Enabling the valorization of guaiacol-based lignin: Integrated chemical and biochemical production of cis,cis-muconic acid using metabolically engineered Amycolatopsis sp ATCC 39116

Metab Eng. 2018 Jan;45:200-210. doi: 10.1016/j.ymben.2017.12.001. Epub 2017 Dec 12.

Abstract

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-1 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.

Keywords: Adipic acid; Amycolatopsis; Aromatics; Blue-white screening; Catechol; Catechol dioxygenase; Guaiacol; Hardwood; Hydrothermal conversion; Lignin; Lignin depolymerization; Muconate cycloisomerase; O-cresol; Phenol; Softwood; Terephthalic acid; gusA.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Actinobacteria* / genetics
  • Actinobacteria* / metabolism
  • Guaiacol / metabolism*
  • Lignin / metabolism*
  • Metabolic Engineering*
  • Sorbic Acid / analogs & derivatives*
  • Sorbic Acid / metabolism

Substances

  • muconic acid
  • Guaiacol
  • Lignin
  • Sorbic Acid