Development of metabolic pathways for the biosynthesis of hydroxyacids and lactones

In this thesis, metabolic routes were developed for the production of hydroxyacids and their lactones in multiple microbial systems. These compounds see widespread use in the production of pharmaceuticals, polymers, and fine chiral intermediates. First in this thesis, strategies and tools for metabolic pathway design are discussed. This is followed by the descriptions of and data for each microbial production system. The compounds produced in this thesis and their highest titers obtained are shown below: 3-Hydroxybutyrate (3HB) 2.9 g/L 3-Hydroxyvalerate (3HV) 5.3 g/L 4-Hydroxyvalerate (4HV) 27.1 g/L 4-Valerolactone (4VL) 8.2 g/L 3,4-Dihydroxybutyrate (DHBA) 3.2 g/L 3-Hydroxybutyrolactone (3-HBL) 2.2 g/L The production of the two hydroxyvalerates was accomplished through the reduction of the renewable substrate levulinate in Pseudomonas putida by endogenous host enzymes followed by the liberation of the hydroxyvalerate product through the recombinant expression of thioesterase B (tesB). The production of 4VL was accomplished from levulinate by adding the lactonase paraoxonase I (PON1) to the P. putida hydroxyvalerate production system. Because 4VL was found to exist in a pH-dependent equilibrium with 4HV, the lactonase was expressed extracytosolically in acidic media to achieve significant titers of 4VL. The addition of a second resin phase to 4VL-producing cultures with a high affinity for 4VL substantially enhanced lactone production. 3HB, 3HV, DHBA, and 3-HBL were all produced in Escherichia coli through the expression of an acetoacetyl-CoA thiolase (thil, bktB, or phaA), a 3-hydroxybutyryl-CoA reductase (phaB or hbd), and tesB.

Department dc:contributor.department

Massachusetts Institute of Technology. Dept. of Chemical Engineering.

Grantor dc:publisher

Massachusetts Institute of Technology

Year dc:date.issued

2010

Author dc:creator

• Martin, Collin H. (Collin Hunter)

Advisor dc:contributor.advisor

• Kristala Jones Prather.

• Chemical Engineering.

Statement dc:rights

• 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.

Licence dc:rights.uri

• http://dspace.mit.edu/handle/1721.1/7582

Language dc:language.iso

eng

Handle dc:identifier.uri

http://hdl.handle.net/1721.1/57867

OAI identifier oai:identifier

oai:dspace.mit.edu:1721.1/57867