Engineered intermodular and intramodular polyketide synthase fusions.
BACKGROUND
Modular polyketide synthases (PKSs) are very large multifunctional enzyme complexes that synthesize a number of medicinally important natural products. The modular arrangement of active sites has made these enzyme systems amenable to combinatorial manipulation for the biosynthesis of novel polyketides. Here, we investigate the involvement of subunit interactions in hybrid and artificially linked PKSs with several series of intermodular and intramodular fusions using the erythromycin (6-deoxyerythronolide B synthase; DEBS) and rapamycin (RAPS) PKSs.


RESULTS
Several two-module and three-module derivatives of DEBS were constructed by fusing module 6 to either module 2 or module 3 at varying junctions. Polyketide production by these intramodular fusions indicated that the core set of active sites remained functional in these hybrid modules, although the ketoreductase domain of module 6 was unable to recognize unnatural triketide and tetraketide substrates. Artificial trimodular PKS subunits were also engineered by covalently linking modules 2 and 3 of DEBS, thereby demonstrating the feasibility of constructing single-chain PKSs. Finally, a series of fusions containing DEBS and RAPS domains in module 2 of an engineered trimodular PKS revealed the structural and functional tolerance for hybrid modules created from distinct PKS gene clusters.


CONCLUSIONS
The general success of the intermodular and intramodular fusions described here demonstrates significant structural tolerance among PKS modules and subunits and suggests that substrate specificity, rather than protein-protein interactions, is the primary determinant of molecular recognition features of PKSs. Furthermore, the ability to artificially link modules may considerably simplify the heterologous expression of modular PKSs in higher eukaryotic systems.
McDaniel
R
R
KOSAN Biosciences, Inc., Burlingame, CA 94010, USA. mcdaniel@kosan.com
Kao
C M
CM
Hwang
S J
SJ
Khosla
C
C
D000595:Amino Acid Sequence; D001483:Base Sequence; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000138:chemical synthesis* / Q000235:genetics* / Q000378:metabolism; D010455:Peptides / Q000138:chemical synthesis / Q000235:genetics; D011090:Polyenes / Q000138:chemical synthesis / Q000737:chemistry; D015202:Protein Engineering / Q000379:methods; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000138:chemical synthesis* / Q000235:genetics / Q000302:isolation & purification; D020123:Sirolimus; D013302:Streptomyces / Q000201:enzymology; D013329:Structure-Activity Relationship
D009097:Multienzyme Complexes; D010455:Peptides; D011090:Polyenes; D011993:Recombinant Fusion Proteins; D020123:Sirolimus