Metabolic engineering of Escherichia coli: construction of an efficient biocatalyst for D-mannitol formation in a whole-cell biotransformation.

A whole-cell biotransformation system for the conversion of d-fructose to d-mannitol was developed in Escherichia coli by constructing a recombinant oxidation/reduction cycle. First, the mdh gene, encoding mannitol dehydrogenase of Leuconostoc pseudomesenteroides ATCC 12291 (MDH), was expressed, effecting strong catalytic activity of an NADH-dependent reduction of D-fructose to D-mannitol in cell extracts of the recombinant E. coli strain. By contrast whole cells of the strain were unable to produce D-mannitol from D-fructose. To provide a source of reduction equivalents needed for d-fructose reduction, the fdh gene from Mycobacterium vaccae N10 (FDH), encoding formate dehydrogenase, was functionally co-expressed. FDH generates the NADH used for d-fructose reduction by dehydrogenation of formate to carbon dioxide. These recombinant E. coli cells were able to form D-mannitol from D-fructose in a low but significant quantity (15 mM). The introduction of a further gene, encoding the glucose facilitator protein of Zymomonas mobilis (GLF), allowed the cells to efficiently take up D-fructose, without simultaneous phosphorylation. Resting cells of this E. coli strain (3 g cell dry weight/l) produced 216 mM D-mannitol in 17 h. Due to equimolar formation of sodium hydroxide during NAD(+)-dependent oxidation of sodium formate to carbon dioxide, the pH value of the buffered biotransformation system increased by one pH unit within 2 h. Biotransformations conducted under pH control by formic-acid addition yielded d-mannitol at a concentration of 362 mM within 8 h. The yield Y(D-mannitol/D-fructose) was 84 mol%. These results show that the recombinant strain of E. coli can be utilized as an efficient biocatalyst for d-mannitol formation.

Biotransformation of glucose to 5-keto-D-gluconic acid by recombinant Gluconobacter oxydans DSM 2343.

For the conversion of glucose to 5-keto-D-gluconate (5-KGA), a precursor of the industrially important L-(+)-tartaric acid, Gluconobacter strains were genetically engineered. In order to increase 5-KGA formation, a plasmid-encoded copy of the gene encoding the gluconate:NADP-5 oxidoreductase (gno) was overexpressed in G. oxydans strain DSM 2434. This enzyme is involved in the nonphosphorylative ketogenic oxidation of glucose and oxidizes gluconate to 5-KGA. As the 5-KGA reductase activity depends on the cofactor NADP+, the sthA gene (encoding Escherichia coli transhydrogenase) was cloned and overexpressed in the GNO-overproducing G. oxydans strain. Growth of the sthA-carrying strains was indistinguishable from the G. oxydans wild-type strain and therefore they were chosen for the coupled overexpression of sthA and gno. G. oxydans strain DSM 2343/pRS201-gno-sthA overproducing both enzymes showed an enhanced accumulation of 5-KGA.

Metabolic engineering of Escherichia coli: construction of an efficient biocatalyst for D-mannitol formation in a whole-cell biotransformation.

A whole-cell biotransformation system for the conversion of D-fructose to D-mannitol was developed in Escherichia coli by construction of a recombinant oxidation/reduction cycle. First, the mdh gene encoding for the mannitol dehydrogenase of Leuconostoc pseudomesenteroides ATCC 12291 (MDH) was expressed, effecting a strong catalytic activity of a NADH-dependent reduction of D-fructose to D-mannitol in cell extracts of the recombinant E. coli strain but not enabling whole cells of the strain to produce D-mannitol from D-fructose. To provide a source for reduction equivalents needed for D-fructose reduction, the fdh gene from Mycobacterium vaccae N10 (FDH) encoding formate dehydrogenase was functionally co-expressed. FDH generates NADH used for D-fructose reduction by dehydrogenation of formate to carbon dioxide. These recombinant E. coli cells were able to form D-mannitol from D-fructose in a low but significant quantity (15 mM). The introduction of a further gene, encoding for the glucose facilitator protein of Zymomonas mobilis (GLF) enabled the cells to efficiently take up D-fructose into the cells, without simultaneous phosphorylation. Resting cells of this E. coli strain (3 g cell dry weight/l) produced 216 mM D-mannitol in 17 hours. Biotransformations conducted under pH-control by formic acid addition yielded D-mannitol at a concentration of 362 mM within 8 hours. The yield Y(D-mannitol D-fructose) was 84 [mol%]. These results show that the recombinant strain of E. coli can be utilized as an efficient biocatalyst for D-mannitol formation.

Isolation of the dxr gene of Zymomonas mobilis and characterization of the 1-deoxy-D-xylulose 5-phosphate reductoisomerase.

