Cascaded Chemical and Biochemical Conversion

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depolymerization techniques available to ... Industrial plastics, ... 5: The cascaded process comprised hydrothermal depolymerization of lignin into a mixture of ...
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