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Related Experiment Videos

Multiple pathways for acetate assimilation in Streptomyces cinnamonensis.

Konstantin Akopiants1, Galina Florova, Chaoxuan Li

  • 1Institute for Structural Biology and Drug Discovery, Virginia Commonwealth University, 800 E. Leigh Street, Suite 212B, Richmond, VA, 23219, USA.

Journal of Industrial Microbiology & Biotechnology
|September 28, 2005
PubMed
Summary

Streptomyces utilize alternative pathways for acetate assimilation, as the glyoxylate cycle is not essential for growth on acetate. New pathways are suggested by mutants unable to use the glyoxylate or butyryl-CoA routes.

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Area of Science:

  • Microbiology
  • Biochemistry
  • Molecular Biology

Background:

  • Acetate assimilation in bacteria typically occurs via the glyoxylate pathway, involving key enzymes isocitrate lyase (ICL) and malate synthase (MS).
  • Streptomycetes possess genes for these enzymes, but their role in acetate assimilation is unclear, with evidence suggesting an alternative butyryl-CoA pathway is critical for growth on acetate in some species.
  • In Streptomyces cinnamonensis, the butyryl-CoA pathway, including crotonyl-CoA reductase (CCR), is vital for producing precursors for monensin A biosynthesis.

Purpose of the Study:

  • To investigate the role of the glyoxylate pathway in acetate assimilation in Streptomyces.
  • To determine if expressing heterologous glyoxylate cycle genes can restore acetate assimilation in a butyryl-CoA pathway mutant.
  • To identify alternative acetate assimilation pathways in Streptomyces.

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Main Methods:

  • Cloning of a malate synthase (MS) homologue gene from Streptomyces cinnamonensis.
  • Reverse transcription and enzyme assays to assess gene expression and enzyme activity.
  • Genetic manipulation, including creating a crotonyl-CoA reductase (CCR) mutant (L1 strain) and introducing heterologous Streptomyces coelicolor glyoxylate cycle genes (aceA and aceB2) via a plasmid (pExIM1).
  • Growth studies on acetate as a sole carbon source.
  • UV mutagenesis to generate new mutants.

Main Results:

  • Native MS and ICL genes/activities were not detected in S. cinnamonensis C730.1 or the L1 mutant during fermentation.
  • The wild-type strain (C730.1) grew on acetate, but the L1 mutant did not, confirming the butyryl-CoA pathway's importance.
  • Expression of heterologous ICL and MS in the L1 mutant (L1/pExIM1) did not restore acetate growth or efficient monensin precursor production, indicating a non-functional glyoxylate cycle.
  • Mutants generated from L1 and L1/pExIM1 strains could grow on acetate despite a blocked butyryl-CoA pathway, suggesting alternative assimilation routes.

Conclusions:

  • The glyoxylate cycle is not essential for acetate assimilation in Streptomyces cinnamonensis.
  • The butyryl-CoA pathway is crucial for acetate assimilation and monensin A precursor biosynthesis in S. cinnamonensis.
  • The identification of acetate-utilizing mutants with blocked butyryl-CoA and non-functional glyoxylate pathways points to the existence of novel acetate assimilation mechanisms in Streptomyces.