Metabolic engineering of Pseudomonas putida for methylmalonyl-CoA biosynthesis to enable complex heterologous secondary metabolite formation

Chem Biol. 2006 Dec;13(12):1253-64. doi: 10.1016/j.chembiol.2006.09.014.

Abstract

An operon consisting of three open reading frames, annotated in silico as methylmalonyl-CoA (mm-CoA) epimerase, mm-CoA mutase (MCM), and meaB, was identified in the sequencing project of the myxobacterium Sorangium cellulosum So ce56. This putative MCM pathway operon was subcloned from a bacterial artificial chromosome by Red/ET recombineering onto a minimal replicon derived from p15A. This plasmid was modified for integration and heterologous expression in Pseudomonas putida to enable the production of complex secondary metabolites requiring mm-CoA as precursor. Methylmalonate was identified in the recombinant P. putida strain by an analysis method based on gas chromatography/mass spectrometry. The engineered strain is able to synthesize polyketides requiring mm-CoA as an extender unit, which was demonstrated by the production of myxothiazol after integration of the biosynthetic gene cluster into the chromosome, followed by induction of expression.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acyl Coenzyme A / biosynthesis
  • Acyl Coenzyme A / genetics*
  • Acyl Coenzyme A / metabolism*
  • Amino Acid Sequence
  • Gene Expression Regulation
  • Gene Transfer Techniques
  • Genetic Engineering / methods*
  • Methacrylates / metabolism
  • Molecular Sequence Data
  • Myxococcales / genetics
  • Operon / genetics
  • Pseudomonas putida / genetics*
  • Pseudomonas putida / metabolism*
  • Sequence Alignment
  • Thiazoles / metabolism

Substances

  • Acyl Coenzyme A
  • Methacrylates
  • Thiazoles
  • methylmalonyl-coenzyme A
  • myxothiazol

Associated data

  • GENBANK/AM260198