Design
Background
The climate crisis is a major challenge humanity faces on the path to sustainable development. Currently, most crop straw is still disposed of by burning, which releases large amounts of harmful substances, causing severe air pollution and contributing significantly to autumn haze in northern China. Straw primarily consists of cellulose, hemicellulose, and lignin. While the utilization methods for cellulose and hemicellulose are relatively well-established, the efficient use of lignin remains challenging. Our project aims to explore green utilization pathways for lignin in straw. Specifically, we employ an integrated chemical and biological approach: first, lignin is converted into vanillic acid through electrochemical catalysis and enzymatic hydrolysis, and then vanillic acid is further transformed into paracetamol (AAP) via a non-natural metabolic pathway. Paracetamol is a classic antipyretic and analgesic drug that can be used to alleviate flu symptoms. This project not only provides new ideas for the high-value utilization of lignin but also helps reduce the environmental pollution caused by straw burning while lessening the environmental burden of traditional chemical production. See more details in our Project Description.
To manage metabolic load and optimize the biosynthetic pathway, we segmented our design into three distinct modules, each tackling a step in the conversion of vanillic acid (derived from lignin degradation) to paracetamol (AAP).
Module 1: Exploration of Pathway Feasibility
Module 2: Enzyme Optimization and Bypass Optimization
Module 3: Design of Temperature-Sensitive Promoter and Enzyme Optimization for Final Product Synthesis
These three modules correspond to the three reaction stages, enabling the conversion from vanillic acid to AAP through distinct component designs in each module. This approach provides an innovative green solution to address the challenges of straw utilization and mitigate the climate crisis.
Module 1: Exploration of Pathway Feasibility
Vanillin acid is a conversion product of lignin through chemical methods and enzymatic hydrolysis. As a terminal metabolite for many organisms, vanillic acid primarily originates from the phenylalanine/tyrosine metabolic pathways in plants and microorganisms[1].We aim to utilize vanillic acid to achieve the production of high-value small compounds containing benzene rings.
In scientific literature research, we observed that chorismic acid and vanillic acid share structural similarities. Notably, chorismic acid can be biologically converted into p-aminobenzoic acid (p-ABA), which is further transformed into paracetamol (AAP)[2].
We hypothesized that the aminodeoxychorismate synthase (PabAB), which converts chorismic acid to p-ABA, might also accept vanillic acid as a substrate. To validate this hypothesis in silico, we performed molecular docking simulations[3].The results were promising: vanillic acid occupied a similar position to chorismic acid in the enzyme's binding pocket with favorable binding energy, suggesting PabAB could catalyze the designed reaction (See Results for details).
Based on these analyses, we plan to first produce p-ABA from vanillic acid, and then synthesize AAP from p-ABA, thereby providing experimental feasibility support for the entire conversion pathway from lignin to paracetamol. Through the innovative application of vanillic acid, our team is the first to use lignin derivative as a substrate for the biosynthesis of p-aminobenzoic acid(p-ABA), demonstrating the immense potential of Escherichia coli in microbial production. We have opened up a new avenue for the use of vanillic acid in biosynthesis, providing fresh perspectives and pathways for future applications.
We constructed pYB1a-pabABC to implement our strategy, which was ultimately proven feasible.(For details, please refer to the Results page.)
Module 2: Enzyme Optimization and Bypass Optimization
The initial conversion of p-ABA to p-AP using MNX1 from Candida parapsilosis CBS604 suffered from low efficiency, creating a bottleneck in our pathway. To optimize production, we identified a more efficient hydroxylase, ABH60, through literature research and experimental validation[4], and replaced MNX1 with it to improve the conversion efficiency.
