TY - JOUR

T1 - From lignin to nylon

T2 - Cascaded chemical and biochemical conversion using metabolically engineered Pseudomonas putida

AU - Kohlstedt, Michael

AU - Starck, Sören

AU - Barton, Nadja

AU - Stotzenberger, Jessica

AU - Selzer, Mirjam

AU - Mehlmann, Kerstin

AU - Schneider, Roland

AU - Pleißner, Daniel

AU - Rinkel, Jan

AU - Dickschat, Jeroen S.

AU - Venus, Joachim

AU - van Duuren, Jozef N. J. H.

AU - Wittmann, Christoph

N1 - Publisher Copyright: © 2018

PY - 2018/5

Y1 - 2018/5

N2 - Cis,cis-muconic acid (MA) is a chemical that is recognized for its industrial value and is synthetically accessible from aromatic compounds. This feature provides the attractive possibility of producing MA from mixtures of aromatics found in depolymerized lignin, the most underutilized lignocellulosic biopolymer. Based on the metabolic pathway, the catechol (1,2-dihydroxybenzene) node is the central element of this type of production process: (i) all upper catabolic pathways of aromatics converge at catechol as the central intermediate, (ii) catechol itself is frequently generated during lignin pre-processing, and (iii) catechol is directly converted to the target product MA by catechol 1,2-dioxygenase. However, catechol is highly toxic, which poses a challenge for the bio-production of MA. In this study, the soil bacterium Pseudomonas putida KT2440 was upgraded to a fully genome-based host for the production of MA from catechol and upstream aromatics. At the core of the cell factories created was a designed synthetic pathway module, comprising both native catechol 1,2-dioxygenases, catA and catA2, under the control of the Pcat promoter. The pathway module increased catechol tolerance, catechol 1,2-dioxygenase levels, and catechol conversion rates. MA, the formed product, acted as an inducer of the module, triggering continuous expression. Cellular energy level and ATP yield were identified as critical parameters during catechol-based production. The engineered MA-6 strain achieved an MA titer of 64.2 g L−1 from catechol in a fed-batch process, which repeatedly regenerated the energy levels via specific feed pauses. The developed process was successfully transferred to the pilot scale to produce kilograms of MA at 97.9% purity. The MA-9 strain, equipped with a phenol hydroxylase, used phenol to produce MA and additionally converted o-cresol, m-cresol, and p-cresol to specific methylated variants of MA. This strain was used to demonstrate the entire value chain. Following hydrothermal depolymerization of softwood lignin to catechol, phenol and cresols, MA-9 accumulated 13 g L−1 MA and small amounts of 3-methyl MA, which were hydrogenated to adipic acid and its methylated derivative to polymerize nylon from lignin for the first time.

AB - Cis,cis-muconic acid (MA) is a chemical that is recognized for its industrial value and is synthetically accessible from aromatic compounds. This feature provides the attractive possibility of producing MA from mixtures of aromatics found in depolymerized lignin, the most underutilized lignocellulosic biopolymer. Based on the metabolic pathway, the catechol (1,2-dihydroxybenzene) node is the central element of this type of production process: (i) all upper catabolic pathways of aromatics converge at catechol as the central intermediate, (ii) catechol itself is frequently generated during lignin pre-processing, and (iii) catechol is directly converted to the target product MA by catechol 1,2-dioxygenase. However, catechol is highly toxic, which poses a challenge for the bio-production of MA. In this study, the soil bacterium Pseudomonas putida KT2440 was upgraded to a fully genome-based host for the production of MA from catechol and upstream aromatics. At the core of the cell factories created was a designed synthetic pathway module, comprising both native catechol 1,2-dioxygenases, catA and catA2, under the control of the Pcat promoter. The pathway module increased catechol tolerance, catechol 1,2-dioxygenase levels, and catechol conversion rates. MA, the formed product, acted as an inducer of the module, triggering continuous expression. Cellular energy level and ATP yield were identified as critical parameters during catechol-based production. The engineered MA-6 strain achieved an MA titer of 64.2 g L−1 from catechol in a fed-batch process, which repeatedly regenerated the energy levels via specific feed pauses. The developed process was successfully transferred to the pilot scale to produce kilograms of MA at 97.9% purity. The MA-9 strain, equipped with a phenol hydroxylase, used phenol to produce MA and additionally converted o-cresol, m-cresol, and p-cresol to specific methylated variants of MA. This strain was used to demonstrate the entire value chain. Following hydrothermal depolymerization of softwood lignin to catechol, phenol and cresols, MA-9 accumulated 13 g L−1 MA and small amounts of 3-methyl MA, which were hydrogenated to adipic acid and its methylated derivative to polymerize nylon from lignin for the first time.

KW - Chemistry

KW - Nylon 6,6

KW - Lignin

KW - Catechol

KW - Phenol

KW - Cresol

KW - Cis,cis-muconic acid

KW - Methyl muconic acid

KW - adipic acid

KW - Methyl adipic acid

KW - Bionylon

KW - Biology

KW - Pseudomonas putida

KW - Funneling

KW - Catechol dioxygenase

KW - Phenol hydroxylase

KW - Synthetic promoter library

KW - Hydrothermal conversion

UR - http://www.scopus.com/inward/record.url?scp=85045437318&partnerID=8YFLogxK

UR - https://www.mendeley.com/catalogue/016f6ca9-6130-33c9-9941-3fb575295cb8/

U2 - 10.1016/j.ymben.2018.03.003

DO - 10.1016/j.ymben.2018.03.003

M3 - Journal articles

C2 - 29548984

VL - 47

SP - 279

EP - 293

JO - Metabolic Engineering

JF - Metabolic Engineering

SN - 1096-7176

IS - May 2018

ER -