Dehydratase mediated 1-propanol production in metabolically engineered Escherichia coli

Abstract

BackgroundWith the increasing consumption of fossil fuels, the question of meeting the global energy demand is of great importance in the near future. As an effective solution, production of higher alcohols from renewable sources by microorganisms has been proposed to address both energy crisis and environmental concerns. Higher alcohols contain more than two carbon atoms and have better physiochemical properties than ethanol as fuel substitutes.ResultsWe designed a novel 1-propanol metabolic pathway by expanding the well-known 1,2-propanediol pathway with two more enzymatic steps catalyzed by a 1,2-propanediol dehydratase and an alcohol dehydrogenase. In order to engineer the pathway into E. coli, we evaluated the activities of eight different methylglyoxal synthases which play crucial roles in shunting carbon flux from glycolysis towards 1-propanol biosynthesis, as well as two secondary alcohol dehydrogenases of different origins that reduce both methylglyoxal and hydroxyacetone. It is evident from our results that the most active enzymes are the methylglyoxal synthase from Bacillus subtilis and the secondary alcohol dehydrogenase from Klebsiella pneumoniae, encoded by mgsA and budC respectively. With the expression of these two genes and the E. coli ydjG encoding methylglyoxal reductase, we achieved the production of 1,2-propanediol at 0.8 g/L in shake flask experiments. We then characterized the catalytic efficiency of three different diol dehydratases on 1,2-propanediol and identified the optimal one as the 1,2-propanediol dehydratase from Klebsiella oxytoca, encoded by the operon ppdABC. Co-expressing this enzyme with the above 1,2-propanediol pathway in wild type E. coli resulted in the production of 1-propanol at a titer of 0.25 g/L.ConclusionsWe have successfully established a new pathway for 1-propanol production by shunting the carbon flux from glycolysis. To our knowledge, it is the first time that this pathway has been utilized to produce 1-propanol in E. coli. The work presented here forms a basis for further improvement in production. We speculate that dragging more carbon flux towards methylglyoxal by manipulating glycolytic pathway and eliminating competing pathways such as lactate generation can further enhance the production of 1-propanol.

Fermentative production of 1-propanol from sugars using wild-type and recombinant Shimwellia blattae

“…Two kinds of engineered biosynthetic pathways of 1-propanol have been reported using E. coli, one employing 2-ketobutyrate and the other employing 1,2-PD as an intermediate Jain and Yan 2011;Kataoka et al 2009;Shen and Liao 2008). In this study, based on the latter pathway, we demonstrated the production of 1-propanol using S. blattae from two kinds of sugars, namely, glucose and L-rhamnose.…”

“…One employs 2-ketobutyrate as an intermediate via the threonine pathway or citramalate pathway Shen and Liao 2008), and the other uses 1,2-propanediol (1,2-PD) as an intermediate (Jain and Yan 2011;Kataoka et al 2009). In both cases, recombinant E. coli strains harboring these artificial pathways were shown to produce 1-propanol.…”

Metabolic engineering of Escherichia coli for the production of 1-propanol

“…To date, 1-propanol has been produced mainly by the strategy of employing ketoacid decarboxylase from Lactococcus lactis, alcohol dehydrogenase 2 from Saccharomyces cerevisiae Shen and Liao, 2008) and evolved citramalate synthase (encoded by cimA). Recently, a new pathway for 1-propanol production by expanding the 1,2-propanediol pathway with combination of 1,2-propanediol dehydratase and alcohol dehydrogenase was also reported (Jain and Yan, 2011). There have so far been fewer reports on the bio-based production of 1-propanol by metabolic engineering.…”

“…Thermobifida fusca, a cellulolytic microorganism, harboring the Clostridium acetobutylicum ATCC 824 alcohol/ aldehyde dehydrogenase also produced 0.48 g L À 1 of 1-propanol from untreated lignocellulosic biomass as a carbon source via 2-ketobutyrate as a metabolic intermediate (Deng and Fong, 2011). Recently, a wild-type E. coli harboring 1,2-propanediol dehydratase from Klebsiella oxytoca was shown to produce 0.25 g L À 1 of 1-propanol following additional engineering of the 1,2-propanediol pathway (Jain and Yan, 2011). Thus, the final 1-propanol titers achieved by employing various engineered microorganism so far are rather low.…”

Glycolysis and Its Metabolic Engineering Applications

“…One pathway was designed by expanding the wellknown 1,2propanediol pathway from methylglyoxal shunt with two more enzy matic steps catalyzed by a 1,2propanediol dehydratase and an alcohol dehydrogenase. 75 The other novel pathway was achieved via extension of threonine pathway from oxaloacetate and decarboxylation of 2ketobutyrate. 76 Synergy of citramalate pathway and threonine pathway achieved 1pro panol production with a titer of 8 g/L after 72 h. 77 A novel 1butanol was also established via a sixstep enzymatic pathway from acetylCoA to butyrylCoA and 1butanol (Figure 1.1).…”