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

该项目旨在开发一种新的生物催化剂系统，用于将有机酸还原为醇，以增加木质纤维素转化为生物燃料或其他有用的基本化学单体时可获得产品的数量。混合有机酸是化学或生物木质素降解最初步骤后的主要产物，但在进一步的过程中表现出有限的可用性，甚至往往对这些过程具有抑制作用。因此，能够将这些酸还原为醇的生物技术转化系统可能会增加可再生资源中更有用产品的产量。我们将采用由非特异性含钨醛氧化还原酶（AOR）和各种醇脱氢酶（ADH）组成的合成生化途径，它应该能够将脂肪酸和芳香酸的复杂混合物转化为相应的醇。AOR是唯一已知的能够将非活性酸还原为相应醛的酶系统，然后通过各种adh的混合物进一步还原为醇，覆盖广泛的底物光谱。我们将为此目的设计一个全细胞系统，该系统由一个细菌宿主组成，该细菌宿主能够在一个广泛的宿主表达质粒上合成含有AOR和几种ADHs基因的适当W-酶（例如Thauera或Aromatoleum物种），以确保在诱导标准条件下，这些酶在预生长细胞中具有高活性。这些细胞将以静止细胞悬浮液的形式用于酸还原，如果需要，也可以修改为渗透细胞或细胞提取系统。为了考虑酸还原所需的氧化还原当量，我们将采用不同的可能的电子给体系统，人工还原剂，如tiiii -柠檬酸盐或二硫代盐，以及天然电子给体，如H2、甲酸盐或谷氨酸盐。能够氧化后一种化合物并为酸还原提供电子的酶已经在预期的宿主细菌中被检测到，并将进一步表征以确保它们参与计划的过程。最后，将通过基于高效液相色谱的分析测试预期的过程，并从仅还原一种确定的有机酸发展到转化木质素水解物中存在的复杂的酸混合物。还将向俄罗斯小组提供相应的制剂，以开发和优化基于膜分离的方案，用于提取和浓缩所生产的醇。

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.