Identification and characterization of novel arsenic-containing antibiotics
Primary Investigator (PI) Name
Masafumi Yoshinaga
Department
CSM – Molecular and Cellular Biology
Abstract
The rise of antimicrobial resistance calls for new and effective antibiotics. Recently, arsinothricin (AST), a novel broad-spectrum arsenic-containing antibiotic, was discovered. AST is a nonproteinogenic analogue of glutamate that is biosynthesized via two steps catalyzed by ArsL and ArsM, unlike its phophorus mimetic, phosphinothricin, which requires over 20 steps. In light of the effectiveness and high selective toxicity of AST, the aim of this project is finding further novel arsenic-containing antibiotics. arsL-guided genome-mining found that many bacteria have arsL-containing biosynthetic gene clusters (BGCs). Their gene compositions are different from the AST BGC, suggesting the potential to produce novel arsenic-containing antibiotics. The BGCs from Anoxybacillus calidus contains two novel genes alongside arsL, named ars1 and ars2, which are annotated to encode 4-carboxymuconolactone decarboxylase and biotin carboxylase, respectively. Given that ArsL converts arsenite [As(III)] to hydroxy AST (AST-OH), the non-methylated AST precursor, it is reasonable to predict that Ars1 decarboxylates AST-OH while Ars2 carboxylates the amino group of the decarboxylated intermediate, leading to the production of an arsenic mimetic of the phosphonate antibiotic fosmidomycin (FM). Thus, the predicted organoarsenical is named arsmidmycin (ASM). A. calidus was found to convert As(III) to multiple arsenic species . The detected arsenic species were crudely purified by size exclusion and cation exchange column chromatography. A series of preliminary experiments demonstrate that 1) the mass of the major arsenic species matches the predicted mass of ASM, and 2) the crudely purified major arsenic species exhibits moderate antibiotic activity, supporting our hypothesis. In parallel, ASM biosynthesis is also being investigated using Escherichia coli heterogeneous expression system, where arsL, ars1 and/or ars2 will be solely expressed or co-expressed. Each construct will be cultured with As(III) and the produced arsenic species will be analyzed to not only complement our hypothesis but also elucidate the ASM biosynthetic pathway.
Disciplines
Biochemistry | Environmental Microbiology and Microbial Ecology | Integrative Biology | Molecular Biology
Identification and characterization of novel arsenic-containing antibiotics
The rise of antimicrobial resistance calls for new and effective antibiotics. Recently, arsinothricin (AST), a novel broad-spectrum arsenic-containing antibiotic, was discovered. AST is a nonproteinogenic analogue of glutamate that is biosynthesized via two steps catalyzed by ArsL and ArsM, unlike its phophorus mimetic, phosphinothricin, which requires over 20 steps. In light of the effectiveness and high selective toxicity of AST, the aim of this project is finding further novel arsenic-containing antibiotics. arsL-guided genome-mining found that many bacteria have arsL-containing biosynthetic gene clusters (BGCs). Their gene compositions are different from the AST BGC, suggesting the potential to produce novel arsenic-containing antibiotics. The BGCs from Anoxybacillus calidus contains two novel genes alongside arsL, named ars1 and ars2, which are annotated to encode 4-carboxymuconolactone decarboxylase and biotin carboxylase, respectively. Given that ArsL converts arsenite [As(III)] to hydroxy AST (AST-OH), the non-methylated AST precursor, it is reasonable to predict that Ars1 decarboxylates AST-OH while Ars2 carboxylates the amino group of the decarboxylated intermediate, leading to the production of an arsenic mimetic of the phosphonate antibiotic fosmidomycin (FM). Thus, the predicted organoarsenical is named arsmidmycin (ASM). A. calidus was found to convert As(III) to multiple arsenic species . The detected arsenic species were crudely purified by size exclusion and cation exchange column chromatography. A series of preliminary experiments demonstrate that 1) the mass of the major arsenic species matches the predicted mass of ASM, and 2) the crudely purified major arsenic species exhibits moderate antibiotic activity, supporting our hypothesis. In parallel, ASM biosynthesis is also being investigated using Escherichia coli heterogeneous expression system, where arsL, ars1 and/or ars2 will be solely expressed or co-expressed. Each construct will be cultured with As(III) and the produced arsenic species will be analyzed to not only complement our hypothesis but also elucidate the ASM biosynthetic pathway.