Lignin conversion to β-ketoadipic acid by Pseudomonas putida via metabolic engineering and bioprocess development

Abstract

Bioconversion of a heterogeneous mixture of lignin-related aromatic compounds (LRCs) to a single product via microbial biocatalysts is a promising approach to valorize lignin. Here, Pseudomonas putida KT2440 was engineered to convert mixed p-coumaroyl- and coniferyl-type LRCs to β-ketoadipic acid, a precursor for performance-advantaged polymers. Expression of enzymes mediating aromatic O-demethylation, hydroxylation, and ring-opening steps was tuned, and a global regulator was deleted. β-ketoadipate titers of 44.5 and 25 grams per liter and productivities of 1.15 and 0.66 grams per liter per hour were achieved from model LRCs and corn stover-derived LRCs, respectively, the latter representing an overall yield of 0.10 grams per gram corn stover-derived lignin. Technoeconomic analysis of the bioprocess and downstream processing predicted a β-ketoadipate minimum selling price of $2.01 per kilogram, which is cost competitive with fossil carbon-derived adipic acid ($1.10 to 1.80 per kilogram). Overall, this work achieved bioproduction metrics with economic relevance for conversion of lignin-derived streams into a performance-advantaged bioproduct.

Fig. 1.. Metabolic pathway for the biological conversion of p-coumarate and ferulate to β-ketoadipate.

The genetic modifications applied to Pseudomonas putida are depicted with an “X” or a filled circle with a “+” for gene deletion or gene overexpression, respectively. N/E, nonenzymatic.

Fig. 2.. Production of β-ketoadipate from p-coumarate by CJ263 in bioreactors using DO-stat and constant fed-batch feeding modes.

The profiles show (A) β-ketoadipate titer, (B) β-ketoadipate instantaneous productivity, (C) p-coumarate accumulation, and (D) 4-hydroxybenzoate accumulation in bioreactors. Data show the average of biological duplicates, and error bars represent the absolute difference between biological duplicates. Single plots with all metabolites and cell growth are provided in fig. S1. Numerical data are provided in data S1.

Fig. 3.. Strain engineering for improved conversion of p-coumarate and ferulate to β-ketoadipate.

Metabolite abundances as a function of time in the base strain CJ263 and further engineered strains including (A) overexpression of vanAB and replacement of pobAR with praI_JJ-1b in CJ263 to generate AW271, (B) overexpression of pcaHG in CJ263 to generate AW297, and (C) deletion of crc in CJ263 to generate AW124. Data show the average of three biological replicates. Error bars represent the SD among biological triplicates. Single plots including all metabolites and cell growth are provided in fig. S3. Numerical data are provided in data S1. Genotypes are provided in Table 1.

Fig. 4.. β-Ketoadipate production from a mixture of p-coumarate and ferulate by the base strain (CJ263) and the integrated engineered strains (AW299 and AW311) in shake flasks.

Profiles show bacterial growth, substrate utilization, accumulation of aromatic intermediates, and β-ketoadipate production by the (A) base strain CJ263 and integrated engineered strains (B) AW299 and (C) AW311. Data show the average of three biological replicates. Error bars represent the SD among biological triplicates. Numerical data are provided in data S1. Genotypes are provided in Table 1. OD₆₀₀, optical density measured as absorbance at 600 nm.

Fig. 5.. β-Ketoadipate production from p-coumarate and ferulate individually or in a mixture by the base strain (CJ263) and the engineered strains AW299 and AW311 in bioreactors.

β-Ketoadipate was produced from (A) p-coumarate, (B) ferulate, or (C) a 3:1 molar mixture of p-coumarate:ferulate. (A) β-Ketoadipate, p-coumarate, and 4-hydroxybenzoate accumulation as a function of time in fed-batch cultivations with constant feeding of p-coumarate. CJ263 is omitted as it did not grow consistently at substrate feeding rates ≥ 18 mmol p-coumarate/liter per hour. (B) β-Ketoadipate, ferulate, and vanillate accumulation as a function of time in fed-batch cultivations with constant feeding of ferulate. AW311 is omitted as it did not grow consistency under the 21 mmol ferulate/liter per hour condition. (C) β-Ketoadipate, p-coumarate, and ferulate as a function of time in fed-batch cultivations with constant feeding of 3:1 (molar) p-coumarate:ferulate. Error bars represent the absolute difference between duplicates or SD for experiments with more than two biological replicates. Single plots with all metabolites and cell growth are provided in figs. S7 to 10. Numerical data and number of replicates for each experiment are provided in data S1.

Fig. 6.. β-Ketoadipate titers, productivities, and yields for AW299 bioreactor cultivations with various LRCs.

Productivities and yields corresponding to the maximum titer are displayed in solid bars; maximum instantaneous productivity and yield data are shown in dotted bars. The data show the average of two biological replicates. Error bars represent the absolute difference between duplicates. Numerical data are provided in data S1. A summary and comparison to previously reported titers and productivities is provided in table S5. n/a, not applicable. APL, alkaline-pretreated liquor.

Fig. 7.. β-Ketoadipate production from corn stover–derived LRCs.

(A) Overall process flow and yields. A summary of yields is provided in table S9. (B) Profile of bioreactor cultivation conducted with AW299 and fed with solid APL extractives. LRC amount (g) fed in each pulse and accumulation of LRCs, aromatic intermediates, and β-ketoadipate are plotted as a function of time. Because of the substrate limitation, a single cultivation was performed. Numerical data are provided in data S1. LRCs quantified in APL and APL extractives are shown in tables S7 and S8.

Fig. 8.. Economic and environmental assessments for the production of 100,000 metric tons of β-ketoadipate per year from LRCs.

(A) Process flow diagram of the β-ketoadipate production process. (B) Breakdown of MSP of β-ketoadipate (βKA). (C) Single-point sensitivity analyses for MSP around main process parameters and configurations. Base case values are shown in parentheses along the y axis. (D) Sensitivity analysis of the isolated effect of productivity and titer on the MSP of β-ketoadipate. (E) Effect of productivity and LRC price on the MSP of β-ketoadipate. (F) Cradle-to-gate LCA of the scaled-up production of LRC-derived β-ketoadipate comparing without (“base”) and with ammonium sulfate recovery as a coproduct for GHG emissions, cumulative fossil energy consumption, and water consumption. Results for the scenarios with ammonium sulfate recovery were derived using the purpose-driven system-level allocation method based on products’ market values. All β-ketoadipate yields are on a molar basis (mol/mol, %). Sensitivity results for coproduct handling approaches, namely, mass-based allocation and coproduct displacement, are presented in fig. S14 and table S14.