دورية أكاديمية
Efficient pathway-driven scyllo -inositol production from myo -inositol using thermophilic cells and mesophilic inositol dehydrogenases: a novel strategy for pathway control.
| العنوان: | Efficient pathway-driven scyllo -inositol production from myo -inositol using thermophilic cells and mesophilic inositol dehydrogenases: a novel strategy for pathway control. |
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| المؤلفون: | Kurashiki R; Department of Engineering, Graduate School of Sustainability Science, Tottori University, Tottori, Japan., Takahashi M; IMRA Japan Co., Ltd., Kariya, Japan., Okumura Y; Department of Engineering, Graduate School of Sustainability Science, Tottori University, Tottori, Japan., Ono T; Department of Engineering, Graduate School of Sustainability Science, Tottori University, Tottori, Japan., Endo H; Department of Chemistry and Biotechnology, Graduate School of Engineering, Tottori University, Tottori, Japan., Makino K; Graduate School of Science, Technology and Innovation, Kobe University, Kobe, Japan., Fukui K; Graduate School of Science, Technology and Innovation, Kobe University, Kobe, Japan., Yokoyama K; Graduate School of Science, Technology and Innovation, Kobe University, Kobe, Japan., Ishikawa S; Graduate School of Science, Technology and Innovation, Kobe University, Kobe, Japan., Yoshida K-i; Graduate School of Science, Technology and Innovation, Kobe University, Kobe, Japan., Ohshiro T; Faculty of Engineering, Tottori University, Tottori, Japan.; Center for Research on Green Sustainable Chemistry, Tottori University, Tottori, Japan., Suzuki H; Faculty of Engineering, Tottori University, Tottori, Japan.; Center for Research on Green Sustainable Chemistry, Tottori University, Tottori, Japan. |
| المصدر: | Applied and environmental microbiology [Appl Environ Microbiol] 2024 Jul 24; Vol. 90 (7), pp. e0028124. Date of Electronic Publication: 2024 Jul 08. |
| نوع المنشور: | Journal Article |
| اللغة: | English |
| بيانات الدورية: | Publisher: American Society for Microbiology Country of Publication: United States NLM ID: 7605801 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1098-5336 (Electronic) Linking ISSN: 00992240 NLM ISO Abbreviation: Appl Environ Microbiol Subsets: MEDLINE |
| أسماء مطبوعة: | Original Publication: Washington, American Society for Microbiology. |
| مواضيع طبية MeSH: | Inositol*/metabolism , Geobacillus*/genetics , Geobacillus*/enzymology , Geobacillus*/metabolism , Bacterial Proteins*/genetics , Bacterial Proteins*/metabolism, Bacillus subtilis/genetics ; Bacillus subtilis/enzymology ; Bacillus subtilis/metabolism ; Sugar Alcohol Dehydrogenases/genetics ; Sugar Alcohol Dehydrogenases/metabolism ; Escherichia coli/genetics ; Escherichia coli/metabolism ; Promoter Regions, Genetic |
| مستخلص: | Mesophilic enzymes, which are active at moderate temperatures, may dominate enzymatic reactions even in the presence of thermophilic crude enzymes. This study was conducted to investigate this hypothesis with mesophilic inositol dehydrogenases (IolG and IolX) produced in Geobacillus kaustophilus HTA426. To ensure the efficient production of mesophilic enzymes, we first screened for promoters induced at moderate temperatures using transcriptome analysis and identified four genes highly expressed at 30°C in the thermophile. We further characterized these promoters using fluorescent reporter assays to determine that the mti3 promoter could direct efficient gene expression at 40°C. We cloned the promoter into an Escherichia coli-Geobacillus shuttle plasmid and confirmed that the resulting vector functioned in G. kaustophilus and other thermophiles. We then used this vector for the cooperative expression of the iolG and iolX genes from Bacillus subtilis 168. G. kaustophilus cells carrying the expression vector were incubated at 60°C for cellular propagation and then at 40°C for the production of IolG and IolX. When the cells were permeabilized, IolG and IolX acted as catalysts to convert exogenous myo -inositol into scyllo -inositol at 30°C. In a scaled-up reaction, 10 g of myo -inositol was converted to 1.8 g of scyllo -inositol, which was further purified to yield 970 mg of pure powder. Notably, myo -inositol was degraded by intrinsic enzymes of G. kaustophilus at 60°C but not at 30°C, supporting our initial hypothesis. We indicate that this approach is useful for preparing enzyme cocktails without the need for purification. Importance: Enzyme cocktails are commonly employed for cell-free chemical synthesis; however, their preparation involves cumbersome processes. This study affirms that mesophilic enzymes in thermophilic crude extracts can function as specific catalysts at moderate temperatures, akin to enzyme cocktails. The catalyst was prepared by permeabilizing cells without the need for concentration, extraction, or purification processes; hence, its preparation was considerably simpler compared with conventional methods for enzyme cocktails. This approach was employed to produce pure scyllo -inositol from an economical substrate. Notably, this marks the first large-scale preparation of pure scyllo -inositol, holding potential pharmaceutical significance as scyllo -inositol serves as a promising agent for certain diseases but is currently expensive. Moreover, this approach holds promise for application in pathway engineering within living cells. The envisioned pathway is designed without chromosomal modification and is simply regulated by switching culture temperatures. Consequently, this study introduces a novel platform for both whole-cell and cell-free synthetic systems. Competing Interests: The authors declare no conflict of interest. |
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| معلومات مُعتمدة: | Noda Institute for Scientific Research (NISR) |
| فهرسة مساهمة: | Keywords: Bacillus; Geobacillus; bioconversion; cell-free synthesis; enzyme cocktail; iol gene; pathway engineering |
| المشرفين على المادة: | 4L6452S749 (Inositol) 1VS4X81277 (scyllitol) 0 (Bacterial Proteins) EC 1.1.- (Sugar Alcohol Dehydrogenases) |
| تواريخ الأحداث: | Date Created: 20240708 Date Completed: 20240724 Latest Revision: 20240726 |
| رمز التحديث: | 20240726 |
| مُعرف محوري في PubMed: | PMC11267878 |
| DOI: | 10.1128/aem.00281-24 |
| PMID: | 38975762 |
| قاعدة البيانات: | MEDLINE |
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