Development of scientific basis for processing of lignin conversion products into fuel components and monomers using membrane and catalytic approaches

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This project intends to develop a new biocatalyst system for the reduction of organic acids to alcohols which may be used to increase the amount of accessible product in the conversion of lignocellulose to biofuel or other useful basic chemical monomers. Mixed organic acids are major products after the initial steps of chemical or biological lignin degradation, but exhibit only limited usability for further processes and are often even inhibitory for those. Therefore, a biotechnological conversion system capable of reducing these acids to alcohols may increase yields of more useful products from renewable sources. We will employ a synthetic biochemical pathway consisting of an unspecific tungsten-containing aldehyde oxidoreductase (AOR) and various alcohol dehydrogenases (ADH), which should be able to convert complex mixtures of aliphatic and aromatic acids to the corresponding alcohols. AOR is the only known enzyme system capable of reducing non-activated acids to the respective aldehydes, which will then be further reduced to alcohols by a mixture of various ADHs, covering a broad substrate spectrum. We will devise a whole-cell system for this purpose, which consists of a bacterial host capable of synthesizing the appropriate W- enzymes (e.g. Thauera or Aromatoleum species) containing the genes for AOR and several ADHs on a broad-host-range expression plasmid to ensure the presence of high activities of these enzymes in pre-grown cells under inducing standard conditions. These cells will be used for acid reduction in the form of resting cell suspensions, which can also be modified to permeabilized cells or cell extract systems if necessary. To account for the necessary redox equivalents for acid reduction, we will employ different possible electron donor systems, artificial reductants like TiIII-citrate or dithionite as well as natural electron donors like H2, formate or glutamate. Enzymes capable of oxidizing the latter compounds and supplying electrons for acid reduction have already been detected in the intended host bacteria and will be further characterized to ensure their participation in the planned process. Finally, the intended process will be tested via HPLC-based analysis and developed from reducing only one defined organic acid to converting the complex mixture of acids present in lignin hydrolysates. The respective preparations will also be made available to the Russian team to develop and optimize membrane-separation based protocols for the extraction and concentration of the produced alcohols.