Alteration of the substrate specificity of a modular polyketide synthase acyltransferase domain through site-specific mutations.
Cassette replacement of acyltransferase (AT) domains in 6-deoxyerythronolide B synthase (DEBS) with heterologous AT domains with different substrate specificities usually yields the predicted polyketide analogues. As reported here, however, several AT replacements in module 4 of DEBS failed to produce detectable polyketide under standard conditions, suggesting that module 4 is sensitive to perturbation of the protein structure when the AT is replaced. Alignments between different modular polyketide synthase AT domains and the Escherichia coli fatty acid synthase transacylase crystal structure were used to select motifs within the AT domain of module 4 to re-engineer its substrate selectivity and minimize potential alterations to protein folding. Three distinct primary regions of AT4 believed to confer specificity for methylmalonyl-CoA were mutated into the sequence seen in malonyl-CoA-specific domains. Each individual mutation as well as the three in combination resulted in functional DEBSs that produced mixtures of the natural polyketide, 6-deoxyerythronolide B, and the desired novel analogue, 6-desmethyl-6-deoxyerythronolide B. Production of the latter compound indicates that the identified sequence motifs do contribute to AT specificity and that DEBS can process a polyketide chain incorporating a malonate unit at module 4. This is the first example in which the extender unit specificity of a PKS module has been altered by site-specific mutation and provides a useful alternate method for engineering AT specificity in the combinatorial biosynthesis of polyketides.
Reeves
C D
CD
Kosan Biosciences, Inc., 3832 Bay Center Place, Hayward, California 94545, USA.
Murli
S
S
Ashley
G W
GW
Piagentini
M
M
Hutchinson
C R
CR
McDaniel
R
R
D000217:Acyltransferases / Q000737:chemistry* / Q000235:genetics*; D019943:Amino Acid Substitution / Q000235:genetics; D008316:Malonyl Coenzyme A / Q000737:chemistry / Q000235:genetics; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000235:genetics*; D016297:Mutagenesis, Site-Directed; D019906:Nuclear Magnetic Resonance, Biomolecular; D011485:Protein Binding / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D013302:Streptomyces / Q000201:enzymology / Q000235:genetics; D013379:Substrate Specificity / Q000235:genetics
D009097:Multienzyme Complexes; D008316:Malonyl Coenzyme A; D000217:Acyltransferases