Biosynthesis pathway of ADP-L-glycero-beta-D-manno-heptose in Escherichia coli.

The steps involved in the biosynthesis of the ADP-L-glycero-beta-D-manno-heptose (ADP-L-beta-D-heptose) precursor of the inner core lipopolysaccharide (LPS) have not been completely elucidated. In this work, we have purified the enzymes involved in catalyzing the intermediate steps leading to the synthesis of ADP-D-beta-D-heptose and have biochemically characterized the reaction products by high-performance anion-exchange chromatography. We have also constructed a deletion in a novel gene, gmhB (formerly yaeD), which results in the formation of an altered LPS core. This mutation confirms that the GmhB protein is required for the formation of ADP-D-beta-D-heptose. Our results demonstrate that the synthesis of ADP-D-beta-D-heptose in Escherichia coli requires three proteins, GmhA (sedoheptulose 7-phosphate isomerase), HldE (bifunctional D-beta-D-heptose 7-phosphate kinase/D-beta-D-heptose 1-phosphate adenylyltransferase), and GmhB (D,D-heptose 1,7-bisphosphate phosphatase), as well as ATP and the ketose phosphate precursor sedoheptulose 7-phosphate. A previously characterized epimerase, formerly named WaaD (RfaD) and now renamed HldD, completes the pathway to form the ADP-L-beta-D-heptose precursor utilized in the assembly of inner core LPS.     

Electrostatic interactions during acidic phospholipid reactivation of DnaA protein, the Escherichia coli initiator of chromosomal replication.

The initiation of Escherichia coli chromosomal replication by DnaA protein is strongly influenced by the tight binding of the nucleotides ATP and ADP. Anionic phospholipids in a fluid bilayer promote the conversion of inactive ADP-DnaA protein to replicatively active ATP-DnaA protein in vitro, and thus likely play a key role in regulating DnaA activity. Previous studies have revealed that, during this reactivation, a specific region of DnaA protein inserts into the hydrophobic portion of the lipid bilayer in an acidic phospholipid-dependent manner. To elucidate the requirement for acidic phospholipids in the reactivation process, the contribution of electrostatic forces in the interaction of DnaA and lipid was examined. DnaA-lipid binding required anionic phospholipids, and DnaA-lipid binding as well as lipid-mediated release of DnaA-bound nucleotide were inhibited by increased ionic strength, suggesting the involvement of electrostatic interactions in these processes. As the vesicular content of acidic phospholipids was increased, both nucleotide release and DnaA-lipid binding increased in a linear, parallel manner. Given that DnaA-membrane binding, the insertion of DnaA into the membrane, and the consequent nucleotide release all require anionic phospholipids, the acidic headgroup may be necessary to recruit DnaA protein to the membrane for insertion and subsequent reactivation for replication.     

Biosynthesis of ethylene glycol in Escherichia coli

Ethylene glycol (EG) is an important platform chemical with steadily expanding global demand. Its commercial production is currently limited to fossil resources; no biosynthesis route has been delineated. Herein, a biosynthesis route for EG production from d-xylose is reported. This route consists of four steps: d-xylose?→?d-xylonate?→?2-dehydro-3-deoxy-d-pentonate?→?glycoaldehyde?→?EG. Respective enzymes, d-xylose dehydrogenase, d-xylonate dehydratase, 2-dehydro-3-deoxy-d-pentonate aldolase, and glycoaldehyde reductase, were assembled. The route was implemented in a metabolically engineered Escherichia coli, in which the d-xylose?→?d-xylulose reaction was prevented by disrupting the d-xylose isomerase gene. The most efficient construct produced 11.7 g?L^?1 of EG from 40.0 g?L^?1 of d-xylose. Glycolate is a carbon-competing by-product during EG production in E. coli; blockage of glycoaldehyde?→?glycolate reaction was also performed by disrupting the gene encoding aldehyde dehydrogenase, but from this approach, EG productivity was not improved but rather led to d-xylonate accumulation. To channel more carbon flux towards EG than the glycolate pathway, further systematic metabolic engineering and fermentation optimization studies are still required to improve EG productivity.     

Biosynthesis of Thiamine Pyrophosphate in Escherichia coli

In Escherichia coli, thiamine pyrophosphate is synthesized from thiamine monophosphate. Free thiamine is not involved as an intermediate in de novo synthesis of thiamine pyrophosphate.     

Biosynthesis of 4-Aminobenzoate in Escherichia coli

Two different mutations (pabA and pabB) affecting 4-aminobenzoate biosynthesis were obtained in strains of Escherichia coli lacking chorismate mutase and anthranilate synthetase activity, thus allowing study of the pathway of biosynthesis of 4-aminobenzoate by use of cell extracts of strains carrying the pab mutations. Two components with approximate molecular weights of 9,000 (component A) and 48,000 (component B) are concerned in the biosynthesis of 4-aminobenzoate from chorismate by E. coli. No diffusible intermediate compound could be detected.     

Biosynthesis of flavone C-glucosides in engineered Escherichia coli

Applied Microbiology and Biotechnology - Two plant-originated C-glucosyltransferases (CGTs) UGT708D1 from Glycine max and GtUF6CGT1 from Gentiana triflora were accessed for glucosylation of...     

