p-Coumaric acid (p-CA), also known as 4-hydroxycinnamic acid, is mainly found in herbal plants, as well as fruits and vegetables [1]. Numerous studies have demonstrated that p-CA exhibits a wide range of pharmacological properties and serves as a common precursor in the biosynthetic pathways of various secondary metabolites [2], [3]. It also finds extensive applications in the food, medical, and chemical industries [4], [5]. Currently, p-CA is produced through plant extraction [6], chemical synthesis [7] and microbial synthesis [8]. Of these, microbial synthesis offers a viable and effective alternative, demonstrating strong potential in terms of shorter production cycles, sustainability, and environmental friendliness [9]. Notably, the pathway involving the conversion of tyrosine to p-CA using tyrosine ammonia-lyase (TAL) is particularly important for microbial p-CA synthesis [10].

Currently, the microbial synthesis of p-CA is predominantly achieved through the heterologous expression of genes encoding TAL in various microbial hosts, including Escherichia coli, Pseudomonas taiwanensis, Corynebacterium glutamicum, Saccharomyces cerevisiae, etc [10], [11], [12], [13], [14]. The p-CA yield ranged from 25.6 mg L−1 to 12.5 g L−1, the engineered strain showed the highest yield in S. cerevisiae [15]. The TAL gene has attracted widespread attention since its isolation from Rhodobacter capsulatus and successful expression in 2002 [16]. Subsequently, Rs-TAL from Rhodobacter sphaeroides [17], Se-sam8 from Saccharothrix espanaensis [18], Rg-TAL from Rhodotorula glutinis [19], Fj-TAL from Flavobacterium johnsoniae [20], as well as Sas-TAL from the actinomycete Saccharothrix sp. NRRL B-16348 and Sts-TAL from Streptomyces sp. NRRL F-4489 [21], were identified in sequence. Among them, Se-sam8 expressed by Saccharothrix espanaensis exhibited a strong substrate affinity for tyrosine [20], [21].

Bacillus subtilis, a Gram-positive bacterium, has been acknowledged as a generally recognized safe (GRAS) strain by the U.S. Food and Drug Administration, owing to its endotoxin-free and non-pathogenic characteristics [22]. Owing to the outstanding characterization of the rapid multiplication rate, short production cycle, and minimal nutrient requirements, B. subtilis has been highly considered to be suitable for large-scale industrial fermentation [23]. However, the p-CA production of engineered B. subtilis has remained to be explored yet.

However, the simple heterologous expression of TAL is often insufficient for high-level production of p-CA. Having established the feasibility of p-CA production in Bacillus subtilis, the focus now shifts to circumventing its limitations via metabolic engineering strategies. As a powerful tool for achieving high product titer, yield, and productivity, dynamic pathway engineering primarily relies on key process signals including fermentation parameters, the concentrations of intermediates or products, and cell density [24]. In addition, cofactors such as NAD (H), NADP (H) and ATP provide energy needed for carbon metabolism [25].

In this study, the TAL gene was heterologously expressed in B. subtilis for the first time, and an engineered strain PBnprE for efficient p-CA synthesis was obtained by number of promoter screening, and the optimization of the fermentation substrates and conditions was used to increase the p-CA production. The effects of PBnprE fermentation broth extracts on inhibiting bacteria and antioxidant activities were also investigated.