Microbial synthesis of 3,4-dihydroxybutyric acid, 3-hydroxybutyrolactone and other 3-hydroxyalkanoic acids

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Chemical Engineering, February 2014. === Cataloged from PDF version of thesis. === Includes bibliographical references (pages 157-162). === Reducing dependence on petroleum feedstocks motivates engineering novel conversion technolo...

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Main Author: Dhamankar, Himanshu Hemant
Other Authors: Kristala L. Jones Prather.
Format: Others
Language:English
Published: Massachusetts Institute of Technology 2014
Subjects:
Online Access:http://hdl.handle.net/1721.1/87535
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spelling ndltd-MIT-oai-dspace.mit.edu-1721.1-875352019-05-02T16:30:05Z Microbial synthesis of 3,4-dihydroxybutyric acid, 3-hydroxybutyrolactone and other 3-hydroxyalkanoic acids Dhamankar, Himanshu Hemant Kristala L. Jones Prather. Massachusetts Institute of Technology. Department of Chemical Engineering. Massachusetts Institute of Technology. Department of Chemical Engineering. Chemical Engineering. Thesis: Ph. D., Massachusetts Institute of Technology, Department of Chemical Engineering, February 2014. Cataloged from PDF version of thesis. Includes bibliographical references (pages 157-162). Reducing dependence on petroleum feedstocks motivates engineering novel conversion technologies to convert biomass as a renewable resource into target value-added products. In this thesis we developed a new pathway for the microbial synthesis of 3-hydroxyalkanoic acids as biomass derived value-added products. 3-hydroxyalkanoic acids (3HAs) find applications as monomers for biodegradable polymers and chiral pharmaceutical building blocks. One part of this thesis focused on investigating the proposed 3-hydroxyalkanoic acid synthesis platform pathway. The platform employs the reactions of the natural polyhydroxyalkanoate synthesis pathway with new substrates, taking advantage of natural enzyme promiscuity for the stereospecific synthesis of a variety of 3HAs of desired carbon chain length and substituents. Using this platform, we have now demonstrated the synthesis of five novel products: 3,4-dihydroxybutyric acid (3,4-DHBA) and 3-hydroxybutyrolactone (3HBL) as pharmaceutical building blocks and 2,3-dihydroxybutyric acid (2,3- DHBA), 3-hydroxy-4-methylvaleric acid (3H4MV) and 3-hydroxyhexanoic acid (3HH) as monomers for novel polymer applications. The synthesis of 2,3-DHBA in particular led to the identification of a novel activity associated with the thiolase enzyme and highlighted the biosynthetic capability of the platform. The experimental study of different pathway enzyme combinations offered insights into their activities and specificities to guide future enzyme selection. In another part of this thesis, we focused specifically on the hydroxyacid 3,4-DHBA and its lactone 3HBL and their synthesis from glucose as a sole carbon source by integrating the 3HA platform with the endogenous glyoxylate shunt. 3HBL has been identified as a top value-added platform chemical from biomass by the US Department of Energy due to its applications as a chiral synthon for a variety of pharmaceuticals, with an estimated wholesale cost of $450/kg. We were successful in establishing the first biosynthetic pathway for the stereospecific synthesis of 3,4-DHBA and 3HBL from glucose in this thesis, achieving up to 24% of the maximum theoretical yield and titers of the order of 1 g/L at the shake flask scale. Overcoming repression of the glyoxylate shunt and independent control of the glycolate and 3HA pathway enzyme expression using two orthogonal expression systems was critical for product synthesis. Additionally, a study of the 3HBL/DHBA fermentation at the shake flask and bench-top bioreactor scales helped gain an understanding of pH as an important factor affecting the synthesis of these products and informed approaches to improve pathway and process performance. by Himanshu H. Dhamankar. Ph. D. 2014-05-23T19:42:50Z 2014-05-23T19:42:50Z 2013 2014 Thesis http://hdl.handle.net/1721.1/87535 879680357 eng M.I.T. theses are protected by copyright. They may be viewed from this source for any purpose, but reproduction or distribution in any format is prohibited without written permission. See provided URL for inquiries about permission. http://dspace.mit.edu/handle/1721.1/7582 162 pages application/pdf Massachusetts Institute of Technology
collection NDLTD
language English
format Others
sources NDLTD
topic Chemical Engineering.
spellingShingle Chemical Engineering.
