Metabolic engineering of Pseudomonas putida for methylmalonyl-CoA biosynthesis to enable complex heterologous secondary metabolite formation.
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.
Gross
Frank
F
Pharmaceutical Biotechnology, Saarland University, P.O. Box 151150, 66041 Saarbrücken, Germany.
Ring
Michael W
MW
Perlova
Olena
O
Fu
Jun
J
Schneider
Susan
S
Gerth
Klaus
K
Kuhlmann
Silvia
S
Stewart
A Francis
AF
Zhang
Youming
Y
Müller
Rolf
R
D000214:Acyl Coenzyme A / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism*; D000595:Amino Acid Sequence; D005786:Gene Expression Regulation; D018014:Gene Transfer Techniques; D005818:Genetic Engineering / Q000379:methods*; D008689:Methacrylates / Q000378:metabolism; D008969:Molecular Sequence Data; D009231:Myxococcales / Q000235:genetics; D009876:Operon / Q000235:genetics; D016958:Pseudomonas putida / Q000235:genetics* / Q000378:metabolism*; D016415:Sequence Alignment; D013844:Thiazoles / Q000378:metabolism
D000214:Acyl Coenzyme A; D008689:Methacrylates; D013844:Thiazoles; C015357:methylmalonyl-coenzyme A; C030517:myxothiazol