Muconic acid (MA) is a bio-based platform chemical that can be converted into the commodity petrochemical building blocks adipic acid or terephthalic acid, or used in emerging, performance-advantaged materials. MA is a metabolic intermediate in the β-ketoadipate pathway, and can be produced from carbohydrates or other traditional carbon sources via the shikimate pathway. MA can also be produced from lignin-derived aromatic compounds with high atom efficiency through aromatic-catabolic pathways. Metabolic engineering efforts to date have developed efficient muconic acid-producing strains of the aromatic-catabolic microbe Pseudomonas putida KT2440, but the titers, productivities, and yields from aromatic compounds in most cases remain below the thresholds needed for industrially-relevant bioreactor cultivations. To that end, this work presents further process and host development towards improving MA titers, yields, and productivities, using the hydroxycinnamic acids, p-coumaric acid and ferulic acid, as model aromatic compounds. Coupling strain engineering and bioprocess development enabled the discovery of new bottlenecks in P. putida that hinder MA production from these compounds. A combination of gene overexpression and removal of a global catabolic regulator resulted in high-yielding strains (100% molar yield). Maximum MA titers of 50 g L⁻¹, which is near the lethal toxicity limit in this bacterium, and productivities over 0.5 g L⁻¹ h⁻¹ were achieved in separate process configurations. Additionally, a high-pH feeding strategy, which could potentially reduce the salt load and enable higher titers by decreasing product dilution, was tested with model compounds and lignin-rich streams from corn stover and a complete conversion of the primary monomeric aromatic compounds to MA was demonstrated, obtaining a titer of 4 g L⁻¹. Overall, this study presents a step forward for the production of value-added chemicals from lignin and highlights critical needs for further strain improvement and bioprocess development that can be applied in the biological valorization of lignin.

中文翻译：

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从芳香族化合物和木质素生产粘康酸的生物工艺开发

粘康酸（MA）是一种基于生物的平台化学品，可以转化为商品石油化工构件己二酸或对苯二甲酸，或用于新兴的性能优越的材料中。MA是β-酮己二酸途径中的代谢中间体，可以通过the草酸途径由碳水化合物或其他传统碳源产生。MA也可以由木质素衍生的芳族化合物通过芳族-代谢途径以高原子效率产生。迄今为止，代谢工程方面的努力已开发出有效的粘康酸微生物假单胞菌假单胞菌产酸菌株。KT2440，但是在大多数情况下，芳族化合物的滴度，生产率和产率仍低于与工业相关的生物反应器培养所需的阈值。为此，这项工作使用羟基肉桂酸，对香豆酸和阿魏酸作为模型芳香族化合物，为提高MA滴度，收率和生产率提出了进一步的方法和宿主开发方法。应变工程学和生物工艺的耦合使发现恶臭假单胞菌的新瓶颈成为可能，这些瓶颈阻碍了这些化合物的MA生产。基因过表达和整体分解代谢调节剂的去除相结合，导致产生高产菌株（100％摩尔产率）。最大MA滴度为50 g L ^-1，其接近该细菌的致死毒性极限，并且在单独的工艺配置中实现了超过0.5 g L ^-1 h ^-1的生产率。此外，还使用模型化合物和玉米秸秆中富含木质素的料流以及主要的单体芳族化合物完全转化为MA的高pH进料策略进行了测试，该策略可以潜在地减少盐负荷并通过降低产品稀释度实现更高的滴度。经证实，获得的滴度为4 g L ^-1。总体而言，这项研究为木质素生产增值化学品迈出了一步，并强调了进一步的菌株改良和生物工艺开发的关键需求，这些需求可用于木质素的生物增值。