While biomass-derived carbohydrates have been predominant substrates for biological production of renewable fuels, chemicals, and materials, organic waste streams are growing in prominence as potential alternative feedstocks to improve the sustainability of manufacturing processes. Catalytic fast pyrolysis (CFP) is a promising approach to generate biofuels from lignocellulosic biomass, but it generates a complex, carbon-rich, and toxic wastewater stream that is challenging to process catalytically but could be biologically upgraded to valuable co-products. In this work, we implemented modular, heterologous catabolic pathways in the Pseudomonas putida KT2440-derived EM42 strain along with the overexpression of native toxicity tolerance machinery to enable utilization of 89% (w/w) of carbon in CFP wastewater. The dmp monooxygenase and meta-cleavage pathway from Pseudomonas putida CF600 were constitutively expressed to enable utilization of phenol, cresols, 2- and 3-ethyl phenol, and methyl catechols, and the native chaperones clpB, groES, and groEL were overexpressed to improve toxicity tolerance to diverse aromatic substrates. Next, heterologous furfural and acetone utilization pathways were incorporated, and a native alcohol dehydrogenase was overexpressed to improve methanol utilization, generating reducing equivalents. All pathways (encoded by genes totaling ~30 kilobases of DNA) were combined into a single strain that can catabolize a mock CFP wastewater stream as a sole carbon source. Further engineering enabled conversion of all aromatic compounds in the mock wastewater stream to (methyl)muconates with a ~90% (mol/mol) yield. Biological upgrading of CFP wastewater as outlined in this work provides a roadmap for future applications in valorizing other heterogeneous waste streams.

虽然生物质衍生的碳水化合物一直是可再生燃料、化学品和材料生物生产的主要底物，但有机废物流正日益成为潜在的替代原料，以提高制造过程的可持续性。催化快速热解 (CFP) 是一种从木质纤维素生物质生产生物燃料的有前景的方法，但它会产生复杂、富含碳和有毒的废水流，对催化处理具有挑战性，但可以通过生物升级为有价值的副产品。在这项工作中，我们在恶臭假单胞菌中实施了模块化的异源分解代谢途径KT2440 衍生的 EM42 菌株以及天然毒性耐受机制的过度表达，使 CFP 废水中碳的利用率达到 89% (w/w)。来自恶臭假单胞菌CF600的dmp单加氧酶和间位裂解途径被组成型表达，以能够利用苯酚、甲酚、2-和 3-乙基苯酚和甲基儿茶酚，以及天然分子伴侣clpB、groES和groEL过表达以提高对多种芳香底物的毒性耐受性。接下来，结合异源糠醛和丙酮利用途径，并过表达天然醇脱氢酶以提高甲醇利用率，产生还原当量。所有途径（由总共约 30 千碱基 DNA 的基因编码）被组合成一个菌株，该菌株可以分解作为唯一碳源的模拟 CFP 废水流。进一步的工程使模拟废水流中的所有芳族化合物能够以~90% (mol/mol) 的产率转化为（甲基）粘康酸盐。本工作中概述的 CFP 废水的生物升级为未来在其他异质废物流中的应用提供了路线图。