Enhanced production of (R)-1,2-propanediol by metabolically engineered Escherichia coli

1,2-Propanediol (1,2-PD) is a major commodity chemical currently derived from propylene. Previously, we have demonstrated the production of enantiomerically pure (R)-1,2-propanediol from glucose by an engineered E. coli expressing genes for NADH-linked glycerol dehydrogenase and methylglyoxal synthase. In this work, we investigate three methods to improve 1,2-PD in E. coli. First, we investigated improving the host by eliminating production of a byproduct, lactate. To do this, we constructed strains with mutations in two enzymes involved in lactate production, lactate dehydrogenase and glyoxalase I. (Surprisingly, when mutations were made in its ability to produce lactate, one strain of E. coli [MM294], produced a small amount of 1,2-PD without any added genes.) Second, we constructed a complete pathway to 1,2-PD from the glycolytic intermediate, dihydroxyacetone phosphate. Our previous 1, 2-PD producing strains relied on at least one endogenous E. coli activity and only produced 0.7 g/L of 1,2-PD. The complete pathway involved the coexpression of methylglyoxal synthase (mgs), glycerol dehydrogenase (gldA), and either yeast alcohol dehydrogenase (adhI) or E. coli 1,2-propanediol oxidoreductase (fucO). Third, we investigated bioprocessing improvements by carrying out a fed-batch fermentation with the best engineered strain (expressing mgs, gldA, and fucO). A final titer of 4.5 g/L of (R)-1,2-PD was produced, with a final yield of 0.19 g of 1,2-PD per gram of glucose consumed. This work provides a basis for further strain and process improvement.     

L-1,2-propanediol exits more rapidly than L-lactaldehyde from Escherichia coli.

Catabolism of the six-carbon compound L-fucose results in formation of dihydroxyacetone phosphate (C-1-to-C-3 fragment) and L-lactaldehyde (C-4-to-C-6 fragment) as intermediates. The fate of lactaldehyde depends on the respiratory growth conditions. Aerobically, lactaldehyde is oxidized to L-lactate by an NAD-linked dehydrogenase (ald product). L-Lactate, in turn, is converted to pyruvate, which enters the general metabolic pool. Anaerobically, lactaldehyde is reduced to L-1,2-propanediol by an NADH-linked oxidoreductase (fucO product). L-1,2-Propanediol is excreted as a terminal fermentation product. In a previous study, we showed that retention of the C-4-to-C-6 fragment of fucose depended on the competition for lactaldehyde by aldehyde dehydrogenase and propanediol oxidoreductase (Y. Zhu and E.C.C. Lin, J. Bacteriol. 169:785-789, 1987). In this study, we compared the wild-type strain and isogenic mutant strains defective in ald, fucO, or both for ability to accumulate radioactivity when incubated with fucose labeled at either the C-1 or the C-6 position. The results showed that although blocking the oxidation of lactaldehyde prevented its assimilation, rapid exit of the 3-carbon unit occurred only when the compound was reduced to propanediol. Moreover, growth experiments on fucose indicated that a double ald fucO mutant accumulated inhibiting concentrations of lactaldehyde. The inner cell membrane therefore appears to be much more permeable to the 3-carbon alcohol than to the 3-carbon aldehyde. The almost instantaneous exit of propanediol appears to be a facilitated process.     

Cross-induction of the L-fucose system by L-rhamnose in Escherichia coli.

Dissimilation of L-fucose as a carbon and energy source by Escherichia coli involves a permease, an isomerase, a kinase, and an aldolase encoded by the fuc regulon at minute 60.2. Utilization of L-rhamnose involves a similar set of proteins encoded by the rha operon at minute 87.7. Both pathways lead to the formation of L-lactaldehyde and dihydroxyacetone phosphate. A common NAD-linked oxidoreductase encoded by fucO serves to reduce L-lactaldehyde to L-1,2-propanediol under anaerobic growth conditions, irrespective of whether the aldehyde is derived from fucose or rhamnose. In this study it was shown that anaerobic growth on rhamnose induces expression of not only the fucO gene but also the entire fuc regulon. Rhamnose is unable to induce the fuc genes in mutants defective in rhaA (encoding L-rhamnose isomerase), rhaB (encoding L-rhamnulose kinase), rhaD (encoding L-rhamnulose 1-phosphate aldolase), rhaR (encoding the positive regulator for the rha structural genes), or fucR (encoding the positive for the fuc regulon). Thus, cross-induction of the L-fucose enzymes by rhamnose requires formation of L-lactaldehyde; either the aldehyde itself or the L-fuculose 1-phosphate (known to be an effector) formed from it then interacts with the fucR-encoded protein to induce the fuc regulon.     

