Fully biological production of adipic acid analogs from branched catechols

Abstract

Microbial production of adipic acid from lignin-derived monomers, such as catechol, is a greener alternative to the petrochemical-based process. Here, we produced adipic acid from catechol using catechol 1,2-dioxygenase (CatA) and a muconic acid reductase (MAR) in Escherichia coli. As the reaction progressed, the pH of the media dropped from 7 to 4-5 and the muconic acid isomerized from the cis,cis (ccMA) to the cis,trans (ctMA) isomer. Feeding experiments suggested that cells preferentially uptook ctMA and that MAR efficiently reduced all muconic isomers to adipic acid. intrigued by the substrate promiscuity of MAR, we probed its utility to produce branched chiral diacids. Using branched catechols likely found in pretreated lignin, we found that while MAR fully reduced 2-methyl-muconic acid to 2-methyl-adipic acid, MAR reduced only one double bond in 3-substituted muconic acids. In the future, MAR's substrate promiscuity could be leveraged to produce chiral-branched adipic acid analogs to generate branched, nylon-like polymers with reduced crystallinity. Abbreviations CatA Catechol-1,2-dioxygenase MAR Muconic acid reductase ccMA cis,cis-Muconic acid ctMA cis,trans-Muconic acid ttMA trans,trans-Muconic acid Adipic acid is used in the production of nylon 6,6, a polyamide present in carpets, textiles and molded plastics. In 2016, the global production of adipic acid was ~ 3.3 million tons per year, with all adipic acid being produced from petroleum 1. This process generated nearly 10% of global nitrogen oxide emissions 2. Renewable production of adipic acid provides a greener alternative, and can be initiated from a variety of feedstocks, including simple sugars and lignin-derived aromatics 1. Independent of the feedstock used, today, the renewable production of adipic acid is a semi-biological process, combining bioproduction of muconic acid followed by its chemical hydrogenation to adipic acid 3,4. For example, Pseudomonas putida has been engineered to convert pretreated lignin to cis,cis-muconic acid (ccMA) at 100% yield from detectable monomers 5. Escherichia coli has a maximum theoretical yield of 83% from glucose through the shikimate pathway 1 , but experimentally a top yield of 22% has been achieved 6. Purification of ccMA from the fermentation broth requires four unit operations to achieve the required purity (99.8%) at 81.4% yield 7. Reduction of ccMA to adipic acid is performed using platinum, rhodium or palladium catalysts, with catalyst cost around $0.30 per kg of adipic acid 7-9 , ~ 19% of the current market value of adipic acid from petroleum 10. Direct production of adipic acid from glucose through a reverse adipate degradation pathway has been achieved, but has a lower maximum theoretical yield (67%) 11. The fully biological production of adipic acid would eliminate (1) the need for a chemical reactor, (2) the catalyst cost, and (3) the cost of purifying muconic acid prior to chemical hydrogenation. Indeed, a recent techno-economic analysis (TEA) accounting for both fixed and variable costs conclud...

Selective Reduction of Carboxylic Acids to Alcohols in the Presence of Alcohols by a Dual Bulky Transition-Metal Complex/Lewis Acid Catalyst

“…Mono- and polycarboxylic acids (mono- and poly-CAs) are ubiquitous in nature and represent a large repository of platform chemical precursors . Several reports have shown that CAs can be derived from biomass-based resources such as lignin and hemicellulose using green and economically feasible methods. , Likewise, many different methods for producing dicarboxylic acids (di-CAs) from biomass have been developed. − Although the produced CAs are valuable by themselves, their corresponding terminal alcohols (ALs) are more interesting as platform chemicals, as they may find useful applications in the production of polymers and as fuel additives to improve the octane number. Moreover, terminal ALs are in general synthetically more useful than the parent CAs.…”

Implementation of Synthetic Pathways to Foster Microbe-Based Production of Non-Naturally Occurring Carboxylic Acids and Derivatives

“…Improvements to this synthetic pathway performed afterwards increased the titers to ~2 mg/L (see details in Supplementary Table S1 and in Table 2). Among the modifications performed in E. coli, the use of an enoate reductase from Clostridium acetobutylicum was the most successful [57]. S1.…”

Engineering the Reductive TCA Pathway to Dynamically Regulate the Biosynthesis of Adipic Acid in Escherichia coli

“…These precursors were then further catalyzed into adipic acid in a hydrogenation process using the precious metal Pt as a catalyst. ,, However, the use of Pt greatly increases production costs. Recently, muconic acid reductase (MAR) has been proven to reduce cis , cis -muconic acid to adipic acid and has been applied for the full biological production of adipic acid. , However, the low titer and high price of cis , cis -muconic acid still hinder the large-scale industrial production of adipic acid. In addition, various biosynthetic routes that are independent of the chemical synthesis processes have been established in recent years, such as the reversal of the β-oxidation pathway , and the ω-oxidation of the fatty acids pathway , and the α-keto acid pathway. , However, these approaches are limited by the low adipic acid titer and the expensive precursors.…”

“…Recently, muconic acid reductase (MAR) has been proven to reduce cis,cis-muconic acid to adipic acid and has been applied for the full biological production of adipic acid. 13,14 However, the low titer and high price of cis,cis-muconic acid still hinder the large-scale industrial production of adipic acid. In addition, various biosynthetic routes that are independent of the chemical synthesis processes have been established in recent years, such as the reversal of the β-oxidation pathway 15,16 and the ω-oxidation of the fatty acids pathway 1,17 and the α-keto acid pathway.…”