Background: Anthranilate is an aromatic amine used industrially as an intermediate for the synthesis of dyes, perfumes, pharmaceuticals and other classes of products. Chemical synthesis of anthranilate is an unsustainable process since it implies the use of nonrenewable benzene and the generation of toxic by-products. In Escherichia coli anthranilate is synthesized from chorismate by anthranilate synthase (TrpED) and then converted to phosphoribosyl anthranilate by anthranilate phosphoribosyl transferase to continue the tryptophan biosynthetic pathway. With the purpose of generating a microbial strain for anthranilate production from glucose, E. coli W3110 trpD9923, a mutant in the trpD gene that displays low anthranilate producing capacity, was characterized and modified using metabolic engineering strategies.

Aims:  A parametric study was conducted to define optimum conditions to achieve high yields in the conversion of tyrosine to eumelanin (EuMel) using recombinant Escherichia coli. Methods and Results:  Escherichia coli W3110 (pTrcMutmelA) expressing the tyrosinase coding gene from Rhizobium etli and glucose‐mineral media were used to transform tyrosine into EuMel. Batch aerobic fermentor cultures were performed to study the effect of temperature, pH and inducer concentration (isopropyl‐d‐thio‐galactopyranoside) on melanin production. Under optimum conditions, 0·1 mmol l−1 of isopropyl‐d‐thio‐galactopyranoside, temperature of 30°C, and changing pH from 7·0 to 7·5 during the production phase, a 100% conversion of tyrosine into EuMel is obtained. Furthermore, tyrosine feeding allowed us to obtain the highest level (6 g l−1) of EuMel produced by recombinant E. coli reported until now. Conclusions:  The most important factors affecting melanin formation and hence influencing the rate and efficiency in the conversion of tyrosine into EuMel in this system, are the temperature and pH. Significance and Impact of the Study:  Maximum theoretical yield was obtained using a simple culture process and mineral media to convert tyrosine (a medium value compound) into melanin, a high value compound. The process reported here avoids the use of purified tyrosinase, expensive chemical methods or the cumbersome extraction of this polymer from animal or plant tissues.

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Pseudomonas aeruginosa produces the biosurfactants rhamnolipids and 3-(3-hydroxyalkanoyloxy)alkanoic acids (HAAs). In this study, we report the production of one family of rhamnolipids, specifically the monorhamnolipids, and of HAAs in a recombinant Escherichia coli strain expressing P. aeruginosa rhlAB operon. We found that the availability in E. coli of dTDP-L: -rhamnose, a substrate of RhlB, restricts the production of monorhamnolipids in E. coli. We present evidence showing that HAAs and the fatty acid dimer moiety of rhamnolipids are the product of RhlA enzymatic activity. Furthermore, we found that in the recombinant E. coli, these compounds have the same chain length of the fatty acid dimer moiety as those produced by P. aeruginosa. These data suggest that it is RhlAB specificity, and not the hydroxyfatty acid relative abundance in the bacterium, that determines the profile of the fatty acid moiety of rhamnolipids and HAAs. The rhamnolipids level produced in recombinant E. coli expressing rhlAB is lower than the P. aeruginosa level and much higher than those reported by others in E. coli, showing that this metabolic engineering strategy lead to an increased rhamnolipids production in this heterologous host.

The glycolytic intermediate phosphoenolpyruvate (PEP) is a precursor of several cellular components, including various aromatic compounds. Modifications to the PEP node such as PEP:sugar phosphotransferase system (PTS) or pyruvate kinase inactivation have been shown to have a positive effect on aromatics production capacity in Escherichia coli and Bacillus subtilis. In this study, pyruvate kinase and PTS-deficient B. subtilis strains were employed for the construction of derivatives lacking shikimate kinase activity that accumulate two industrially valuable chemicals, the intermediates of the common aromatic pathway, shikimic and dehydroshikimic acids. The pyruvate kinase-deficient strain (CLC6-PYKA) showed the best production parameters under resting-cell conditions. Compared to the PTS-deficient strain, the shikimic and dehydroshikimic acids specific production rates for CLC6-PYKA were 1.8- and 1.7-fold higher, respectively. A batch fermentor culture using complex media supplemented with 83 g/l of glucose was developed with strain CLC6-PYKA, where final titers of 4.67 g/l (shikimic acid) and 6.2 g/l (dehydroshikimic acid) were produced after 42 h.