Dhamankar, Himanshu Hemant
Microbial synthesis of 3,4-dihydroxybutyric acid, 3-hydroxybutyrolactone and other 3-hydroxyalkanoic acids
description Thesis: Ph. D., Massachusetts Institute of Technology, Department of Chemical Engineering, February 2014. === Cataloged from PDF version of thesis. === Includes bibliographical references (pages 157-162). === Reducing dependence on petroleum feedstocks motivates engineering novel conversion technologies to convert biomass as a renewable resource into target value-added products. In this thesis we developed a new pathway for the microbial synthesis of 3-hydroxyalkanoic acids as biomass derived value-added products. 3-hydroxyalkanoic acids (3HAs) find applications as monomers for biodegradable polymers and chiral pharmaceutical building blocks. One part of this thesis focused on investigating the proposed 3-hydroxyalkanoic acid synthesis platform pathway. The platform employs the reactions of the natural polyhydroxyalkanoate synthesis pathway with new substrates, taking advantage of natural enzyme promiscuity for the stereospecific synthesis of a variety of 3HAs of desired carbon chain length and substituents. Using this platform, we have now demonstrated the synthesis of five novel products: 3,4-dihydroxybutyric acid (3,4-DHBA) and 3-hydroxybutyrolactone (3HBL) as pharmaceutical building blocks and 2,3-dihydroxybutyric acid (2,3- DHBA), 3-hydroxy-4-methylvaleric acid (3H4MV) and 3-hydroxyhexanoic acid (3HH) as monomers for novel polymer applications. The synthesis of 2,3-DHBA in particular led to the identification of a novel activity associated with the thiolase enzyme and highlighted the biosynthetic capability of the platform. The experimental study of different pathway enzyme combinations offered insights into their activities and specificities to guide future enzyme selection. In another part of this thesis, we focused specifically on the hydroxyacid 3,4-DHBA and its lactone 3HBL and their synthesis from glucose as a sole carbon source by integrating the 3HA platform with the endogenous glyoxylate shunt. 3HBL has been identified as a top value-added platform chemical from biomass by the US Department of Energy due to its applications as a chiral synthon for a variety of pharmaceuticals, with an estimated wholesale cost of $450/kg. We were successful in establishing the first biosynthetic pathway for the stereospecific synthesis of 3,4-DHBA and 3HBL from glucose in this thesis, achieving up to 24% of the maximum theoretical yield and titers of the order of 1 g/L at the shake flask scale. Overcoming repression of the glyoxylate shunt and independent control of the glycolate and 3HA pathway enzyme expression using two orthogonal expression systems was critical for product synthesis. Additionally, a study of the 3HBL/DHBA fermentation at the shake flask and bench-top bioreactor scales helped gain an understanding of pH as an important factor affecting the synthesis of these products and informed approaches to improve pathway and process performance. === by Himanshu H. Dhamankar. === Ph. D.
author2 Kristala L. Jones Prather.
author_facet Kristala L. Jones Prather.
Dhamankar, Himanshu Hemant
author Dhamankar, Himanshu Hemant
author_sort Dhamankar, Himanshu Hemant
title Microbial synthesis of 3,4-dihydroxybutyric acid, 3-hydroxybutyrolactone and other 3-hydroxyalkanoic acids
title_short Microbial synthesis of 3,4-dihydroxybutyric acid, 3-hydroxybutyrolactone and other 3-hydroxyalkanoic acids
title_full Microbial synthesis of 3,4-dihydroxybutyric acid, 3-hydroxybutyrolactone and other 3-hydroxyalkanoic acids
title_fullStr Microbial synthesis of 3,4-dihydroxybutyric acid, 3-hydroxybutyrolactone and other 3-hydroxyalkanoic acids
title_full_unstemmed Microbial synthesis of 3,4-dihydroxybutyric acid, 3-hydroxybutyrolactone and other 3-hydroxyalkanoic acids
title_sort microbial synthesis of 3,4-dihydroxybutyric acid, 3-hydroxybutyrolactone and other 3-hydroxyalkanoic acids
publisher Massachusetts Institute of Technology
publishDate 2014
url http://hdl.handle.net/1721.1/87535
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