MNX1 enzyme
MNX1 is a flavin-dependent monooxygenase from Candida parapsilosis CBS604, which can catalyze the oxidative decarboxylation of 4-hydroxybenzoic acid and its derivatives to produce the corresponding hydroquinone compounds[5].(Eppink et al., 1997)
Based on this reported function, we hypothesized that MNX1 could also act on structurally similar p-aminobenzoic acid (p-ABA) via oxidative decarboxylation to generate the target intermediate p-aminophenol (p-AP).Therefore, we introduced and expressed the MNX1 gene in our experimental system to achieve the key conversion step from p-ABA to p-AP, thereby advancing the entire pathway toward the synthesis of paracetamol (AAP).
ABH60 enzyme
P-Aminobenzoic acid hydroxylase (ABH60) is a FAD (flavin adenine dinucleotide)-dependent monooxygenase initially discovered in the edible mushroom Agaricus bisporus. This enzyme is involved in the synthesis of characteristic aromatic compounds in mushrooms and is capable of hydroxylating substrates containing a benzene ring. In our pathway design, we replaced MNX1 with ABH60, which significantly improved conversion efficiency. However, this optimization revealed a new problem: the native E. coli NhoA, preferentially acetylated our substrate (p-ABA) instead of p-AP when both substrates were present, diverting flux away from our desired product, AAP.
To eliminate this competing pathway, we employed two strategies. First, we used CRISPR interference (CRISPRi) to repress the expression of the endogenous nhoA gene. Following the success of this approach, we proceeded to permanently remove the gene using CRISPR-Cas9 mediated knockout in Escherichia coli.
CRISPRi
CRISPR-Cas9
Module 3: Design of Temperature-Sensitive Promoter and Enzyme Optimization for Final Product Synthesis
To ensure the smooth progression of the conversion step from p-AP to AAP, we designed a temperature-sensitive promoter to repress expression at 30°C and induce expression at 37°C, a temperature-control feature consistent with our intended fermentation conditions.
The use of this temperature-sensitive promoter significantly reduced the metabolic flux toward by-product formation, directing most of the substrate toward our target product, AAP (paracetamol).This design not only increased the yield of the target product but also reduced by-product generation, providing a reliable foundation for subsequent experiments and product yield optimization. The temperature-sensitive promoter played a key regulatory role in the entire biosynthetic pathway, enabling controllable metabolic flux and further ensuring the feasibility and efficiency of the project design.
Meanwhile, we compared and experimentally evaluated the enzymes NhoA and PANAT, finding that PANAT exhibited superior conversion efficiency. Therefore, PANAT was selected for the subsequent production of AAP. Compared with nhoA, PANAT not only acetylates common NAT substrates but also effectively catalyzes reactions involving folate precursors and the folate catabolite 4-aminobenzoylglutamate.
NhoA enzyme
N-Hydroxyarylamine O-acetyltransferase (NhoA) belongs to the arylamine N-acetyltransferase (NAT) protein family and is well-conserved in both bacteria and eukaryotes. Enzymes of this class typically exhibit N-hydroxyarylamine O-acetyltransferase (OAT) activity and are primarily involved in the metabolic activation of nitrosoaromatic compounds and aromatic amines in bacteria. The nhoA gene is endogenous to Escherichia coli.
PANAT enzyme
PANAT is an enzyme derived from the NAT (arylamine N-acetyltransferase) gene of Pseudomonas aeruginosa. This gene was cloned and expressed as an N-terminal His-tagged protein in Escherichia coli, and its catalytic properties have been studied using various substrates and systematically compared with other prokaryotic NAT enzymes. Results indicate that PANAT exhibits high specific activity in vitro toward a wide range of arylamine substrates—often an order of magnitude higher than recombinant NAT enzymes from Mycobacterium smegmatis or Salmonella typhimurium. To date, identified substrates of PANAT include the anti-tuberculosis drug isoniazid, the inflammatory bowel disease therapeutic 5-aminosalicylic acid, as well as several significant environmental pollutants such as 3,4-dichloroaniline and 2-aminofluorene[6]. The introduction of the PANAT gene provided a novel solution for optimizing the final product yield.
