Metabolic engineering of Corynebacterium glutamicum for the production of cis, cis-muconic acid from lignin

Abstract

Background: Cis, cis-muconic acid (MA) is a dicarboxylic acid of recognized industrial value. It provides direct access to adipic acid and terephthalic acid, prominent monomers of commercial plastics.

Results: In the present work, we engineered the soil bacterium Corynebacterium glutamicum into a stable genome-based cell factory for high-level production of bio-based MA from aromatics and lignin hydrolysates. The elimination of muconate cycloisomerase (catB) in the catechol branch of the β-ketoadipate pathway provided a mutant, which accumulated MA at 100% molar yield from catechol, phenol, and benzoic acid, using glucose as additional growth substrate. The production of MA was optimized by constitutive overexpression of catA, which increased the activity of the encoded catechol 1,2-dioxygenase, forming MA from catechol, tenfold. Intracellular levels of catechol were more than 30-fold lower than extracellular levels, minimizing toxicity, but still saturating the high affinity CatA enzyme. In a fed-batch process, the created strain C. glutamicum MA-2 accumulated 85 g L^-1 MA from catechol in 60 h and achieved a maximum volumetric productivity of 2.4 g L^-1 h^-1. The strain was furthermore used to demonstrate the production of MA from lignin in a cascade process. Following hydrothermal depolymerization of softwood lignin into small aromatics, the MA-2 strain accumulated 1.8 g L^-1 MA from the obtained hydrolysate.

Conclusions: Our findings open the door to valorize lignin, the second most abundant polymer on earth, by metabolically engineered C. glutamicum for industrial production of MA and potentially other chemicals.

Keywords: Adipic acid; Aromatics; Bio-plastic; Catechol dioxygenase; Lignin; Metabolic engineering; Muconate cycloisomerase; Terephthalic acid.

Fig. 1

Catabolic pathway for aromatic compounds in C. glutamicum. The displayed reactions comprise the degradation routes for benzoic acid, catechol, and phenol. BenABC benzoate 1,2-diogygenase, BenD benzoate cis-diol dehydrogenase, CatA catechol 1,2-dioxygenase, CatB muconate cycloisomerase, Phe phenol 2-monooxygenase

Fig. 2

Kinetics and stoichiometry of cis–cis-muconic acid (MA) production, using the first generation producer Corynebacterium glutamicum MA-1. The aromatics benzoic acid (a), catechol (b) and phenol (c) were used for production. In addition, glucose was added as growth substrate. The data represent the culture profiles until the depletion of the aromatic. The full culture profiles are given in the supplement (Additional file 1: Figure S4). The concentration of glucose was corrected to represent the amount consumed during this phase. The data comprise mean values and deviations from three biological replicates

Fig. 3

Culture profile of the second generation producer MA-2, using 10 mM catechol for production (a), and of the first generation producer MA-1, using 10 mM catechol for production plus 2 mM benzoic acid for induction of catA expression (b). In addition, glucose was added as growth substrate. The data represent the culture profiles until the depletion of the aromatic. The full culture profiles are given in the supplement (Additional file 1: Figure S7). The concentration of glucose was corrected to represent the amount consumed during this phase. The data comprise mean values and deviations from three biological replicates

Fig. 4

Production of cis–cis-muconic acid (MA) with feeding of catechol. The production was conducted in a shake flask. Metabolically engineered Corynebacterium glutamicum MA-2 was grown on glucose minimal medium for the first 2 h. Then, the feeding was started. Every hour, pulses with 5 mM catechol and 0.5 g L⁻¹ glucose, were added. In addition, the pH was controlled above 7.0 by manual control, adding appropriate volumes of 10 mM NaOH. The black arrow indicates the time point, when the glucose feed was stopped. The data represent mean values and standard deviations from three biological replicates

Fig. 5

Fed-batch production of cis–cis-muconic acid (MA) from catechol by metabolically engineered Corynebacterium glutamicum MA-2. Substrate consumption, growth and MA formation (a). Pulse-wise feeding of catechol (b). Glucose was added continuously to maintain the glucose level in the range between about 5–15 g L⁻¹ (a). The vertical lines represent individual catechol feed pulses (b). The feed frequency was variably adjusted, depending on the signal of dissolved oxygen, which precisely indicated the time point of catechol depletion. As example, feeding was halted once during the initial phase, corresponding to transient catechol accumulation and was accelerated later in response to the faster conversion. The data represent mean values from two replicates. The fermentation data, specifying the MA level and the biomass concentration in g L⁻¹ (Additional file 1: Figure S8A), the volumetric productivity (Additional file 1: Figure S8B) and the different yields (Additional file 1: Figure S8C), are given in the supplement

Fig. 6

Production of cis, cis-muconic (MA) from softwood lignin hydrolysate, using the engineered producer Corynebacterium glutamicum MA-2 with glucose as a growth substrate and pulsed feeding of the hydrolysate. Feeding was stopped after 12 pulses due to limited availability of the hydrolysate. The data represent mean values and deviations from three replicates