Outlook

I. Channel Expansion: From "Single Drug" to "Aromatic Drug Library”

1. Expand the high-value conversion directions of vanillic acid:

Building upon the existing technological foundation for converting vanillic acid into aromatic intermediates, subsequent efforts will focus on synthesizing other important benzene-ring-containing pharmaceuticals. For instance, salicylic acid—a precursor to the antipyretic and analgesic drug aspirin—can be synthesized from vanillic acid. This can be achieved by introducing salicylate hydroxylase to catalyze the demethylation and carboxyl group modification of vanillic acid, thereby converting it into salicylic acid.

Additionally, dopamine synthesis may be attempted. Based on the p-aminophenol (p-AP) synthesis pathway, the incorporation of dopamine β-hydroxylase could enable the directed biosynthesis of dopamine from vanillic acid.

Beyond these two compounds, another target is vitamin K3. By engineering the substrate specificity of PabABC enzymes and introducing methyltransferases, a novel pathway from vanillic acid to vitamin K3 can be constructed. This approach aims to establish a versatile synthesis platform for producing “multiple classes of aromatic drugs from lignin,” positioning vanillic acid as a key intermediate in the valorization of lignin-derived aromatics.

II. Efficiency Improvement: Directed Evolution of Enzymes and Iterative Optimization of Strains

1. Directed evolution of key enzymes:

For core enzymes such as PabABC (vanillic acid → p-ABA) and ABH60 (p-ABA → p-AP), the catalytic efficiency is improved through the following methods: Based on error-prone PCR and high-throughput screening, mutants with 2-3 times increased enzyme activity were obtained; Combining molecular docking technology, perform site-directed mutagenesis on the active center of the enzyme to enhance the affinity between the enzyme and vanillic acid, thereby further improving the substrate conversion rate.

2. Iterative modification of strain chassis:

Based on the existing BW-ΔnhoA strain, by using CRISPR-Cas9 technology to knock out more genes in competitive pathways, and simultaneously integrating auto-inducible promoters, an efficient strain with "no need for exogenous inducers and autonomous regulation of pathway expression" can be achieved, thereby reducing the operational costs of industrial production.

III. Process Scale-Up: From "Laboratory" to "Industrialization”

1. Large-scale optimization of fermentation processes:

The current experiment is based on a 250 mL conical flask. Subsequently, it will be scaled up to 5 L and 50 L fermenters. Parameters such as stirring rate, feeding strategy, and real-time pH regulation will be optimized to address mass and heat transfer issues in large-scale fermentation, ensuring that the yield remains consistent with the laboratory level.

Developing a lignin pretreatment process: In collaboration with biorefineries, optimize the chemical depolymerization conditions of lignin to improve the extraction efficiency of vanillic acid, reduce the cost of raw material pretreatment, and provide a stable raw material supply for industrialization.

2. Development of product separation and purification technology:

Currently, the product is detected by HPLC, and a low-cost separation process will be developed subsequently, such as: ion exchange resins are used to adsorb charged intermediates such as p-AP and AAP, achieving efficient separation; Membrane separation technology is used to remove bacterial cells and impurities from fermentation broth, simplifying purification steps and reducing downstream costs in industrial production.

IV. Technology Integration: Collaborative Breakthroughs Across Fields

1. Combine synthetic biology with AI technology:

Utilize AI tools (such as AlphaFold3) to predict the three-dimensional structures of enzymes like PabABC and ABH60, simulate the binding process between enzymes and substrates, guide the design of site-directed mutagenesis, and shorten the enzyme evolution cycle;

Construct a metabolic flux prediction model, analyze the impact of different fermentation conditions on product yield through AI, realize intelligent optimization of fermentation parameters, and reduce the cost of experimental trial and error.

2. Promote the collaborative implementation of industry, academia, and research:

Establish cooperation with pharmaceutical enterprises and pulp mills to jointly build a "lignin-pharmaceutical transformation" joint laboratory, transform laboratory technologies into industrial demonstration production lines, and optimize technical parameters according to enterprise needs to accelerate the commercial application of technologies. Ultimately, the long-term goals of "reducing industrial waste, greening pharmaceutical production, and maximizing economic benefits" will be achieved.