Development of metabolic pathways for the biosynthesis of hydroxyacids and lactones

Abstract

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.

(cont.) Supplying glucose to E. coli expressing these enzymes resulted in 3HB production, while supplying glucose and propionate results in 3HV production. Supplying glucose and glycolate resulted in DHBA production with some 3-HBL, but only with the help of a fourth gene - propionyl-CoA transferase (pct). Removing the tesB gene from this four-gene system substantially increases 3-HBL titers at the expense of DHBA. This work represents the first successful production of DHBA and 3-HBL in a biological system from carbohydrate-based substrates. In each of these systems, several broadly-applicable tools and strategies were developed to enhance product titer or discover new metabolic activities. In the P. putida system, cytosolic and extracytosolic biocatalysis were combined in a single metabolic pathway to realize lactone production. This catalytic strategy, termed integrated bioprocessing, is applicable to other metabolic pathways whose production suffers due to a suboptimal cytosolic enzyme. Also in the P. putida system, two-phase cultures were used to sequester the lactone product away from the lactonase, helping to drive lactonehydroxyacid equilibrium towards the lactone. This methodology allows one to overcome equilibrium-based limitations of product titer. Finally in the E. coli work, a promiscuous pathway normally used for polyhydroxyalkanoate synthesis was exploited to give a wide range of hydroxyacid products. This substrate promiscuity was critical in achieving the production of new compounds biologically and thus substrate promiscuity was identified as a key component for metabolic pathway design and construction.