Metabolic Engineering of Anthocyanin Biosynthesis in Escherichia coli

Anthocyanins are red, purple, or blue plant pigments that belong to the family of polyphenolic compounds collectively called flavonoids. Their demonstrated antioxidant properties and economic importance to the dye, fruit, and cut-flower industries have driven intensive research into their metabolic biosynthetic pathways. In order to produce stable, glycosylated anthocyanins from colorless flavanones such as naringenin and eriodictyol, a four-step metabolic pathway was constructed that contained plant genes from heterologous origins: flavanone 3β-hydroxylase from Malus domestica, dihydroflavonol 4-reductase from Anthurium andraeanum, anthocyanidin synthase (ANS) also from M. domestica, and UDP-glucose:flavonoid 3-O-glucosyltransferase from Petunia hybrida. Using two rounds of PCR, each one of the four genes was first placed under the control of the trc promoter and its own bacterial ribosome-binding site and then cloned sequentially into vector pK184. Escherichia coli cells containing the recombinant plant pathway were able to take up either naringenin or eriodictyol and convert it to the corresponding glycosylated anthocyanin, pelargonidin 3-O-glucoside or cyanidin 3-O-glucoside. The produced anthocyanins were present at low concentrations, while most of the metabolites detected corresponded to their dihydroflavonol precursors, as well as the corresponding flavonols. The presence of side product flavonols is at least partly due to an alternate reaction catalyzed by ANS. This is the first time plant-specific anthocyanins have been produced from a microorganism and opens up the possibility of further production improvement by protein and pathway engineering.     

Biosynthesis of bioactive O-methylated flavonoids in Escherichia coli

Two bioactive O-methylflavonoids, sakuranetin (7-O-methylnaringenin) and ponciretin (7-O-methylnaringenin), were synthesized in Escherichia coli. Sakuranetin inhibits germination of Magnaporthe grisea, and ponciretin is a potential inhibitor of Helicobacter pylori. To achieve this, we reconstructed the naringenin biosynthesis pathway in E. coli. First, the shikimic acid pathway, which leads to the biosynthesis of tyrosine, was engineered in E. coli to increase the amount of available tyrosine. Second, several genes for the biosynthesis of ponciretin and sakuranetin such as tyrosine ammonia lyase (TAL), 4-coumaroyl CoA ligase (4CL), chalcone synthase (CHS), and O-methyltransferase (OMT) were overexpressed. In order to increase the supply the Coenzyme A (CoA), one gene (icdA, isocitrate dehydrogenase) was deleted. Using these strategies, we synthesized ponciretin and sakuranetin from glucose in E. coli at the concentration of 42.5 mg/L and 40.1 mg/L, respectively.     

SecG function and phospholipid metabolism in Escherichia coli

SecG is an auxiliary protein in the Sec-dependent protein export pathway of Escherichia coli. Although the precise function of SecG is unknown, it stimulates translocation activity and has been postulated to enhance the membrane insertion-deinsertion cycle of SecA. Deletion of secG was initially reported to result in a severe export defect and cold sensitivity. Later results demonstrated that both of these phenotypes were strain dependent, and it was proposed that an additional mutation was required for manifestation of the cold-sensitive phenotype. The results presented here demonstrate that the cold-sensitive secG deletion strain also contains a mutation in glpR that causes constitutive expression of the glp regulon. Introduction of both the glpR mutation and the secG deletion into a wild-type strain background produced a cold-sensitive phenotype, confirming the hypothesis that a second mutation (glpR) contributes to the cold-sensitive phenotype of secG deletion strains. It was speculated that the glpR mutation causes an intracellular depletion of glycerol-3-phosphate due to constitutive synthesis of GlpD and subsequent channeling of glycerol-3-phosphate into metabolic pathways. In support of this hypothesis, it was demonstrated that addition of glycerol-3-phosphate to the growth medium ameliorated the cold sensitivity, as did introduction of a glpD mutation. This depletion of glycerol-3-phosphate is predicted to limit phospholipid biosynthesis, causing an imbalance in the levels of membrane phospholipids. It is hypothesized that this state of phospholipid imbalance imparts a dependence on SecG for proper function or stabilization of the translocation apparatus.     

ATP requirement for acidic resistance in Escherichia coli

ATP participates in many cellular metabolic processes as a major substrate to supply energy. Many systems for acidic resistance (AR) under extremely acidic conditions have been reported, but the role of ATP has not been examined. To clarify whether or not ATP is necessary for the AR in Escherichia coli, the AR of mutants deficient in genes for ATP biosynthesis was investigated in this study. The deletion of purA or purB, each of which encodes enzymes to produce AMP from inosinate (IMP), markedly decreased the AR. The content of ATP in these mutants decreased rapidly at pH 2.5 compared to that of the wild type. The AR was again decreased significantly by the mutation of adk, which encoded an enzyme to produce ADP from AMP. The DNA damage in the purA and purB mutants was higher than that in the wild type. These results demonstrated that metabolic processes that require ATP participate in survival under extremely acidic conditions, and that one such system is the ATP-dependent DNA repair system.     

Specific inhibition of phospholipid synthesis in plsA mutants of Escherichia coli.

plsA mutants of Escherichia coli are temperature-sensitive strains which possess two enzymes of abnormal thermolability, sn-glycerol 3-phosphate acyltransferase and adenylate kinase. Phospholipid synthesis is inhibited after shift of plsA mutants to temperatures at the lower end of the nonpermissive temperature range. This inhibition is not due to inactivation of the adenylate kinase activity since nucleic acid (and hence adenosine 5'-triphosphate) synthesis is inhibited only slightly. These results show that in vivo inactivation of the sn-glycerol 3-phosphate acyltransferase can be observed under conditions which allow normal adenylate kinase function.