From Lignin to Nylon: Cascaded Chemical and Biochemical Conversion Using Metabolically Engineered Pseudomonas putida M. Kohlstedt1 (
[email protected]), S. Starck1, N. Barton1, J. Stolzenberger1, M. Selzer1, K. Mehlmann2, R. Schneider2, D. Pleissner2, J. Rinkel3, J.S. Dickschat3, J. Venus2, J.B.J.H. van Duuren1, C. Wittmann1 1Institute
of Systems Biotechnology, Saarland University, Germany, 2Leibniz Institute for Agricultural Engineering and Bioeconomy, Potsdam, Germany,
3Kekulé-Institute
for Organic Chemistry and Biochemistry, University of Bonn, Germany
Products derived from MA
Metabolic Funneling
Lignin feedstocks • a major constituent of biomass (15-40%)
Muconic acid (cis,cis / cis,trans / trans,trans-MA)
• largest renewable source of aromatic building blocks on the planet
Adipic acid
Terephthalic acid
Trimellitic acid
(2.8 Mio t/a)
(71 Mio t/a)
(100 K t/a)
• by-product of lignocellulosic biorefineries and pulp and paper manufacturing
Institute of Systems Biotechnology
• usually burned for energy generation
• Nylon-6,6 (7.2 Mio t/a) • • • • •
• efficient catalytic and hydrothermal depolymerization techniques available to decompose lignin into well-defined aromatic monomers, like catechol, phenol or cresols at high yield (over 60%)
Polyethylene terephthalate Dimethyl terephthalate Polytrimethylene terephthalate Trimellitic anhydride Industrial plastics,
• • • • • •
Resins Polyester polyols Food ingredients Pharmaceuticals Plasticizers Cosmetics
Fig. 1: Metabolic engineering strategy
Results Recovery of the energy level boosts MA titer 64 g/L ccMA from Catechol
Enhancing catechol conversion and strain robustness
Fig. 1: Advanced production of ccMA from catechol by P. putida MA-1 using a fedbatch bioreactor process (A) with transient cell regeneration (B) for enery charge recovery (C).
2KGlcnt_ex
Glcnt_ex Gluc_pp
Central metabolism:
Glcnt_pp PQQH2
2 ATP
Fig. 3: Producer strains exhibit higher tolerance towards catechol (A) and enhanced catechol degradation capacity (B).
2KGlcnt_pp FADH 2 ATP
ATP
NADPH
2K6PG
6PG
G6P
NADPH
CO2
E4P
NADPH
P5P
F6P ATP
Glucose
ATP, NADPH, Biomass
2KDPG FBP
DHAP
S7P
G3P
PEP
3PG
ATP
ATP NADH
PYR
CO 2 ATP
CO 2 NADH
AcCoA
CO 2
Aromatic catabolic pathways: B
CIT
OAA NADPH NADH
MAL
CO2
ICIT
GLYOX CO 2
NADPH
AKG
FUM CO 2
Benzoate
C
FADH2
NADH, O2, Fe2+
ATP NADH
Catechol
benABCD Benzoate dioxygenase Benzoate dehydrogenase
β-ketoadipate pathway Succinyl-CoA + Acetyl-CoA catBC
O2, Fe3+
catA catA2
NADH, O2, Fe2+
Phenol
SUC
dmpKLMNOP
ccMA
Catechol-1,2 dioxygenase
Muconate cycloisomerase Muconolactone Δ-isomerase
Phenol hydroxylase from Pseudomonas CF600
Genetic organization:
Pben
Production from aromatic mixtures
Efficient lignin hydrolysate conversion
Pcat
13 g/L ccMA from Lignin
ben operon
cat operon
Fig. 2: Production of ccMA from mixtures of catechol and phenol by P. putida MA-9 (left), Synthetic promoters using mCherry for fine-tuned enzyme expression in P. putida KT2440 (right)
Fig. 4: Fed-batch profile using hydrothermally decomposed lignin as aromatic feed.
First time demonstration of the entire value chain from lignin to nylon Lignin Depolymerisation Hydrothermal decomposition
Hydrogenation & Polymerisation Hydrogenation
Concentration
Lignin
Polycondensation with HDMA
Evaporation
Adipic acid
Nylon-6,6
Bioconversion & Downstream Processing Fermentation
Culture harvesting
Biomass separation
Summary – Key findings
Vacuum filtration
Decoloration using activated carbon
Precipitation via acidification
Lyophilisation
• Genomic integration of phenol hydroxylase DmpKLMNOP from P. putida CF600 extends the substrate spectrum to phenol • Additional expression of catechol-1,2 dioxygenase CatA2 under the strong cat promoter significantly enhances catechol conversion and strain tolerance • ccMA was produced at high concentration, yield (up to 100%) and purity (>98%) from benzoic acid, catechol, phenol, and from depolymerized lignin at the laboratory- and at 50-L pilotscale • First case example of a lignin-to-Nylon-6,6 value chain Publications: [1] Poblete & Kohlstedt et al. (2017), „Host organism P. putida“ in: Industrial Biotechnology: Microorganisms, Wiley.
Muconic acid Fig. 5: The cascaded process comprised hydrothermal depolymerization of lignin into a mixture of aromatics, containing mainly catechol, phenol and small amounts of cresols; biochemical conversion of the aromatics to ccMA by the advanced producer MA-9; purification of ccMA; hydrogenation to adipic acid; and final polymerization to nylon 6,6.
Acknowledgments The authors gratefully acknowledge the financial support by the BMBF through funding of the VIP project “Bio2Nylon”.
[2] Kohlstedt et al. (2018), Metab, Eng. 47:279-293. [3] Silva-Rocha & de Lorenzo (2012) PlosONE 7:e34675
