Efficient pathway-driven scyllo -inositol production from myo -inositol using thermophilic cells and mesophilic inositol dehydrogenases: a novel strategy for pathway control
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 cata...
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| Published in | Applied and Environmental Microbiology Vol. 90; no. 7; p. e0028124 |
|---|---|
| Main Authors | , , , , , , , , , , , |
| Format | Journal Article |
| Language | English |
| Published |
United States
American Society for Microbiology
24.07.2024
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| Subjects | |
| Online Access | Get full text |
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| Abstract | 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. |
|---|---|
| AbstractList | 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. 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.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.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.IMPORTANCEEnzyme 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. 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 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 promoter could direct efficient gene expression at 40°C. We cloned the promoter into an shuttle plasmid and confirmed that the resulting vector functioned in and other thermophiles. We then used this vector for the cooperative expression of the and genes from 168. 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 -inositol into -inositol at 30°C. In a scaled-up reaction, 10 g of -inositol was converted to 1.8 g of -inositol, which was further purified to yield 970 mg of pure powder. Notably, -inositol was degraded by intrinsic enzymes of 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. 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 -inositol from an economical substrate. Notably, this marks the first large-scale preparation of pure -inositol, holding potential pharmaceutical significance as -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. 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.IMPORTANCEEnzyme 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. 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. |
| Author | Kohei Makino Kaho Fukui Yuta Okumura Tatsuya Ono Kyosuke Yokoyama Ryota Kurashiki Hirofumi Endo Takashi Ohshiro Shu Ishikawa Masahiro Takahashi Ken-ichi Yoshida Hirokazu Suzuki |
| Author_xml | – sequence: 1 givenname: Ryota surname: Kurashiki fullname: Kurashiki, Ryota organization: Department of Engineering, Graduate School of Sustainability Science, Tottori University, Tottori, Japan – sequence: 2 givenname: Masahiro surname: Takahashi fullname: Takahashi, Masahiro organization: IMRA Japan Co., Ltd., Kariya, Japan – sequence: 3 givenname: Yuta surname: Okumura fullname: Okumura, Yuta organization: Department of Engineering, Graduate School of Sustainability Science, Tottori University, Tottori, Japan – sequence: 4 givenname: Tatsuya surname: Ono fullname: Ono, Tatsuya organization: Department of Engineering, Graduate School of Sustainability Science, Tottori University, Tottori, Japan – sequence: 5 givenname: Hirofumi surname: Endo fullname: Endo, Hirofumi organization: Department of Chemistry and Biotechnology, Graduate School of Engineering, Tottori University, Tottori, Japan – sequence: 6 givenname: Kohei surname: Makino fullname: Makino, Kohei organization: Graduate School of Science, Technology and Innovation, Kobe University, Kobe, Japan – sequence: 7 givenname: Kaho surname: Fukui fullname: Fukui, Kaho organization: Graduate School of Science, Technology and Innovation, Kobe University, Kobe, Japan – sequence: 8 givenname: Kyosuke surname: Yokoyama fullname: Yokoyama, Kyosuke organization: Graduate School of Science, Technology and Innovation, Kobe University, Kobe, Japan – sequence: 9 givenname: Shu surname: Ishikawa fullname: Ishikawa, Shu organization: Graduate School of Science, Technology and Innovation, Kobe University, Kobe, Japan – sequence: 10 givenname: Ken-ichi orcidid: 0000-0002-3383-4664 surname: Yoshida fullname: Yoshida, Ken-ichi organization: Graduate School of Science, Technology and Innovation, Kobe University, Kobe, Japan – sequence: 11 givenname: Takashi surname: Ohshiro fullname: Ohshiro, Takashi organization: Faculty of Engineering, Tottori University, Tottori, Japan, Center for Research on Green Sustainable Chemistry, Tottori University, Tottori, Japan – sequence: 12 givenname: Hirokazu orcidid: 0000-0002-9507-6467 surname: Suzuki fullname: Suzuki, Hirokazu organization: Faculty of Engineering, Tottori University, Tottori, Japan, Center for Research on Green Sustainable Chemistry, Tottori University, Tottori, Japan |
| BackLink | https://cir.nii.ac.jp/crid/1872555066415616768$$DView record in CiNii https://www.ncbi.nlm.nih.gov/pubmed/38975762$$D View this record in MEDLINE/PubMed |
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| ContentType | Journal Article |
| Copyright | Copyright © 2024 American Society for Microbiology. Copyright © 2024 American Society for Microbiology. 2024 American Society for Microbiology. |
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| Editor | Atomi, Haruyuki |
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| Issue | 7 |
| Keywords | iol gene pathway engineering bioconversion Bacillus Geobacillus cell-free synthesis enzyme cocktail |
| Language | English |
| License | All Rights Reserved. https://doi.org/10.1128/ASMCopyrightv2 All Rights Reserved. |
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| Snippet | Enzyme cocktails are commonly employed for cell-free chemical synthesis; however, their preparation involves cumbersome processes. This study affirms that... Mesophilic enzymes, which are active at moderate temperatures, may dominate enzymatic reactions even in the presence of thermophilic crude enzymes. This study... |
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| SubjectTerms | Bacillus subtilis - enzymology Bacillus subtilis - genetics Bacillus subtilis - metabolism Bacterial Proteins - genetics Bacterial Proteins - metabolism Biotechnology Escherichia coli - genetics Escherichia coli - metabolism Geobacillus - enzymology Geobacillus - genetics Geobacillus - metabolism Inositol - metabolism Promoter Regions, Genetic Sugar Alcohol Dehydrogenases - genetics Sugar Alcohol Dehydrogenases - metabolism |
| Title | 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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