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A Validamycin Shunt Pathway for Valienamine Synthesis in Engineered Streptomyces hygroscopicus 5008.
- Source :
-
ACS synthetic biology [ACS Synth Biol] 2020 Feb 21; Vol. 9 (2), pp. 294-303. Date of Electronic Publication: 2020 Jan 24. - Publication Year :
- 2020
-
Abstract
- Valienamine is the key functional component of many natural glycosidase inhibitors, including the crop protectant validamycin A and the clinical antidiabetic agent acarbose. Due to its important biomedical activity, it is also the prominent lead compound for the exploration of therapeutic agents, such as the stronger α-glucosidase inhibitor voglibose. Currently, the main route for obtaining valienamine is a multistep biosynthetic process involving the synthesis and degradation of validamycin A. Here, we established an alternative, vastly simplified shunt pathway for the direct synthesis of valienamine based on an envisioned non-natural transamination in the validamycin A producer Streptomyces hygroscopicus 5008. We first identified candidate aminotransferases for the non-natural ketone substrate valienone and conducted molecular evolution in vitro . The WecE enzyme from Escherichia coli was verified to complete the envisioned step with >99.9% enantiomeric excess and was further engineered to produce a 32.6-fold more active mutant, VarB, through protein evolution. Subsequently, two copies of VarB were introduced into the host, and the new shunt pathway produced 0.52 mg/L valienamine after a 96-h fermentation. Our study thus illustrates a dramatically simplified alternative shunt pathway for valienamine production and introduces a promising foundational platform for increasing the production of valienamine and its valuable N-modified derivatives for use in pharmaceutical applications.
- Subjects :
- Binding Sites
Catalytic Domain
Cyclohexenes chemistry
Cyclohexenes metabolism
Escherichia coli enzymology
Escherichia coli Proteins chemistry
Escherichia coli Proteins genetics
Escherichia coli Proteins metabolism
Hexosamines chemistry
Hexosamines metabolism
Inositol chemistry
Inositol metabolism
Kinetics
Molecular Docking Simulation
Mutagenesis, Site-Directed
Streptomyces metabolism
Transaminases chemistry
Transaminases genetics
Transaminases metabolism
Hexosamines biosynthesis
Inositol analogs & derivatives
Streptomyces chemistry
Subjects
Details
- Language :
- English
- ISSN :
- 2161-5063
- Volume :
- 9
- Issue :
- 2
- Database :
- MEDLINE
- Journal :
- ACS synthetic biology
- Publication Type :
- Academic Journal
- Accession number :
- 31940432
- Full Text :
- https://doi.org/10.1021/acssynbio.9b00319