Cloning, sequence analysis, and expression in Escherichia coli of the gene encoding monovalent cation-activated levodione reductase from Corynebacterium aquaticum M-13

The gene encoding (6R)-2,2,6-trimethyl-1,4-cyclohexanedione (levodione) reductase was cloned from the genomic DNA of the soil isolate bacterium Corynebacterium aquaticum M-13. The gene contained an open reading frame consisting of 801 nucleotides corresponding to 267 amino acid residues. The deduced amino acid sequence showed approximately 35% identity with other short chain alcohol dehydrogenase/reductase (SDR) superfamily enzymes. The probable NADH-binding site and three catalytic residues (Ser-Tyr-Lys) were conserved. The enzyme was sufficiently produced in recombinant Escherichia coli cells using an expression vector pKK223-3, and purified to homogeneity by two-column chromatography steps. The enzyme purified from E. coli catalyzed stereo- and regio-selective reduction of levodione, and was strongly activated by monovalent cations, such as K+, Na+, and NH4+, as was the case of that from C. aquaticum M-13. To our knowledge, this is the first sequencing report of a monovalent cation-activated SDR enzyme.     

A simple method for the rapid determination of the stereospecificity of NAD-dependent dehydrogenases applied to mammalian IMP dehydrogenase and bacterial NADH peroxidase

The stereospecificity of IMP dehydrogenase (IMP:NAD+ oxidoreductase, EC 1.1.1.205) from two different sources was determined. The enzyme preparations were obtained from murine lymphoblasts and from Escherichia coli. Both enzymes transferred the 2-3H of IMP to the pro-S position of carbon atom C-4 of the nicotinamide ring in NAD. Thus, B-sided stereospecificity is common to the enzyme from two very different species. In addition, the studies described here demonstrate that alcohol dehydrogenase and NADH peroxidase, used as auxiliary enzymes, in combination with a microdistillation procedure, should permit rapid determination of the stereospecificity of any NAD-dependent dehydrogenase for which the appropriate tritiated substrate is available.     

Gene order of the TOL catabolic plasmid upper pathway operon and oxidation of both toluene and benzyl alcohol by the xylA product.

TOL plasmid pWW0 specifies enzymes for the oxidative catabolism of toluene and xylenes. The upper pathway converts the aromatic hydrocarbons to aromatic carboxylic acids via corresponding alcohols and aldehydes and involves three enzymes: xylene oxygenase, benzyl alcohol dehydrogenase, and benzaldehyde dehydrogenase. The synthesis of these enzymes is positively regulated by the product of xylR. Determination of upper pathway enzyme levels in bacteria carrying Tn5 insertion mutant derivatives of plasmid pWW0-161 has shown that the genes for upper pathway enzymes are organized in an operon with the following order: promoter-xylC (benzaldehyde dehydrogenase gene[s])-xylA (xylene oxygenase gene[s])-xylB (benzyl alcohol dehydrogenase gene). Subcloning of the upper pathway genes in a lambda pL promoter-containing vector and analysis of their expression in Escherichia coli K-12 confirmed this order. Two distinct enzymes were found to attack benzyl alcohol, namely, xylene oxygenase and benzyl alcohol dehydrogenase; and their catalytic activities were additive in the conversion of benzyl alcohol to benzaldehyde. The fact that benzyl alcohol is both a product and a substrate of xylene oxygenase indicates that this enzyme has a relaxed substrate specificity.     

Identification of an enzyme system for daidzein-to-equol conversion in Slackia sp. strain NATTS

An Escherichia coli library comprising 8,424 strains incorporating gene fragments of the equol-producing bacterium Slackia sp. strain NATTS was constructed and screened for E. coli strains having daidzein- and dihydrodaidzein (DHD)- metabolizing activity. We obtained 3 clones that functioned to convert daidzein to DHD and 2 clones that converted DHD to equol. We then sequenced the gene fragments inserted into plasmids contained by these 5 clones. All of the gene fragments were contiguous, encoding three open reading frames (ORF-1, -2, and -3). Analysis of E. coli strains containing an expression vector incorporating one of the orf-1, -2, or -3 genes revealed that (i) the protein encoded by orf-1 was involved in the conversion of cis/trans-tetrahydrodaidzein (cis/trans-THD) to equol, (ii) the protein encoded by orf-2 was involved in the conversion of DHD to cis/trans-THD, and (iii) the protein encoded by orf-3 was involved in the conversion of daidzein to DHD. ORF-1 had a primary amino acid structure similar to that of succinate dehydrogenase. ORF-2 was presumed to be an enzyme belonging to the short-chain dehydrogenase/reductase superfamily. ORF-3 was predicted to have 42% identity to the daidzein reductase of Lactococcus strain 20-92 and belonged to the NADH:flavin oxidoreductase family. These findings showed that the daidzein-to-equol conversion reaction in the Slackia sp. NATTS strain proceeds by the action of these three enzymes.     

