Chemo-enzymatic synthesis of catechol and investigation of biosynthetic nitration for synthesis of arylnitro molecules

Sustainable development is a worldwide movement of the chemical industry and requires environmentally benign chemical manufacturing and minimizing the use of nonrenewable resources. Biocatalytic synthesis, in the forms of enzymatic synthesis and microbial synthesis, is attractive for industrial organic synthesis. However, organic synthesis using biocatalysis to meet the requirements of sustainable development encounters two challenges. One is inhibition or even toxicity of molecules involved in the biosynthetic reactions towards enzymes and/or living cells. The other is the limited number of enzymes available for chemical conversion.; As a case study of the challenges presented by the microbial toxicity of many aromatic chemicals, the synthesis of catechol from D-glucose is examined. One route, which led to an increase of catechol productivity, is to reduce the concentration of catechol synthesized in culture medium by resin-based extractive fermentation and hence minimizing the catechol toxicity towards E. coli used as biocatalyst. Other routes include hybrid microbial synthesis and chemical synthesis to completely avoid interfacing catechol and microbes. E. coli strains were constructed to accumulate non-toxic shikimate pathway intermediates, 3-dehydroquinic acid and 3-dehydroshikimic acid, which were chemically converted to catechol by heating in near critical H2O. The less-toxic protocatechuic acid, which was also converted to catechol in near critical H2O, was synthesized either chemically from 3-dehydroquinic acid and 3-dehydroshikimic acid, or microbially from glucose via regular or resin-based extractive fed-batch fermentation. By comparison, catechol was synthesized from glucose with the highest yield 43% (mol/mol) via intermediacy of protocatechuic acid obtained from extractive fermentation. Elucidation of biosynthetic pathways may lead to the discovery of enzymes with new functionalities. Enzymatic routes can then be established for organic synthesis. Arylnitro-containing antibiotics, pyrrolomycin A was isolated and characterized from Actinosporogum vitaminophilus and dioxapyrrolomycin from Streptomycses sp. UpJohn UC11065. Experiments were designed to investigate the biosynthetic pathway of pyrrolomycin A, with emphasis on the mechanism of biosynthetic nitration. One of the hypothesized mechanisms, direct nitration, was found unlikely involved in the biosynthesis of pyrrolomycin A. However, evidence from an indirect method suggested bacterial nitric oxide synthase (NOS) was most likely responsible for biosynthetic nitration in' the synthesis of pyrrolomycin A. Further genetic information is required to full understand bacterial biosynthetic nitration.