The gene encoding the second enzyme of the 2C-methyl-D-erythritol 4-phosphate (MEP) pathway for isopentenyl diphosphate biosynthesis, 1-deoxy-D-xylulose 5-phosphate (DXP) reductoisomerase, was cloned and sequenced from Zymomonas mobilis. The deduced amino acid sequence showed the highest identity (48.2%) to the DXP reductoisomerase of Escherichia coli. Biochemical characterization of the purified DXP reductoisomerase showed a strict dependence of the enzyme on NADPH and divalent cations (Mn(2+), Co(2+) or Mg(2+)). The enzyme is a dimer with a molecular mass of 39 kDa per subunit and has a specific activity of 19.5 U mg protein(-1). Catalysis of the intramolecular rearrangement and reduction of DXP to MEP is competitively inhibited by the antibiotic fosmidomycin with a K(i) of 0.6 microM.

Exceptional characteristics of heterotetrameric (alpha 2 beta 2) E1p of the pyruvate dehydrogenase complex from Zymomonas mobilis: expression from an own promoter and a lipoyl domain in E1 beta.

In the pyruvate dehydrogenase complex (PDHC) of Zymomonas mobilis the beta subunit of the pyruvate dehydrogenase (E1p) as well as the acetyltransferase (E2p) contain an N-terminal lipoyl domain. Both lipoyl domains were acetylated in vitro using 2-14C-pyruvate as a substrate, demonstrating that both lipoyl domains can accept acetyl groups from the E1 component. As previously shown the structural genes (pdhA alpha beta, pdhB, lpd) encoding the pyruvate dehydrogenase complex of Z. mobilis are located in two distinct gene clusters, pdhA alpha beta and pdhB-orf2-lpd (U. Neveling et al. (1998) J. Bacteriol. 180, 1540-1548). Analysis of pdh gene expression using lacZ fusions revealed that the DNA fragments upstream of pdhA alpha, pdhB and lpd each have promoter activities. These pdh promoter activities were 7-30-fold higher in Z. mobilis than in Escherichia coli.

Membrane D-lactate oxidase in Zymomonas mobilis: evidence for a branched respiratory chain.

Respiratory chain composition of the ethanol-producing bacterium Zymomonas mobilis was studied. Its membrane D-lactate oxidase was characterised. With NADH, but not D-lactate as substrate, a cytochrome o-like component was seen in CO difference spectra. Chlorpromazine specifically inhibited reduction of cytochrome d, while myxothiazol eliminated the cytochrome o-like features in CO difference spectra. It is suggested that electrons from NADH are distributed between branches terminated by the cytochrome o-like component, cytochrome a, and cytochrome d. With D-lactate, electrons are transported to cytochrome a, or an unidentified CN(-)-sensitive oxidase, and cytochrome d.

Gene and subunit organization of bacterial pyruvate dehydrogenase complexes.

Pyruvate dehydrogenase complexes of bacterial origin are compared with respect to subunit composition, organization of the corresponding genes, and the number and location of lipoyl domains. Special attention is given to two unusual examples of pyruvate dehydrogenase complexes, formed by Zymomonas mobilis and Thiobacillus ferrooxidans.

Purification of the pyruvate dehydrogenase multienzyme complex of Zymomonas mobilis and identification and sequence analysis of the corresponding genes.

The pyruvate dehydrogenase (PDH) complex of the gram-negative bacterium Zymomonas mobilis was purified to homogeneity. From 250 g of cells, we isolated 1 mg of PDH complex with a specific activity of 12.6 U/mg of protein. Analysis of subunit composition revealed a PDH (E1) consisting of the two subunits E1alpha (38 kDa) and E1beta (56 kDa), a dihydrolipoamide acetyltransferase (E2) of 48 kDa, and a lipoamide dehydrogenase (E3) of 50 kDa. The E2 core of the complex is arranged to form a pentagonal dodecahedron, as shown by electron microscopic images, resembling the quaternary structures of PDH complexes from gram-positive bacteria and eukaryotes. The PDH complex-encoding genes were identified by hybridization experiments and sequence analysis in two separate gene regions in the genome of Z. mobilis. The genes pdhAalpha (1,065 bp) and pdhAbeta (1,389 bp), encoding the E1alpha and E1beta subunits of the E1 component, were located downstream of the gene encoding enolase. The pdhB (1,323 bp) and lpd (1,401 bp) genes, encoding the E2 and E3 components, were identified in an unrelated gene region together with a 450-bp open reading frame (ORF) of unknown function in the order pdhB-ORF2-lpd. Highest similarities of the gene products of the pdhAalpha, pdhAbeta, and pdhB genes were found with the corresponding enzymes of Saccharomyces cerevisiae and other eukaryotes. Like the dihydrolipoamide acetyltransferases of S. cerevisiae and numerous other organisms, the product of the pdhB gene contains a single lipoyl domain. The E1beta subunit PDH was found to contain an amino-terminal lipoyl domain, a property which is unique among PDHs.