Identity of Escherichia coli D-1-amino-2-propanol:NAD+ oxidoreductase with E. coli glycerol dehydrogenase but not with Neisseria gonorrhoeae 1,2-propanediol:NAD+ oxidoreductase.

The properties of D-1-amino-2-propanol oxidoreductase from wild-type Escherichia coli have been compared with those of a glycerol dehydrogenase from mutant E. coli 424 and of a 1,2-propanediol oxidoreductase from Neisseria gonorrhoeae. Several independent lines of evidence indicate that the former two enzymes are identical. (i) Both enzymatic activities purified to virtual homogeneity in an identical manner, and the ratio of specific activities (glycerol/aminopropanol) remained constant at all stages. (ii) When electrophoresed, both purified enzymes showed a major as well as a minor band of protein coincident with activity, and these two bands from each enzyme had the same mobility. (iii) The subunit molecular weights and isoelectric points were identical for each enzyme, and (iv) kinetic constants (Km and Vmax values) determined with three different substrates were the same. The somewhat greater stability of the glycerol dehydrogenase to controlled heat denaturation at 74 degrees C was the only difference observed between these two enzymes. In contrast, D-1-amino-2-propanol oxidoreductase was found to be immunochemically and kinetically distinct from the 1,2-propanediol oxidoreductase from N. gonorrhoeae.     

Characterization of the genes of the 2,3-butanediol operons from Klebsiella terrigena and Enterobacter aerogenes.

The genes involved in the 2,3-butanediol pathway coding for alpha-acetolactate decarboxylase, alpha-acetolactate synthase (alpha-ALS), and acetoin (diacetyl) reductase were isolated from Klebsiella terrigena and shown to be located in one operon. This operon was also shown to exist in Enterobacter aerogenes. The budA gene, coding for alpha-acetolactate decarboxylase, gives in both organisms a protein of 259 amino acids. The amino acid similarity between these proteins is 87%. The K. terrigena genes budB and budC, coding for alpha-ALS and acetoin reductase, respectively, were sequenced. The 559-amino-acid-long alpha-ALS enzyme shows similarities to the large subunits of the Escherichia coli anabolic alpha-ALS enzymes encoded by the genes ilvB, ilvG, and ilvI. The K. terrigena alpha-ALS is also shown to complement an anabolic alpha-ALS-deficient E. coli strain for valine synthesis. The 243-amino-acid-long acetoin reductase has the consensus amino acid sequence for the insect-type alcohol dehydrogenase/ribitol dehydrogenase family and has extensive similarities with the N-terminal and internal regions of three known dehydrogenases and one oxidoreductase.     

Cloning, expression, and sequence analysis of the Bacillus methanolicus C1 methanol dehydrogenase gene.

The gene (mdh) coding for methanol dehydrogenase (MDH) of thermotolerant, methylotroph Bacillus methanolicus C1 has been cloned and sequenced. The deduced amino acid sequence of the mdh gene exhibited similarity to those of five other alcohol dehydrogenase (type III) enzymes, which are distinct from the long-chain zinc-containing (type I) or short-chain zinc-lacking (type II) enzymes. Highly efficient expression of the mdh gene in Escherichia coli was probably driven from its own promoter sequence. After purification of MDH from E. coli, the kinetic and biochemical properties of the enzyme were investigated. The physiological effect of MDH synthesis in E. coli and the role of conserved sequence patterns in type III alcohol dehydrogenases have been analyzed and are discussed.     

Propanediol oxidoreductases of Escherichia coli, Klebsiella pneumoniae and Salmonella typhimurium. Aspects of interspecies structural and regulatory differentiation.

