Cis,cis-muconic acid production from lignin related molecules byAcinetobacter baylyi ADP1

Abstract

Background: Cis,cis-muconic acid (ccMA), an important platform chemical, can be produced from lignin related molecules (LRM) via a specific two-branch catabolic route known as the β-ketoadipate pathway, which is present in certain soil bacteria. This pathway enables high production yields because ccMA is a native intermediate in one of its branches. However, commonly obtained LRM, such as p-coumaric and ferulic acid, are typically metabolized through the branch that lacks the ccMA intermediate. To redirect these LRM toward ccMA production, the two branches must be functionally integrated. This is usually achieved by introducing a non-native enzymatic activity, specifically protocatechuate decarboxylase (PCADC), which catalyzes the conversion of protocatechuate to catechol. Nevertheless, this conversion often represents the rate-limiting step in the production process.

Results: Here, we established a growth-coupled selection system for screening PCADCs using the soil bacterium Acinetobacter baylyi ADP1 as the host. In this system, cell growth depends on the in vivo performance of PCADC, thereby enabling the selection of the optimal candidate for further ccMA production. In total, five PCADC candidates were screened. AGDC1, a gallic acid decarboxylase from the yeast Blastobotrys adeninivorans, was selected for production studies. In fed-batch cultivations, the engineered strain expressing AGDC1 achieved an 83% molar yield of ccMA from ferulate and p-coumarate, that were found in lignin hydrolysate derived from straw.

Conclusion: In this study, we established a growth-based selection system for PCADCs. The outcome of the selection system was further validated in production cultivations using an engineered A. baylyi ADP1 strain. This study not only confirms the feasibility of AGDC1 in ccMA production in bacterial systems but also provides a practical screening system for future improvements.

Supplementary Information: The online version contains supplementary material available at 10.1186/s12934-025-02780-3.

Keywords: Acinetobacter baylyi ADP1; cis; cis-muconic acid; Gallic acid decarboxylase; Growth-coupled selection; Lignin related molecules; Protocatechuate decarboxylase.

Fig. 1

(A) Modified metabolic pathway for the growth-coupled selection of the PCADC candidates. Arrow with solid line indicates native pathway; arrow with dashed line indicates heterologous activity; X indicates pathway blocked by the gene deletions. (B) Growth of ASA903, ASA904, ASA905, ASA906, ASA907, wild-type ADP1 in MSM supplemented with 10 mM 4-hydroxybenzoate as a sole carbon source. The experiment was repeated using independent biological triplicates. The averages of the measurements, with error bars representing standard deviations are shown. The y-axis is shown on the log₁₀ scale

Fig. 2

Growth and ccMA production kinetics of (A) ASA916 and (B) ASA917 in batch cultivations with p-coumarate. The experiment was repeated using independent biological triplicates, and the averages of the measurements, with error bars representing standard deviations are shown

Fig. 3

Growth of ASA917 and the kinetics of substrate utilization and product formation. (A) growth and gluconate, (B) ferulate, p-coumarate, and intermediates (vanillate, 4-hydroxybenzoate, protocatechuate, catechol), and (C) products (cis,cis- and cis,trans-muconate)