Multimodular polyketide synthases (PKSs) are enzymatic assembly lines that synthesize many antibiotics. We seek to understand and engineer such systems, using the 6-deoxyerythronolide B synthase (DEBS) as a prototype. This revised application addresses the reviewers'major concerns by focusing primarily on two aspects of DEBS that are arguably of greatest interest to biosynthetic engineers: programmed unidirectional channeling of intermediates along the assembly line, and extender unit specificity.
Our Specific Aims are:
Aim 1 : Reconstitution and analysis of DEBS: Although DEBS has been studied for two decades, in vitro reconstitution and analysis of the entire system has not been reported. Kinetic analysis of 6-deoxyerythronolide B synthesis will not only lay the quantitative foundation for Aims 2 and 3, but will also answer basic questions of broad relevance to all of nature's PKS assembly lines.
Aim 2 : Understanding and engineering protein-protein interactions in assembly line PKSs: Our studies have suggested that three types of non-covalent protein-protein interactions can collectively explain how PKS assembly lines have been evolutionarily programmed for unidirectional chain growth. We will perform two sets of experiments to test this hypothesis. First, we will construct and analyze mutants in which the directionality of polyketide chain growth is altered. Second, by directed manipulation of protein-protein interactions, we will improve the efficiency of representative chimeric PKSs that show measurable but sub-optimal turnover.
Aim 3 : Understanding and engineering extender unit specificity of assembly line PKSs: Acyl transferase (AT) domains catalyze extender unit incorporation in each module of an assembly line PKS. Although a few cases of engineering AT specificity via homology-based approaches have been reported, the resulting PKSs are kinetically impaired and the underlying causes are unclear. We propose a fundamentally different strategy to engineer AT specificity. First, we will investigate the mechanistic basis for acyl-CoA and ACP specificity via kinetic analysis of three AT domains whose structures we have solved. The resulting methods and insights will enable quantitative interrogation of AT mutants whose design is guided by either structure-based or statistics-based principles. Finally, by re-introducing the most promising AT mutants into the DEBS assembly line, we will evaluate the consequences of AT domain engineering on the turnover rate of the full system.
Aim 4 : Preparation and evaluation of 15-propargyl-erythromycin A analogues: While not directly related to our primary future directions, during the past funding cycle we discovered 15-propargyl erythromycin A, a unique orthogonally functionalized antibiotic with similar activity to erythromycin A. With modest effort, we propose t prepare a few derivatives for ongoing studies on macrolide-ribosome interactions in our collaborator's lab. Our studies will not only open new doors for biosynthetic engineers who seek to manipulate antibiotic structure by genetic engineering, but will also highlight the broader relevance of polyketide biosynthetic research.

Public Health Relevance

Polyketide synthases are enzyme systems that synthesize complex antibiotics in an assembly line manner. By studying and engineering the erythromycin polyketide synthase, we seek to understand the operating principles of nature's assembly lines, and how they can be redesigned to make new antibiotics.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM087934-20
Application #
8719127
Study Section
Special Emphasis Panel (ZRG1-BCMB-K (02))
Program Officer
Gerratana, Barbara
Project Start
1995-07-05
Project End
2017-05-31
Budget Start
2014-06-01
Budget End
2015-05-31
Support Year
20
Fiscal Year
2014
Total Cost
$386,059
Indirect Cost
$138,847
Name
Stanford University
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
009214214
City
Stanford
State
CA
Country
United States
Zip Code
94305
Mathews, Irimpan I; Allison, Kim; Robbins, Thomas et al. (2017) The Conformational Flexibility of the Acyltransferase from the Disorazole Polyketide Synthase Is Revealed by an X-ray Free-Electron Laser Using a Room-Temperature Sample Delivery Method for Serial Crystallography. Biochemistry 56:4751-4756
Xie, Xinqiang; Khosla, Chaitan; Cane, David E (2017) Elucidation of the Stereospecificity of C-Methyltransferases from trans-AT Polyketide Synthases. J Am Chem Soc 139:6102-6105
Xie, Xinqiang; Garg, Ashish; Khosla, Chaitan et al. (2017) Mechanism and Stereochemistry of Polyketide Chain Elongation and Methyl Group Epimerization in Polyether Biosynthesis. J Am Chem Soc 139:3283-3292
Xie, Xinqiang; Garg, Ashish; Khosla, Chaitan et al. (2017) Elucidation of the Cryptic Methyl Group Epimerase Activity of Dehydratase Domains from Modular Polyketide Synthases Using a Tandem Modules Epimerase Assay. J Am Chem Soc 139:9507-9510
Xie, Xinqiang; Garg, Ashish; Keatinge-Clay, Adrian T et al. (2016) Epimerase and Reductase Activities of Polyketide Synthase Ketoreductase Domains Utilize the Same Conserved Tyrosine and Serine Residues. Biochemistry 55:1179-86
Klaus, Maja; Ostrowski, Matthew P; Austerjost, Jonas et al. (2016) Protein-Protein Interactions, Not Substrate Recognition, Dominate the Turnover of Chimeric Assembly Line Polyketide Synthases. J Biol Chem 291:16404-15
Robbins, Thomas; Kapilivsky, Joshuah; Cane, David E et al. (2016) Roles of Conserved Active Site Residues in the Ketosynthase Domain of an Assembly Line Polyketide Synthase. Biochemistry 55:4476-84
Kuo, James; Lynch, Stephen R; Liu, Corey W et al. (2016) Partial In Vitro Reconstitution of an Orphan Polyketide Synthase Associated with Clinical Cases of Nocardiosis. ACS Chem Biol 11:2636-41
Lowry, Brian; Li, Xiuyuan; Robbins, Thomas et al. (2016) A Turnstile Mechanism for the Controlled Growth of Biosynthetic Intermediates on Assembly Line Polyketide Synthases. ACS Cent Sci 2:14-20
Robbins, Thomas; Liu, Yu-Chen; Cane, David E et al. (2016) Structure and mechanism of assembly line polyketide synthases. Curr Opin Struct Biol 41:10-18

Showing the most recent 10 out of 43 publications