The enzyme propanediol oxidoreductase, which converts the lactaldehyde formed in the metabolism of fucose and rhamnose into propane-1,2-diol under anaerobic conditions, was investigated in Escherichia coli, Klebsiella pneumoniae and Salmonella typhimurium. Structural analysis indicated that the enzymes of E. coli and K. pneumoniae have the same Mr and pI, whereas that of Salm. typhimurium also has the same Mr but a slightly different pI. One-dimensional peptide mapping showed identity between the E. coli and K. pneumoniae enzymes when digested with alpha-chymotrypsin, Staphylococcus aureus V8 proteinase or subtilisin. In the case of Salm. typhimurium, this held only for the subtilisin-digested enzymes, indicating that the hydrophobic regions were preserved to a considerable extent. Anaerobically, the three species induced an active propanediol oxidoreductase when grown on fucose or rhamnose. An inactive propanediol oxidoreductase was induced in Salm. typhimurium by either fucose or rhamnose under aerobic conditions, and this was activated once anaerobiosis was established. An inactive propanediol oxidoreductase was also induced in E. coli under aerobic conditions, but only by growth on fucose. The inactive enzyme was not induced by either of the sugars in K. pneumoniae.     

Constitutive activation of the fucAO operon and silencing of the divergently transcribed fucPIK operon by an IS5 element in Escherichia coli mutants selected for growth on L-1,2-propanediol.

L-1,2-Propanediol is an irretrievable end product of L-fucose fermentation by Escherichia coli. Selection for increased aerobic growth rate on propanediol results in the escalation of basal synthesis of the NAD+-linked oxidoreductase encoded by fucO, a member of the fuc regulon for the utilization of L-fucose. In general, when fucO becomes constitutively expressed, two other simultaneous changes occur: the fucA gene encoding fuculose-1-phosphate aldolase becomes constitutively expressed and the fucPIK operon encoding fucose permease, fucose isomerase, and fuculose kinase becomes noninducible. In the present study, we show that fucO and fucA form an operon which is divergently transcribed from the adjacent fucPIK operon. In propanediol-positive and fucose-negative mutants the cis-controlling region shared by the operons fucAO and fucPIK is lengthened by 1.2 kilobases. DNA hybridization identified the insertion element to be IS5. This element, always oriented in the same direction with the left end (the BglII end) proximal to fucA, apparently causes constitutive expression of fucAO and noninducibility of fucPIK. The DNA of the fucAO operon and a part of the adjacent fucP was sequenced.     

Cloning,Sequence Analysis,and Expression in Escherichia coli of the Gene Encoding Monovalent Cation-activated Levodione Reductase from Corynebacterium aquaticum M-13

The gene encoding (6R)-2,2,6-trimethyl-1,4-cyclohexanedione (levodione) reductase was cloned from the genomic DNA of the soil isolate bacterium Corynebacterium aquaticum M-13. The gene contained an open reading frame consisting of 801 nucleotides corresponding to 267 amino acid residues. The deduced amino acid sequence showed approximately 35% identity with other short chain alcohol dehydrogenase/reductase (SDR) superfamily enzymes. The probable NADH-binding site and three catalytic residues (Ser-Tyr-Lys) were conserved. The enzyme was sufficiently produced in recombinant Escherichia coli cells using an expression vector pKK223-3, and purified to homogeneity by two-column chromatography steps. The enzyme purified from E. coli catalyzed stereo- and regio-selective reduction of levodione, and was strongly activated by monovalent cations, such as K⁺, Na⁺, and NH₄ ⁺, as was the case of that from C. aquaticum M-13. To our knowledge, this is the first sequencing report of a monovalent cation-activated SDR enzyme.     

Gel electrophoretic analysis of Zymomonas mobilis glycolytic and fermentative enzymes: identification of alcohol dehydrogenase II as a stress protein.

The 13 major enzymes which compose the glycolytic and fermentative pathways in Zymomonas mobilis are particularly abundant and represent one-half of the soluble protein in exponential-phase cells. One- and two-dimensional polyacrylamide gel electrophoresis maps were developed for 12 of these enzymes. Assignments were made by comigration with purified proteins, comparison with overexpressed genes in recombinant strains, and Western blots (immunoblots). Although most glycolytic enzymes appeared resistant to turnover and accumulated in stationary-phase cells, the protein levels of pyruvate kinase, alcohol dehydrogenase I, and glucokinase declined. Alcohol dehydrogenase II was identified as a major stress protein and was induced both by exposure to ethanol and by elevated temperature (45 degrees C). This enzyme, encoded by the adhB gene, is expressed from tandem promoters which share partial sequence identity with the Escherichia coli consensus sequence for heat shock proteins.