Dehydratase mediated 1-propanol production in metabolically engineered Escherichia coli

Microbial Cell Factories Dehydratase mediated 1-propanol production in metabolically engineered Escherichia coli Rachit Jain 0 Yajun Yan 0 0 Biochemical Engineering Program, Faculty of Engineering, University of Georgia , Athens, GA 30602 USA Background: With 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. Results: We 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. Conclusions: We 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 1propanol 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. - Background The excessive utilization of petroleum plays a major role in the release of the green house gas-carbon dioxide contributing to global warming. Renewable energy sources provide a wide platform of resources to address the problem of increasing energy demand. The manufacture of biofuels such as higher chain alcohols from renewable sources provides an alternative energy source which possesses the advantage of having desirable fuel properties and uncomplicated transportability [1-4]. The synthesis of various higher chain alcohols has been achieved by constructing biosynthetic pathways in E. coli and other microorganisms [1-7]. Here, we describe the design of a new pathway for 1-propanol synthesis and its validation in E. coli. In petrochemical industry, 1-propanol is produced from ethene by a reaction with carbon monoxide and hydrogen to give propionaldehyde, which is then hydrogenated [8]. 1-propanol is also produced as a by-product when potatoes or grains are fermented during the commercial manufacture of ethanol [8,9]. The general use of 1-propanol is in the manufacture of drugs and cosmetics such as lotions, soaps, and nail polishes. It also finds applications in the manufacture of flexographic printing ink and textiles [8,9]. Recently, the use of 1-propanol as a potential fuel substitute to petroleum has promoted the interest in its production via biological approaches. In 2008, Atsumi et al. and Shen et al. reported the production of 1-propanol from glucose by metabolic engineering of E. coli. Their work relied on the keto-acid pathway in E. coli with 2-ketobutyrate as a key intermediate [1,7]. The 2-ketobutyrate was converted to 1-propanol by the action of a keto acid decarboxylase and an alcohol dehydrogenase. Wild type E. coli carrying this pathway was able to produce around 0.15 g/L of 1-propanol. With the elimination of the genes metA, tdh, ilvB, ilvl and adhE encoding the enzymes o-succinyltransferase, threonine dehydrogenase, acetohydroxy acid synthase and alcohol dehydrogenase respectively, the production of 1-propanol achieved was 1 g/L. Atsumi et al. [2] reported higher levels of 1-propanol production in E. coli using cimA encoding a citramalate synthase from Methanoccus jannaschii. They established a direct route for the conversion of pyruvate to 2-ketobutyrate. With the utilization of citramalate pathway and incorporating an evolutionary strategy based on growth they were able to overcome feedback inhibition by isoleucine. Using wild type cimA they achieved 0.3 g/L of 1-propanol production. With the development of cimA variants, the production of 1-propanol was 9 times higher compared to the wild type cimA. We developed a new approach for the biosynthesis of 1propanol by extending the well-known 1,2-propanediol pathway. As the pathway scheme shown in Figure 1, the intermediate of glycolysis dihydroxyacetone phosphate is converted to methylglyoxal by the action of the enzyme methylglyoxal synthase. The methylglyoxal generated is further reduced to either hydroxyacetone or lactaldehyde via two different routes. The formation of hydroxyacetone is catalyzed by the enzyme methylglyoxal reductase which is a primary alcohol dehydrogenase, while a secondary alcohol dehydrogenase such as glycerol dehydrogenase reduces methylglyoxal into lactaldehyde. Both hydroxyacetone and lactaldehyde can be further reduced to 1,2-propanediol by either a secondary alcohol dehydrogenase or a primary alcohol dehydrogenase. The dehydration of 1,2propanediol into 1-propanal can be achieved by a diol dehydratase. The conversion of 1-propanal to 1-propanol is also catalyzed by a primary alcohol dehydrogenase. The pathway that leads to the synthesis of 1,2-propanediol has been introduced into both E. coli and Saccharomyces cerevisiae. By over-expressing the E. coli genes mgsA and gldA and relying on the native expression of other enzymes, Altaras et al. achieved the production of 0.7 g/L of 1,2-propanediol in E. coli [10]. 1.08 g/L 1,2-propanediol production in E. coli was reported by BerriosRivera et al. by utlizing Clostridium acetobutylicum mgsA and E. coli gldA in a strain defecient in lactate production and using an initial glucose concentration of 101.68 mM [11]. Enhanced production of 1,2-propanediol in E. coli was also reported by Altaras et al. [12]. The study involved expression of more complete pathway by addition of fucO gene (1,2-propanediol oxidoreduc (...truncated)