REGENTS OF THE UNIVERSITY OF COLORADO, THE
New enzymes can evolve by recruitment of enzymes that have a promiscuous ability to catalyze a newly needed reaction. This project addresses the early stages of evolution of a new activity using the promiscuous N-acetyl glutamyl phosphate (NAGP) reductase activity of E. coli glutamyl phosphate (GP) reductase. The experiments will combine in vitro evolution methods for generating and identifying enzymes with enhanced NAGP reductase activities with classical enzymological approaches for characterizing the catalytic abilities of the improved enzymes. The first objective is to examine how the potential for evolution of promiscuous activities depends on both the level of the promiscuous activity and its evolvability - i.e. whether there is an evolutionary trajectory to an improved enzyme that avoids intermediates that decrease the fitness of the organism. The second objective is to explore how a requirement to maintain GP reductase activity constrains the emergence of NAGP reductase activity before gene duplication provides a gene copy encoding an enzyme that can evolve to specialize in the new function. The third objective is to dissect the mechanistic reasons for improvements in NAGP reductase activity in evolved variants using kinetic approaches that allow measurement of rate constants for individual steps in the overall reaction.
Broader Impact. This work will provide new insights into the processes that have governed the evolution of new enzymes in microbes since the origin of life and continue to affect the evolution of new enzymes in microbes exposed to novel environmental stresses such as the presence of toxic pollutants or antibiotics. In addition, work on the evolvability of promiscuous activities in homologous enzymes will be of great interest to protein engineers who aim to evolve new green catalysts for industrial purposes. Other broader impacts of this work include development of a workshop for high school teachers on molecular evolution, development of a lab on microbial evolution for a summer camp for minority middle school and high school girls, and training of undergraduates.
Choose a quarter and click "Go."
| AWARD OVERVIEW |
| Award Number |
0919617 |
Funding Agency |
National Science Foundation |
| Total Award Amount |
$895,017 |
Project Location - City |
Boulder |
| Award Date |
07/20/2009 |
Project Location - State |
CO |
| Project Status |
More than 50% Completed |
Project Location - Zip |
80309-0572
|
| Jobs Reported |
3.00 |
Congressional District |
02 |
| Project Location - Country |
US |
|
|
Recipient Information
(Grants)
| Recipient Information (Grants) |
|
Recipient Name
|
REGENTS OF THE UNIVERSITY OF COLORADO, THE |
| Recipient DUNS Number |
007431505
|
| Recipient Address |
3100 MARINE ST 572 UCB |
| Recipient City |
BOULDER |
| Recipient State |
Colorado |
| Recipient Zip |
80303-1058 |
| Recipient Congressional District |
02 |
| Recipient Country |
USA |
Required to Report Top 5
Highly Compensated Officials |
No |
Projects and Jobs Information
| Projects and Jobs Information |
| Project Title |
Effects of Genetic Background and Gene Sharing on the Evolvability of a Promiscuous Enzyme Activity |
| Project Status |
More than 50% Completed |
| Final Project Report Submitted |
No |
| Project Activities Description |
Research & Public Policy Analysis |
| Quarterly Activities/Project Description |
The goal of this project is to examine the evolvability of the promiscuous N-acetyl glutamyl phosphate (NAGP) reductase activity of glutamyl phosphate (GP) reductase (ProA). We previously found that a mutation that changes Glu383 to Ala results in an inefficient generalist enzyme that can support slow growth of E. coli on M9/glucose. We are exploring the mechanisms by which cells can improve fitness when growth is limited by this inefficient enzyme. Our pilot experiments have shown that strains expressing E383A ProA can improve fitness either by amplifying a large region around proA or via point mutations that increase expression of either proA or the proBA operon. We expect that beneficial mutations may also occur elsewhere in the genome.
We are constructing strains carrying a gene encoding E383A ProA for long-term adaptation experiments. We have introduced a temperature-sensitive allele of recA so that we can prevent changes in copy number (a process that is mediated by RecA) during analytical procedures by raising the temperature. We are also introducing genes encoding YFP or GFP to facilitate tracking of beneficial mutations.
We are using a complementary approach to identify mutations in the coding region of proA that provide increased NAGP reductase activity. We have generated libraries of mutant proA genes using error-prone PCR. This library will be introduced into strains that lack argC. Colonies that carry an allele of proA that has improved NAGP reductase activity will grow fastest. Second-round libraries will be constructed using the plasmids recovered from the fastest growing cells. This experiment will be repeated until we no longer achieve improvements in NAGP reductase activity. This experiment will provide information about the types of mutations that can lead to improved NAGP reductase activity for comparison with the mutations that arise in vivo during long-term adaptation experiment described above.
|
| Jobs Created |
3.00 |
| Description of Jobs Created |
Post Doctorate |
Purchaser Information
(Grants)
| Purchaser Information |
| Contracting Office ID |
Not Reported |
| Contracting Office Name |
Not Available |
| Contracting Office Region |
Not Available |
| TAS Major Program |
49-0101 |
| Award Information |
| Award Date |
07/20/2009 |
| Award Number |
0919617 |
| Order Number |
|
| Award Type |
Grants |
| Funding Agency ID |
49 |
| Funding Agency Name |
National Science Foundation |
| Funding Office Name |
Not Available |
| Awarding Agency ID |
49 |
| Awarding Agency Name |
National Science Foundation |
| Amount of Award |
$895,017 |
| Funds Invoiced/Received |
$838,227 |
| Expenditure Amount |
$840,577 |
| Infrastructure Expenditure Amount |
$0 |
| Infrastructure Purpose and Rationale |
Not Reported |
| Infrastructure Point of Contact Name |
Not Reported |
| Infrastructure Point of Contact Email |
Not Reported |
| Infrastructure Point of Contact Phone |
Not Reported |
| Infrastructure Point of Contact Address |
Not Reported |
| Infrastructure Point of Contact City |
Not Reported |
| Infrastructure Point of Contact State |
Not Reported |
| Infrastructure Point of Contact Zip |
Not Reported |
Product or Service Information
(Grants)
| Product or Service Information |
| Primary Activity Code |
**K |
| Activity Description |
Research & Public Policy Analysis |
| Sub-Awards Information |
| Sub-awards to Organizations |
0 |
| Sub-award Amounts to Organizations |
$0 |
| Sub-Awards to Individuals |
0 |
| Sub-Award Amounts to Individuals |
$0 |
| Number of Sub-awards less than $25,000/award |
0 |
| Amount of Sub-awards less than $25,000/award |
$0 |
| Number of payments to vendors greater than $25,000 |
0 |
| Total Amount of payments to vendors greater than $25,000/award |
$0 |
| Number of payments to vendors less than $25,000/award |
384 |
| Total Amount of payments to vendors less than $25,000/award |
$106,520 |
| Location Information |
| Latitude, Longitude |
40º 0' 22",
-105º 15' 55" |
| Congressional District |
02 |
| Address 1 |
3100 Marine St - Room 479 |
| Address 2 |
572 UCB |
| City |
Boulder |
| County |
Boulder |
| State |
CO |
| Zip |
80309-0572 |
|
 |