초록
<P><B>Abstract</B></P> <P>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 <I>Amycolatopsis</I> sp. ATCC 39116 to produce <I>cis,cis</I>-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, <I>p</I>-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 <I>Amycolatopsis</I> sp. ATCC 39116 MA-2, which accumulated 3.1gL<SUP>-1</SUP> 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, <I>Amycolatopsis</I> sp. ATCC 39116 MA-2 was able to produce 2-methyl MA from <I>o</I>-cresol (2-methyl phenol), which opens possibilities towards polymers with novel architecture and properties.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Conjugation and blue-white screening enable genomic modification of <I>Amycolatopsis</I> sp. ATCC 39116. </LI> <LI> Engineered strains produce <I>cis,cis</I>-muconic acid (MA) from guaiacol and other lignin-aromatics. </LI> <LI> MA production is demonstrated for a guaiacol-rich true lignin hydrolysate. </LI> <LI> The microbe produces 2-methyl MA from <I>o</I>-cresol towards polymers with novel architecture. </LI> </UL> </P>