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Lignin is nature's second most abundant polymer and displays a largely unexploited renewable resource for value-added bio-production. None of the lignin-based fermentation processes so far managed to use guaiacol (2-methoxy phenol), the predominant aromatic monomer in depolymerized lignin. In this work, we describe metabolic engineering of Amycolatopsis sp. ATCC 39116 to produce cis,cis-muconic acid (MA), a precursor of recognized industrial value for commercial plastics, from guaiacol. The microbe utilized a very broad spectrum of lignin-based aromatics, such as catechol, guaiacol, phenol, toluene, p-coumarate, and benzoate, tolerated them in elevated amounts and even preferred them over sugars. As a next step, we developed a novel approach for genomic engineering of this challenging, GC-rich actinomycete. The successful introduction of conjugation and blue-white screening, using β-glucuronidase, enabled tailored genomic modifications within ten days. Successive deletion of two putative muconate cycloisomerases from the genome provided the mutant Amycolatopsis sp. ATCC 39116 MA-2, which accumulated 3.1gL MA from guaiacol within 24h, achieving a yield of 96%. The mutant was found also capable to produce MA from a guaiacol-rich true lignin hydrolysate, obtained from pine through hydrothermal conversion. This provides an important proof-of-concept to successfully coupling chemical and biochemical process steps into a value chain from the lignin polymer to an industrial chemical. In addition, Amycolatopsis sp. ATCC 39116 MA-2 was able to produce 2-methyl MA from o-cresol (2-methyl phenol), which opens possibilities towards polymers with novel architecture and properties.

Lignin is an abundant and heterogeneous waste byproduct of the cellulosic industry, which has the potential of being transformed into valuable biochemicals via microbial fermentation. In this study, we applied a fast‐pyrolysis process using softwood lignin resulting in a two‐phase bio‐oil containing monomeric and oligomeric aromatics without syringol. We demonstrated that an additional hydrodeoxygenation step within the process leads to an enhanced thermochemical conversion of guaiacol into catechol and phenol. After steam bath distillation, Pseudomonas putida KT2440‐BN6 achieved a percent yield of cis, cis‐muconic acid of up to 95 mol% from catechol derived from the aqueous phase. We next established a downstream process for purifying cis, cis‐muconic acid (39.9 g/L) produced in a 42.5 L fermenter using glucose and benzoate as carbon substrates. On the basis of the obtained values for each unit operation of the empirical processes, we next performed a limited life cycle and cost analysis of an integrated biotechnological and chemical process for producing adipic acid and then compared it with the conventional petrochemical route. The simulated scenarios estimate that by attaining a mixture of catechol, phenol, cresol, and guaiacol (1:0.34:0.18:0, mol ratio), a titer of 62.5 (g/L) cis, cis‐muconic acid in the bioreactor, and a controlled cooling of pyrolysis gases to concentrate monomeric aromatics in the aqueous phase, the bio‐based route results in a reduction of CO2‐eq emission by 58% and energy demand by 23% with a contribution margin for the aqueous phase of up to 88.05 euro/ton. We conclude that the bio‐based production of adipic acid from softwood lignins brings environmental benefits over the petrochemical procedure and is cost‐effective at an industrial scale. Further research is essential to achieve the proposed cis, cis‐muconic acid yield from true lignin‐derived aromatics using whole‐cell biocatalysts.

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The cover image is based on the Original Article Limited life cycle and cost assessment for the bioconversion of lignin‐derived aromatics into adipic acid by Jozef B.J.H. van Duuren, Stephan Scholl, Nadja Barton et al., https://doi.org/10.1002/bit.27299.