Design and synthesis of pathway genes for polyketide biosynthesis

S. PEIRÚ, H. GRAMAJO AND HG. MENZELLA

METHODS IN ENZYMOLOGY.

ELSEVIER ACADEMIC PRESS INC

Año: 2009 vol. 459 p. 319 - 337

0076-6879

In this chapter we describe novel methods for the design and assembly of synthetic pathways for the synthesis of polyketides and tailoring sugars. First, a generic design for type I polyketide synthase genes is presented that allows their facile assembly for the expression of chimeric enzymes in an engineered Escherichia coli host. The sequences of the synthetic genes are based on naturally occurring polyketide synthase genes but they are redesigned by custom-made software to optimize codon usage to maximize expression inhost. The sequences of the synthetic genes are based on naturally occurring polyketide synthase genes but they are redesigned by custom-made software to optimize codon usage to maximize expression in E. coli and to provide a standard set of restriction sites to allow combinatorial assembly into unnatural enzymes. The methodology has been validated by building a large number of bimodular mini-PKSs that make easily assayed triketide products. Learning from the successful bimodules, a conceptual advance was made by assembling genes encoding functional trimodular enzymes, capable of making tetraketide products. Second, methods for the rapid assembly and exchange of sugar pathway genes into functional operonsand to provide a standard set of restriction sites to allow combinatorial assembly into unnatural enzymes. The methodology has been validated by building a large number of bimodular mini-PKSs that make easily assayed triketide products. Learning from the successful bimodules, a conceptual advance was made by assembling genes encoding functional trimodular enzymes, capable of making tetraketide products. Second, methods for the rapid assembly and exchange of sugar pathway genes into functional operons