Summary
Our project designs a four-step modular biosynthetic pathway to convert vanillic acid into acetaminophen (AAP), following the route: vanillic acid → p-ABA → p-AP → AAP. Through pathway exploration and optimization, we first achieved the conversion of vanillic acid to p-ABA. Subsequently, by employing gene disruption and knockout strategies, along with the design of temperature-sensitive promoters, we successfully facilitated the synthesis of AAP from p-ABA. To avoid the formation of the byproduct p-acetaminophenylbenzoic acid, we knocked out the endogenous nhoA gene in E. coli. Furthermore, to improve conversion efficiency, we optimized enzyme usage by replacing the inefficient MNX1 enzyme with the more effective ABH60 enzyme, significantly enhancing the production of the intermediate p-AP. To ensure efficient conversion of p-AP to AAP, we engineered temperature-sensitive promoters, optimized metabolic flux, and minimized byproduct formation. Additionally, replacing the NhoA enzyme with PANAT further increased AAP yield.
Future expectations
Currently, our design primarily focuses on the utilization of vanillic acid. By converting lignin into vanillic acid and further synthesizing paracetamol (AAP), we have achieved preliminary high-value utilization of lignin and exploration of green sustainability. In the future, we plan to expand the utilization pathways of other high-value benzene ring-containing compounds derived from lignin to achieve more green chemistry and sustainable development goals. By designing and optimizing metabolic pathways for a broader range of substrates, we aim to develop novel biosynthetic routes to convert lignin into various functional compounds, thereby fully realizing its economic and environmental potential.
Furthermore, we also intend to partially incorporate vanillic acid into paracetamol-based pharmaceuticals to improve their palatability and user experience. This approach could not only enhance the taste of the medication but may also provide potential auxiliary benefits to drug stability and efficacy. Through these designs, we hope to extend the project’s research outcomes to practical applications, establishing a complete innovation chain from basic research to sustainable production and drug improvement.
Reference
[1] Funk, C., & Brodelius, P. E. (1990). Phenylpropanoid Metabolism in Suspension Cultures of Vanilla planifolia Andr. PLANT PHYSIOLOGY, 94(1), 95–101. https://doi.org/10.1104/pp.94.1.95
[2]Shen, X., Chen, X., Wang, J., Sun, X., Dong, S., Li, Y., Yan, Y., Wang, J., & Yuan, Q. (2021). Design and construction of an artificial pathway for biosynthesis of acetaminophen in Escherichia coli. Metabolic Engineering, 68, 26–33. https://doi.org/10.1016/j.ymben.2021.09.001
[3]Parsons, J. F., Jensen, P. Y., Pachikara, A. S., Howard, A. J., Eisenstein, E., & Ladner, J. E. (2002). Structure of Escherichia coli Aminodeoxychorismate Synthase: Architectural Conservation and Diversity in Chorismate-Utilizing Enzymes,. Biochemistry, 41(7), 2198–2208. https://doi.org/10.1021/bi015791b
[4]Johnson, N. W., Valenzuela-Ortega, M., Thorpe, T. W., Era, Y., Kjeldsen, A., Mulholland, K., & Wallace, S. (2025). A biocompatible Lossen rearrangement in Escherichia coli. Nature Chemistry. https://doi.org/10.1038/s41557-025-01845-5
[5]Shen, X., Wang, J., Wang, J., Chen, Z., Yuan, Q., & Yan, Y. (2017). High-level De novo biosynthesis of arbutin in engineered Escherichia coli. Metabolic Engineering, 42, 52–58. https://doi.org/10.1016/j.ymben.2017.06.001
[6]Westwood, I. M., Holton, S. J., Rodrigues-Lima, F., Dupret, J., Bhakta, S., Noble, M. E. M., & Sim, E. (2005). Expression, purification, characterization and structure of Pseudomonas aeruginosa arylamine N-acetyltransferase. Biochemical Journal, 385(2), 605–612. https://doi.org/10.1042/bj20041330