1,3-Propanediol production by Escherichia coli expressing genes from the Klebsiella pneumoniae dha regulon

Abstract

The dha regulon in KkebsieUa pneumoniae enables the organism to grow anaerobically on glycerol and produce 1,3-propanediol (1,3-PD). Escherichia coli, which does not have a dha system, is unable to grow anaerobically on glycerol without an exogenous electron acceptor and does not produce 1,3-PD. A genomic library of K. pneumoniae ATCC 25955 constructed in E. coli AG1 was enriched for the ability to grow anaerobically on glycerol and dihydroxyacetone and was screened for the production of 1,3-PD. The cosmid pTC1 (42.5 kb total with an 18.2-kb major insert) was isolated from a 1,3-PD-producing strain of E. coli and found to possess enzymatic activities associated with four genes of the dha regulon: glycerol dehydratase (dhaB), 1,3-PD oxidoreductase (dhaT), glycerol dehydrogenase (dhaD), and dihydroxyacetone kinase (dhaK). All four activities were inducible by the presence of glycerol. When E. coli AG1/pTC1 was grown on complex medium plus glycerol, the yield of 1,3-PD from glycerol was 0.46 mol/mol. The major fermentation by-products were formate, acetate, and D-lactate. 1,3-PD is an intermediate in organic synthesis and polymer production. The 1,3-PD fermentation provides a useful model system for studying the interaction of a biochemical pathway in a foreign host and for developing strategies for metabolic pathway engineering. Metabolic pathway engineering (MPE, also metabolic engineering), the modification, design, and construction of biochemical pathways, is an emerging discipline of potential importance to the chemical, biochemical, food, and environmental industries. MacQuitty (19) has called MPE the fourth wave of biotechnology following classical fermentation, recombinant DNA technology, and protein engineering. Recent progress in MPE has been reviewed by Bailey (2). We have selected the conversion of glycerol to 1,3propanediol (1,3-PD) as a model system for the study of MPE. Our reasons are as follows. (i) The pathway is relatively simple, consisting of only two enzymes, a dehydratase (glycerol dehydratase [EC 4.2.1.30] or diol dehydratase [EC 4.2.1.28] and 1,3-PD oxidoreductase [EC 1.1.1.202]); (ii) the pathway possesses features of a more complex metabolic network (e.g., the dehydratase is a multicomponent enzyme and requires coenzyme B12, and

Kinetic, dynamic, and pathway studies of glycerol metabolism byKlebsiella pneumoniae in anaerobic continuous culture: III. Enzymes and fluxes of glycerol dissimilation and 1,3-propanediol formation

“…They followed the same oscillation patterns as CO 2 evolution and growth rate (data not shown). A possible reason for the simultaneous oscillations of these three enzymes may be that their genes belong to one and the same regulon, dha (Forage and Lin, 1982;Tong et al, 1991), and thus subject to the same regulations. The magnitudes of the oscillations of these enzymes corresponded to their concentration levels under steady state: GDH displayed a minimum of 2.3 and a maximum of 4.6 U/mg protein; PDOR a minimum of 1.2 and a maximum of 4.3 U/mg protein; and GDHt a minimum of 0.8 and a maximum of 2.0 U/mg protein.…”

“…In this work, we report on experimental results concerning the effects of culture conditions on the in vitro and in vivo activities of glycerol dehydrogenase, glycerol dehydratase, and 1,3-propanediol oxidoreductase under both steadystate and oscillation conditions. The genes for these three enzymes and the enzyme dihydroxyacetone kinase are encoded in one and the same regulon named dha (Forage and Lin, 1982;Tong et al, 1991) and have been cloned and sequenced for K. pneumoniae (Cameron et al, 1998;Tong et al, 1991) and C. freundii (Daniel and Gottschalk, 1992;Daniel et al, 1995). The transient behavior of the culture after step changes of substrate concentration in medium is also studied.…”

Metabolic Engineering of Propanediol Pathways

“…Native E. coli does not ferment glycerol to 1,3-PD. We have shown that E. coli transformed with the K. pneumoniae dhaT and dhaB genes is able to coferment glycerol and glucose (8,12). The glycerol is converted primarily to 1,3-PD, and the glucose is used for growth and regeneration of reducing potential.…”

“…In addition, coenzyme-B 12 (5 mg/L) was added at the beginning of each fermentation in darkness to prevent light inactivation. No 1,3-PD was detected in the fermentation broth after 48 h. To determine if the enzymes were expressed, the fermentation was repeated, but this time without coenzyme-B 12 , which has been reported to contribute to the irreversible inactivation of glycerol dehydratase (18,19). After 36 h, the cells were harvested and crude cell extracts were made by vortexing with glass beads according to the method of Jazwinski (20).…”

“…Control experiments with E. coli containing a plasmid with a mutation in the aldose reductase gene produced none or, at most, a small amount of 1,2-PD (occasionally an HPLC peak that coeluted with 1,2-PD was detected; in all cases, the peak corresponded to a 1,2-PD concentration of less than 0.05 g/L). To determine the stereochemistry of the fermentation 1,2-PD, we purified some of the product by extraction (8) and analyzed it by GC with a chiral capillary column (Chrompack, Inc., Raritan, NJ). The product was approximately 90% (R)-1,2-PD.…”