-1, and the diversity of FTCPs was mainly attributed to a mechanism of clubroot disease biocontrol. In the five selected medium MOLP, PSA, LB, Landy and LD, the most suitable for growth of strain medium is MOLP, and the least for strains longevity is the Landy sucrose medium. However, the lipopeptide highest yield is in Landy sucrose medium. The lipopeptides in five medium were analyzed with HPLC, and the results showed that lipopeptides component were same, while their contents from B. subtilis XF-1 fermented in five medium were different. We found that it is the lipopeptides content but ingredients of XF-1 could be impacted by medium and lacking of nutrition seems promoting lipopeptides secretion from XF-1. The volatile components with inhibition fungal Cylindrocarpon spp. activity which were collect in sealed vesel were detected with metheds of HS-SPME-GC-MS in eight biocontrol Bacillus species and four positive mutant strains of XF-1 mutagenized with chemical mutagens, respectively. They have same main volatile components including pyrazine, aldehydes, oxazolidinone and sulfide which are composed of 91.62% in XF-1, in which, the most abundant is the pyrazine type composition with 47.03%, and in second place is the aldehydes with 23.84%, and the third place is oxazolidinone with 15.68%, and the smallest ones is the sulfide with 5.07%.

  • PDF

인삼내생균 Bacillus velezensis CH-15의 인삼뿌리썩음병 방제 효과 (Biological Efficacy of Endophytic Bacillus velezensis CH-15 from Ginseng against Ginseng Root Rot Pathogens)

  • 김도현;;이정관;이승호
    • 식물병연구
    • /
    • 제28권1호
    • /
    • pp.19-25
    • /
    • 2022
  • 인삼은 수천 년 동안 동아시아에서 재배된 중요한 약용 식물이다. 일반적으로 같은 포장에서 4-6년 동안 재배되기 때문에 다양한 병원균에 노출된다. 그 중 인삼뿌리썩음병이 가장 큰 피해를 주는 주요 원인이다. 본 연구에서는 건강한 인삼에서 내생균을 분리하고, 인삼뿌리썩음병원균에 대해 길항작용을 갖는 내생균을 선발하였다. 분리된 17개 균주 중, 3개 균주가 인삼뿌리썩음병원균에 길항작용을 나타냈으며 인삼뿌리썩음병균이 생산하는 곰팡이독소인 라디시콜에 내성을 보였다. 우수한 길항효과와 라디시콜 저항성을 갖는 Bacillus velezensis CH-15를 선발하여 추가 실험을 수행하였다. 인삼 뿌리에 CH-15를 접종하면 병원균의 균사 생장을 억제할 뿐만 아니라 질병의 진행도 억제하였다. CH-15는 또한 바실로마이신 D, 이투린 A, 바실리신, 서팩틴 생합성 유전자를 갖고 있었다. 또한 CH-15 배양여액은 병원균 생장과 분생포자 발아를 억제하였다. 본 연구는 내생균 CH-15가 인삼뿌리썩음병 병원체에 대해 길항작용을 하고 인삼뿌리썩음병의 진행을 억제함을 보여주었으며 이 균주가 인삼뿌리썩음병을 억제하는 미생물 제제가 될 수 있을 것으로 기대한다.