| NSF Org: |
MCB Division of Molecular and Cellular Biosciences |
| Recipient: |
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| Initial Amendment Date: | July 14, 2017 |
| Latest Amendment Date: | July 14, 2017 |
| Award Number: | 1716594 |
| Award Instrument: | Standard Grant |
| Program Manager: |
Anthony Garza
aggarza@nsf.gov (703)292-2489 MCB Division of Molecular and Cellular Biosciences BIO Directorate for Biological Sciences |
| Start Date: | August 1, 2017 |
| End Date: | January 31, 2022 (Estimated) |
| Total Intended Award Amount: | $750,000.00 |
| Total Awarded Amount to Date: | $750,000.00 |
| Funds Obligated to Date: |
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| History of Investigator: |
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| Recipient Sponsored Research Office: |
21 N PARK ST STE 6301 MADISON WI US 53715-1218 (608)262-3822 |
| Sponsor Congressional District: |
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| Primary Place of Performance: |
1550 Linden Drive Madison WI US 53706-1521 |
| Primary Place of
Performance Congressional District: |
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| Unique Entity Identifier (UEI): |
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| Parent UEI: |
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| NSF Program(s): | Systems and Synthetic Biology |
| Primary Program Source: |
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| Program Reference Code(s): |
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| Program Element Code(s): |
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| Award Agency Code: | 4900 |
| Fund Agency Code: | 4900 |
| Assistance Listing Number(s): | 47.074 |
ABSTRACT
Non-ribosomal peptides are naturally produced by many bacteria and have been an excellent source of therapeutics, most notably new antibiotics. The synthesis of many of these peptides involves a class of enzymes called non-ribosomal peptide synthetases. These enzymes function in a manner analogous to an assembly line with enzyme workstations or 'modules' that coordinately build the peptide one amino acid at a time. The goal of this project is to learn the rules for mixing and matching the workstations from different non-ribosomal peptide synthetases. Ultimately, this would allow researchers to construct hybrid enzymes that synthesize novel peptides and then screen the peptides for therapeutic properties. This research will provide cross-disciplinary training due to the collaborative effort between faculty in the biological sciences and engineering sciences. This cross-disciplinary approach will be integrated into a formal undergraduate capstone course, an undergraduate summer research program, and outreach to K-12 students. This approach will also expose the students to basic scientific questions (e.g. substrate recognition) and show the students how this information can be applied to an important goal (e.g. production of designer molecules).
During non-ribosomal peptide assembly, the adenylation (A) domains recognize specific amino acids and tethers them to partner peptidyl carrier protein (PCP) domains. Condensation (C) domains subsequently form amide bonds between two neighboring PCP-tethered amino acids in a directional manner, forming the peptide backbone. Structural and biochemical studies have identified A domain specificity codes that define the amino acid recognized by the domain. While this code has proved enormously useful in predicting the amino acid activated, a number of studies have shown that altering the residues that define this code nearly always fail to switch substrate specificity. Furthermore, complete domain or module swaps generating chimeric non-ribosomal peptide synthetases generally fail to function efficiently, likely due to improper protein-protein interactions or substrate specificity of the associated C domains. This project will investigate the reprogramming of both A and C domains of non-ribosomal peptide synthetases using in vivo directed evolution approaches. Using these approaches, this study aims to define a means for generating functional chimeric A domains, identify new A domain specificity codes for altering amino acid recognition, and understand the residues governing substrate recognition by C domains. Results from these studies will aid our ability to rationally reprogram the biosynthesis of this important class of natural products to generate designer molecules for a variety of applied purposes, while also gaining insights into how nature has accomplished this goal.
PUBLICATIONS PRODUCED AS A RESULT OF THIS RESEARCH
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PROJECT OUTCOMES REPORT
Disclaimer
This Project Outcomes Report for the General Public is displayed verbatim as submitted by the Principal Investigator (PI) for this award. Any opinions, findings, and conclusions or recommendations expressed in this Report are those of the PI and do not necessarily reflect the views of the National Science Foundation; NSF has not approved or endorsed its content.
Project Outcomes Summary for NSF award 1716594
Project Title: An evolutionary approach to enable reprogramming of nonribosomal peptide synthase enzymology
Summary:
We developed genetic selections and metabolic engineering tools and strains for the analysis, evolution, and engineering of nonribosomal peptide synthetase-base natural products. In Escherichia coli, we developed a genetic selection system that enables us to decipher the functionality of the nonribosomal peptide synthetase (NRPS) associated with enterobactin biosynthesis. We exploited this genetic selection to investigate the flexibility of the L-serine specificity code of the EntF adenylation (A) domain. Using site-saturation mutagenesis coupled with our genetic selection, we performed the most comprehensive analysis of an A domain specificity code to date. From this analysis, we determined the code was more expansive than seen in Nature, with 152 new L-serine specificity codes being identified. Analysis of a subset of these A domain variants determined that retained their specificity for L-serine and had a five-fold variation in in vivo function. In vitro biochemical analysis of these variants showed they had changed their affinity for L-serine or changed the reaction rate. Molecular modeling of the active sites suggested interactions between the enzyme and L-serine were reduced in these variants while the interactions between the specificity code residues were increased. This work laid the groundwork for further studies on specificity code flexibility. Further mutagenesis on an additional 60 million EntF variants determined that changes to the specificity code alone are not sufficient for changing substrate specificity of targeted A domains. Since the enterbactin system was limited to L-serine recognition, we used genome mining, metabolomics, and structural analysis to identify a siderophore in Agrobacterium fabrum strain C58 that extends the use of a genetic selection to include additional NRPS domains as well as polyketide synthase (PKS) domains. This work discovered previously unknown enzymology associated with NRPS and PKS enzymology. To extend our approach to additional organisms, we targeted the further development of Pseudomonas putida KT2440 as a microbial chassis to enable the reprogramming of NRPS enzymology. We developed a series of genetic tools for P. putida to capture and modify biosynthetic gene clusters from other organisms. As a proof-of-principle, we used these tools to enable the robust heterologous production of the NRPS-derived prodigiosin from Serratia marcescens and glidobactin A from Schlegelella brevitalea DSM7029 and Photorhabdus luminescens subp. laumondii TT01 in P. putida KT2440. These efforts showed that we could produce 1.1 g/L of prodigiosin and 470 mg/L of glydobactin A in P. putida KT2440. Therefore, we have developed the necessary genetic tools and strains for taking an evolutionary approach to enabling the reprogramming of NRPS enzymology in diverse microorganisms.
These studies resulted in six publications (four research papers and two reviews). Additionally, the Thomas laboratory trained 1.5 Ph.D. students and four BS students (two received undergraduate fellowships, one student was a member of an underrepresented group, and another student was a military veteran). The Pfleger laboratory trained one Ph.D. student, one BS student, and one high school apprentice on these projects.
Last Modified: 04/13/2022
Modified by: Michael G Thomas
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