Redesign, synthesis and functional expression of the 6-deoxyerythronolide B polyketide synthase gene cluster.
A generic design of Type I polyketide synthase genes has been reported in which modules, and domains within modules, are flanked by sets of unique restriction sites that are repeated in every module [1]. Using the universal design, we synthesized the six-module DEBS gene cluster optimized for codon usage in E. coli, and cloned the three open reading frames into three compatible expression vectors. With one correctable exception, the amino acid substitutions required for restriction site placements were compatible with polyketide production. When expressed in E. coli the codon-optimized synthetic gene cluster produced significantly more protein than did the wild-type sequence. Indeed, for optimal polyketide production, PKS expression had to be down-regulated by promoter attenuation to achieve balance with expression of the accessory proteins needed to support polyketide biosynthesis.
Menzella
Hugo G
HG
Kosan Biosciences, Inc., 3832 Bay Center Place, Hayward, CA, 94545, USA. santi@kosan.com
Reisinger
Sarah J
SJ
Welch
Mark
M
Kealey
James T
JT
Kennedy
Jonathan
J
Reid
Ralph
R
Tran
Chau Q
CQ
Santi
Daniel V
DV
D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics / Q000502:physiology; D005798:Genes, Bacterial / Q000235:genetics; D009097:Multienzyme Complexes / Q000378:metabolism; D005810:Multigene Family; D010957:Plasmids / Q000235:genetics; D048630:Polyketide Synthases / Q000235:genetics* / Q000378:metabolism
D009097:Multienzyme Complexes; D048630:Polyketide Synthases