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Title: Comprehensive in Vitro Analysis of Acyltransferase Domain Exchanges in Modular Polyketide Synthases and Its Application for Short-Chain Ketone Production

Abstract

Type I modular polyketide synthases (PKSs) are polymerases that utilize acyl-CoAs as substrates. Each polyketide elongation reaction is catalyzed by a set of protein domains called a module. Each module usually contains an acyltransferase (AT) domain, which determines the specific acyl-CoA incorporated into each condensation reaction. Although a successful exchange of individual AT domains can lead to the biosynthesis of a large variety of novel compounds, hybrid PKS modules often show significantly decreased activities. Using monomodular PKSs as models, we have systematically analyzed in this paper the segments of AT domains and associated linkers in AT exchanges in vitro and have identified the boundaries within a module that can be used to exchange AT domains while maintaining protein stability and enzyme activity. Importantly, the optimized domain boundary is highly conserved, which facilitates AT domain replacements in most type I PKS modules. To further demonstrate the utility of the optimized AT domain boundary, we have constructed hybrid PKSs to produce industrially important short-chain ketones. Our in vitro and in vivo analysis demonstrated production of predicted ketones without significant loss of activities of the hybrid enzymes. Finally, these results greatly enhance the mechanistic understanding of PKS modules and prove the benefit ofmore » using engineered PKSs as a synthetic biology tool for chemical production.« less

Authors:
 [1];  [2];  [3];  [3];  [4];  [5];  [6];  [7]
+ Show Author Affiliations
  1. Univ. of California, Berkeley, CA (United States)
  2. Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Sandia National Lab. (SNL-CA), Livermore, CA (United States)
  3. Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States)
  4. Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
  5. Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Univ. of California, Berkeley, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
  6. Univ. of California, Berkeley, CA (United States); Synthetic Biology Research Center, Emeryville, CA (United States)
  7. Univ. of California, Berkeley, CA (United States); Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Synthetic Biology Research Center, Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
Publication Date:
Research Org.:
Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Biological and Environmental Research (BER); USDOD Defense Advanced Research Projects Agency (DARPA) (United States); National Science Foundation (NSF)
OSTI Identifier:
1393117
Grant/Contract Number:  
AC02-05CH11231; HR001148071; MCB-1341894; EEC-0540879
Resource Type:
Accepted Manuscript
Journal Name:
ACS Synthetic Biology
Additional Journal Information:
Journal Volume: 6; Journal Issue: 1; Journal ID: ISSN 2161-5063
Publisher:
American Chemical Society (ACS)
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; acyltransferase domain; protein engineering; substrate specificity; synthetic biology tool; Type I modular polyketide synthase

Citation Formats

Yuzawa, Satoshi, Deng, Kai, Wang, George, Baidoo, Edward E. K., Northen, Trent R., Adams, Paul D., Katz, Leonard, and Keasling, Jay D. Comprehensive in Vitro Analysis of Acyltransferase Domain Exchanges in Modular Polyketide Synthases and Its Application for Short-Chain Ketone Production. United States: N. p., 2016. Web. doi:10.1021/acssynbio.6b00176.
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Yuzawa, Satoshi, Deng, Kai, Wang, George, Baidoo, Edward E. K., Northen, Trent R., Adams, Paul D., Katz, Leonard, & Keasling, Jay D. Comprehensive in Vitro Analysis of Acyltransferase Domain Exchanges in Modular Polyketide Synthases and Its Application for Short-Chain Ketone Production. United States. https://doi.org/10.1021/acssynbio.6b00176
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Yuzawa, Satoshi, Deng, Kai, Wang, George, Baidoo, Edward E. K., Northen, Trent R., Adams, Paul D., Katz, Leonard, and Keasling, Jay D. Mon . "Comprehensive in Vitro Analysis of Acyltransferase Domain Exchanges in Modular Polyketide Synthases and Its Application for Short-Chain Ketone Production". United States. https://doi.org/10.1021/acssynbio.6b00176. https://www.osti.gov/servlets/purl/1393117.
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@article{osti_1393117,
title = {Comprehensive in Vitro Analysis of Acyltransferase Domain Exchanges in Modular Polyketide Synthases and Its Application for Short-Chain Ketone Production},
author = {Yuzawa, Satoshi and Deng, Kai and Wang, George and Baidoo, Edward E. K. and Northen, Trent R. and Adams, Paul D. and Katz, Leonard and Keasling, Jay D.},
abstractNote = {Type I modular polyketide synthases (PKSs) are polymerases that utilize acyl-CoAs as substrates. Each polyketide elongation reaction is catalyzed by a set of protein domains called a module. Each module usually contains an acyltransferase (AT) domain, which determines the specific acyl-CoA incorporated into each condensation reaction. Although a successful exchange of individual AT domains can lead to the biosynthesis of a large variety of novel compounds, hybrid PKS modules often show significantly decreased activities. Using monomodular PKSs as models, we have systematically analyzed in this paper the segments of AT domains and associated linkers in AT exchanges in vitro and have identified the boundaries within a module that can be used to exchange AT domains while maintaining protein stability and enzyme activity. Importantly, the optimized domain boundary is highly conserved, which facilitates AT domain replacements in most type I PKS modules. To further demonstrate the utility of the optimized AT domain boundary, we have constructed hybrid PKSs to produce industrially important short-chain ketones. Our in vitro and in vivo analysis demonstrated production of predicted ketones without significant loss of activities of the hybrid enzymes. Finally, these results greatly enhance the mechanistic understanding of PKS modules and prove the benefit of using engineered PKSs as a synthetic biology tool for chemical production.},
doi = {10.1021/acssynbio.6b00176},
journal = {ACS Synthetic Biology},
number = 1,
volume = 6,
place = {United States},
year = {Mon Aug 22 00:00:00 EDT 2016},
month = {Mon Aug 22 00:00:00 EDT 2016}
}
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https://doi.org/10.1021/acssynbio.6b00176
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