Complete Reconstitution of a Highly Reducing Iterative Polyketide Synthase
Suzanne M. Ma,1
Jesse W.-H. Li,2
Jin W. Choi,3
Hui Zhou,1
K. K. Michael Lee,3
Vijayalakshmi A. Moorthie,2
Xinkai Xie,1
James T. Kealey,4
Nancy A. Da Silva,3
John C. Vederas,2,*
Yi Tang1,*
Highly reducing iterative polyketide synthases are large, multifunctional
enzymes that make important metabolites in fungi, such as lovastatin,
a cholesterol-lowering drug from
Aspergillus terreus. We report
efficient expression of the lovastatin nonaketide synthase (LovB)
from an engineered strain of
Saccharomyces cerevisiae, as well
as complete reconstitution of its catalytic function in the
presence and absence of cofactors (the reduced form of nicotinamide
adenine dinucleotide phosphate and
S-adenosylmethionine) and
its partner enzyme, the enoyl reductase LovC. Our results demonstrate
that LovB retains correct intermediates until completion of
synthesis of dihydromonacolin L, but off-loads incorrectly processed
compounds as pyrones or hydrolytic products. Experiments replacing
LovC with analogous MlcG from compactin biosynthesis demonstrate
a gate-keeping function for this partner enzyme. This study
represents a key step in the understanding of the functions
and structures of this family of enzymes.
2 Department of Chemistry, University of Alberta, Edmonton, Alberta, T6G 2G2, Canada.
3 Department of Chemical Engineering and Materials Science, University of California, Irvine, CA 92697, USA.
4 Amyris Biotechnologies, 5885 Hollis Street, Suite 100, Emeryville, CA 94608, USA.
* To whom correspondence should be addressed. E-mail: john.vederas{at}ualberta.ca (J.C.V.); yitang{at}ucla.edu (Y.T.)
