Engineered biosynthesis of regioselectively modified aromatic polyketides using bimodular polyketide synthases

被引:72
作者
Tang, Y
Lee, TS
Khosla, C [1 ]
机构
[1] Stanford Univ, Dept Chem Engn, Stanford, CA USA
[2] Stanford Univ, Dept Chem, Stanford, CA USA
[3] Stanford Univ, Dept Biochem, Stanford, CA USA
来源
PLOS BIOLOGY | 2004年 / 2卷 / 02期
关键词
D O I
10.1371/journal.pbio.0020031
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Bacterial aromatic polyketides such as tetracycline and doxorubicin are a medicinally important class of natural products produced as secondary metabolites by actinomyces bacteria. Their backbones are derived from malonyl-CoA units by polyketide synthases (PKSs). The nascent polyketide chain is synthesized by the minimal PKS, a module consisting of four dissociated enzymes. Although the biosynthesis of most aromatic polyketide backbones is initiated through decarboxylation of a malonyl building block (which results in an acetate group), some polyketides, such as the estrogen receptor antagonist R1128, are derived from nonacetate primers. Understanding the mechanism of nonacetate priming can lead to biosynthesis of novel polyketides that have improved pharmacological properties. Recent biochemical analysis has shown that nonacetate priming is the result of stepwise activity of two dissociated PKS modules with orthogonal molecular recognition features. In these PKSs, an initiation module that synthesizes a starter unit is present in addition to the minimal PKS module. Here we describe a general method for the engineered biosynthesis of regioselectively modified aromatic polyketides. When coexpressed with the R1128 initiation module, the actinorhodin minimal PKS produced novel hexaketides with propionyl and isobutyryl primer units. Analogous octaketides could be synthesized by combining the tetracenomycin minimal PKS with the R1128 initiation module. Tailoring enzymes such as ketoreductases and cyclases were able to process the unnatural polyketides efficiently. Based upon these findings, hybrid PKSs were engineered to synthesize new anthraquinone antibiotics with predictable functional group modifications. Our results demonstrate that (i) bimodular aromatic PKSs present a general mechanism for priming aromatic polyketide backbones with nonacetate precursors; (ii) the minimal PKS controls polyketide chain length by counting the number of atoms incorporated into the backbone rather than the number of elongation cycles; and (iii) in contrast, auxiliary PKS enzymes such as ketoreductases, aromatases, and cyclases recognize specific functional groups in the backbone rather than overall chain length. Among the anthracyclines engineered in this study were compounds with (i) more superior activity than R1128 against the breast cancer cell line MCF-7 and (ii) inhibitory activity against glucose-6-phosphate translocase, an attractive target for the treatment of Type II diabetes.
引用
收藏
页码:227 / 238
页数:12
相关论文
共 41 条
[1]  
ARION WJ, 1989, METHOD ENZYMOL, V174, P58
[2]   BIOSYNTHESIS OF ANTHRAQUINONES BY INTERSPECIES CLONING OF ACTINORHODIN BIOSYNTHESIS GENES IN STREPTOMYCETES - CLARIFICATION OF ACTINORHODIN GENE FUNCTIONS [J].
BARTEL, PL ;
ZHU, CB ;
LAMPEL, JS ;
DOSCH, DC ;
CONNORS, NC ;
STROHL, WR ;
BEALE, JM ;
FLOSS, HG .
JOURNAL OF BACTERIOLOGY, 1990, 172 (09) :4816-4826
[3]   CLONING, SEQUENCING AND DEDUCED FUNCTIONS OF A CLUSTER OF STREPTOMYCES GENES PROBABLY ENCODING BIOSYNTHESIS OF THE POLYKETIDE ANTIBIOTIC FRENOLICIN [J].
BIBB, MJ ;
SHERMAN, DH ;
OMURA, S ;
HOPWOOD, DA .
GENE, 1994, 142 (01) :31-39
[4]   A chain initiation factor common to both modular and aromatic polyketide synthases [J].
Bisang, C ;
Long, PF ;
Cortés, J ;
Westcott, J ;
Crosby, J ;
Matharu, AL ;
Cox, RJ ;
Simpson, TJ ;
Staunton, J ;
Leadlay, PF .
NATURE, 1999, 401 (6752) :502-505
[5]   Biochemistry - Harnessing the biosynthetic code: Combinations, permutations, and mutations [J].
Cane, DE ;
Walsh, CT ;
Khosla, C .
SCIENCE, 1998, 282 (5386) :63-68
[6]   Identification of a cyclohexylcarbonyl CoA biosynthetic gene cluster and application in the production of doramectin [J].
Cropp, TA ;
Wilson, DJ ;
Reynolds, KA .
NATURE BIOTECHNOLOGY, 2000, 18 (09) :980-983
[7]   Mechanistic analysis of a type II polyketide synthase.: Role of conserved residues in the β-ketoacyl synthase-chain length factor heterodimer [J].
Dreier, J ;
Khosla, C .
BIOCHEMISTRY, 2000, 39 (08) :2088-2095
[8]   NOVEL AVERMECTINS PRODUCED BY MUTATIONAL BIOSYNTHESIS [J].
DUTTON, CJ ;
GIBSON, SP ;
GOUDIE, AC ;
HOLDOM, KS ;
PACEY, MS ;
RUDDOCK, JC ;
BULOCK, JD ;
RICHARDS, MK .
JOURNAL OF ANTIBIOTICS, 1991, 44 (03) :357-365
[9]   RELAXED SPECIFICITY OF THE OXYTETRACYCLINE POLYKETIDE SYNTHASE FOR AN ACETATE PRIMER IN THE ABSENCE OF A MALONAMYL PRIMER [J].
FU, H ;
EBERTKHOSLA, S ;
HOPWOOD, DA ;
KHOSLA, C .
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, 1994, 116 (14) :6443-6444
[10]   Genetic contributions to understanding polyketide synthases [J].
Hopwood, DA .
CHEMICAL REVIEWS, 1997, 97 (07) :2465-2497