Identification of a thiamin-dependent synthase in Escherichia coli required for the formation of the 1-deoxy-D-xylulose 5-phosphate precursor to isoprenoids, thiamin, and pyridoxol.

In Escherichia coli, 1-deoxy-D-xylulose (or its 5-phosphate, DXP) is the biosynthetic precursor to isopentenyl diphosphate [Broers, S. T. J. (1994) Dissertation (Eidgenössische Technische Hochschule, Zürich)], thiamin, and pyridoxol [Himmeldirk, K., Kennedy, I. A., Hill, R. E., Sayer, B. G. & Spenser, I. D. (1996) Chem. Commun. 1187-1188]. Here we show that an open reading frame at 9 min on the chromosomal map of E. coli encodes an enzyme (deoxyxylulose-5-phosphate synthase, DXP synthase) that catalyzes a thiamin diphosphate-dependent acyloin condensation reaction between C atoms 2 and 3 of pyruvate and glyceraldehyde 3-phosphate to yield DXP. We have cloned and overexpressed the gene (dxs), and the enzyme was purified 17-fold to a specific activity of 0.85 unit/mg of protein. The reaction catalyzed by DXP synthase yielded exclusively DXP, which was characterized by 1H and 31P NMR spectroscopy. Although DXP synthase of E. coli shows sequence similarity to both transketolases and the E1 subunit of pyruvate dehydrogenase, it is a member of a distinct protein family, and putative DXP synthase sequences appear to be widespread in bacteria and plant chloroplasts.

Biochemical characterization and sequence analysis of the gluconate:NADP 5-oxidoreductase gene from Gluconobacter oxydans.

Gluconate:NADP 5-oxidoreductase (GNO) from the acetic acid bacterium Gluconobacter oxydans subsp. oxydans DSM3503 was purified to homogeneity. This enzyme is involved in the nonphosphorylative, ketogenic oxidation of glucose and oxidizes gluconate to 5-ketogluconate. GNO was localized in the cytoplasm, had an isoelectric point of 4.3, and showed an apparent molecular weight of 75,000. In sodium dodecyl sulfate gel electrophoresis, a single band appeared corresponding to a molecular weight of 33,000, which indicated that the enzyme was composed of two identical subunits. The pH optimum of gluconate oxidation was pH 10, and apparent Km values were 20.6 mM for the substrate gluconate and 73 microM for the cosubstrate NADP. The enzyme was almost inactive with NAD as a cofactor and was very specific for the substrates gluconate and 5-ketogluconate. D-Glucose, D-sorbitol, and D-mannitol were not oxidized, and 2-ketogluconate and L-sorbose were not reduced. Only D-fructose was accepted, with a rate that was 10% of the rate of 5-ketogluconate reduction. The gno gene encoding GNO was identified by hybridization with a gene probe complementary to the DNA sequence encoding the first 20 N-terminal amino acids of the enzyme. The gno gene was cloned on a 3.4-kb DNA fragment and expressed in Escherichia coli. Sequencing of the gene revealed an open reading frame of 771 bp, encoding a protein of 257 amino acids with a predicted relative molecular mass of 27.3 kDa. Plasmid-encoded gno was functionally expressed, with 6.04 U/mg of cell-free protein in E. coli and with 6.80 U/mg of cell-free protein in G. oxydans, which corresponded to 85-fold overexpression of the G. oxydans wild-type GNO activity. Multiple sequence alignments showed that GNO was affiliated with the group II alcohol dehydrogenases, or short-chain dehydrogenases, which display a typical pattern of six strictly conserved amino acid residues.

Incapability of Gluconobacter oxydans to produce tartaric acid.

The dependence of tartaric acid production by Gluconobacter oxydans ssp. oxydans ATCC 19357 and G. oxydans ssp. suboxydans ATCC 621 on vanadate was investigated. It was found with both organisms that tartaric acid could only be produced in a medium containing vanadate (NH(4)VO(3)). A proposed intermediate of the tartaric acid metabolism in G. oxydans, 5-ketogluconic acid, was tested on its reactivity in the presence of the oxidizing catalyst vanadate. It could be shown that 5-ketogluconic acid and the catalyst vanadate, but not the activity of G. oxydans, were responsible for the formation of tartaric acid. G. oxydans was not able to produce tartaric acid by itself. The stereochemical identity of the formed tartaric acid could be identified as the L-(+)-type. Oxalic acid was formed from 5-ketogluconic acid with vanadate in the absence and in the presence of G. oxydans. The ratio of oxalic acid to tartaric acid was 1:1.