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The excretion of the aromatic amino acid L: -tyrosine was achieved by manipulating three gene targets in the wild-type Escherichia coli K12: The feedback-inhibition-resistant (fbr) derivatives of aroG and tyrA were expressed on a low-copy-number vector, and the TyrR-mediated regulation of the aromatic amino acid biosynthesis was eliminated by deleting the tyrR gene. The generation of this L: -tyrosine producer, strain T1, was based only on the deregulation of the aromatic amino acid biosynthesis pathway, but no structural genes in the genome were affected. A second tyrosine over-producing strain, E. coli T2, was generated considering the possible limitation of precursor substrates. To enhance the availability of the two precursor substrates phosphoenolpyruvate and erythrose-4-phosphate, the ppsA and the tktA genes were over-expressed in the strain T1 background, increasing L: -tyrosine production by 80% in 50-ml batch cultures. Fed-batch fermentations revealed that L: -tyrosine production was tightly correlated with cell growth, exhibiting the maximum productivity at the end of the exponential growth phase. The final L: -tyrosine concentrations were 3.8 g/l for E. coli T1 and 9.7 g/l for E. coli T2 with a yield of L: -tyrosine per glucose of 0.037 g/g (T1) and 0.102 g/g (T2), respectively.

The development of non-petrochemical sources for the plastics industry continues to progress as large multinationals focus on renewable resources to replace fossil carbon. Many bacteria are known to accumulate polyoxoesters as water-insoluble granules in the cytoplasm. The thermoplastic and/or elastomeric behaviour of these biodegradable polymers holds promise for the development of various technological applications. Here, we report the synthesis and characterization of microbial polythioesters (PTEs), a novel class of biopolymers of general technological relevance. Biosynthesis of PTE homopolymers was achieved using a recombinant strain of Escherichia coli that expressed a non-natural pathway consisting of a butyrate kinase, a phosphotransbutyrylase, and a PHA synthase. Different homopolymers were produced, consisting of either 3-mercaptopropionate, 3-mercaptobutyrate, or 3-mercaptovalerate repeating units, if the respective mercaptoalkanoic acids were provided as precursor substrates to the fermentative process. The PTEs contributed up to 30% (w/w) of the cellular dry weight and were identified as hydrophobic inclusions in the cytoplasm. The chemical and stereochemical homogeneity of the purified PTEs were identified by different methods, and the estimated physical properties were compared to the oxypolyester equivalents, revealing low crystalline order and, for the poly(3-mercaptopropionate) improved thermal stability. The ability to produce PTEs through a biosynthetic route opens up new avenues in the field of biomaterials.

This is the first report on the biosynthesis of a hitherto unknown, sulfurcontaining polyester and also the first report on a bacterial polymer containing sulfur in the backbone. The Gram-negative polyhydroxyalkanoate (PHA)-accumulating bacterium Ralstonia eutropha synthesized a copolymer of 3-hydroxybutyrate and 3-mercaptopropionate, poly(3HB-co-3MP), when 3-mercaptopropionic acid or 3,3'-thiodipropionic acid was provided as carbon source in addition to fructose or gluconic acid under nitrogen-limited growth conditions. The peculiarity of this polymer was the occurrence of thioester linkages derived from the thiol groups of 3MP and the carboxyl groups of 3MP or 3HB, respectively, which occurred in addition to the common oxoester bonds of PHAs. Depending on the cultivation conditions and the feeding regime, poly(3HB-co-3MP) contributed up to 19 % of the cellular dry weight, with a molar fraction of 3MP of up to 43 %. The chemical structure of poly(3HB-co-3MP) was confirmed by GC/MS, IR spectroscopy, 1 H-and 13 C-NMR spectroscopy, and elemental sulfur analysis. The identification of this novel biopolymer reveals a new quality regarding the substrate range of PHA synthases and their capability for the synthesis of technically interesting polymers.

In order to get insights into the feedback regulation by tyrosine of the Escherichia coli chorismate mutase/ prephenate dehydrogenase (CM/PDH), which is encoded by the tyrA gene, feedback-inhibition-resistant (fbr) mutants were generated by error-prone PCR. The tyrA fbr mutants were selected by virtue of their resistance toward m-fluoro-D,L-tyrosine, and seven representatives were characterized on the biochemical as well as on the molecular level. The PDH activities of the purified His 6 -tagged TyrA proteins exhibited up to 35% of the enzyme activity of TyrA WT , but tyrosine did not inhibit the mutant PDH activities. On the other hand, CM activities of the TyrA fbr mutants were similar to those of the TyrA WT protein. Analyses of the DNA sequences of the tyrA genes revealed that tyrA fbr contained amino acid substitutions either at Tyr263 or at residues 354 to 357, indicating that these two sites are involved in the feedback inhibition by tyrosine.Chorismate is a central intermediate of the shikimate pathway and branch point for five different metabolic pathways in microorganisms (10). The aromatic amino acids L-phenylalanine and L-tyrosine are formed from chorismate via prephenate, which undergoes either decarboxylation/dehydration or decarboxylation/dehydrogenation, followed by a transamination to form the respective amino acids (Fig. 1).Chorismate mutase/prephenate dehydrogenase (CM/PDH, TyrA) is a bifunctional enzyme occurring as a homodimer with a molecular weight of approximately 78,000 and is involved in tyrosine biosynthesis in Escherichia coli (13). The rearrangement of chorismate to prephenate is catalyzed by the N-terminal CM domain of TyrA, whereas the C-terminal PDH domain catalyzes the oxidative decarboxylation of prephenate to 4-hydroxyphenylpyruvate. Analogously, the PDT domain of the bifunctional CM/PDT (PheA) yields phenylpyruvate, which is subsequently transaminated to phenylalanine.Aromatic amino acid biosynthesis is tightly regulated, in particular, at the chorismate branch point and at the first committed step, i.e., at 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthase, which catalyzes the condensation of phosphoenolpyruvate and erythrose-4-phosphate. The DAHP synthase occurs as three isoforms, each feedback regulated by either phenylalanine, tyrosine, or tryptophan (20). Furthermore, the TyrR protein, binding any one of the three aromatic amino acids, provides transcriptional control of eight different promoters related to aromatic amino acid biosynthesis (21).Due to the industrial importance of L-phenylalanine, for example, as a precursor for the sweetener aspartame (L-aspartyl-L-phenylalanine methyl ester), much more effort has been devoted to the investigation of the phenylalanine biosynthesis pathway than to the tyrosine branch (1). For the biotechnological production of phenylalanine, engineered E. coli strains were employed, exhibiting, among other characteristics, alleviated feedback inhibition by the end product (23, 24). As a consequence, different feedback-inhibition-...

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Various samples of polythioesters with different contents of 3-mercaptopropionic acid (3MP) or 3-mercaptobutyric acid (3MB) as one comonomer and with 3-hydroxybutyric acid (3HB) as the second constituent were produced by cultivating cells of Ralstonia eutropha strain H16 in mineral salts medium containing 3MP or 3MB plus gluconate as carbon sources. Fermentations were done also at the 30-L scale. The various samples were cast as films from chloroform and the following were recorded: melting point, solid-state NMR, X-ray diffraction. The copolyester poly(3HB-co-3MP) displayed mutiple melting peaks corresponding to separate phases rich in 3MP and 3HB. The copolyester poly(3HB-co-3MB) displayed very low crystallinity and melting points higher than that of poly(3HB) when the 3HB content was 40% or less.