Methyl salicylate (MeSA) is a volatile methylated aromatic ester widely used in agriculture, food, and pharmaceuticals. Microbial biosynthesis offers a sustainable alternative to plant extraction and petrochemical synthesis; however, efficient MeSA production remains limited by the low catalytic efficiency of carboxyl methyltransferases, insufficient intracellular supply of S-adenosylmethionine (SAM), and severe product volatilization during fermentation. In this study, we systematically screened salicylate carboxymethyltransferase and identified CbSAMT as a relatively efficient catalyst for salicylic acid (SA). To further improve catalytic performance, we engineered CbSAMT through fusion-protein design. Remarkably, the catalytic efficiency of 7 × His-CbSAMT was 12-fold higher than that of the wild-type. Molecular dynamics simulations and computational analyses revealed that the polyhistidine tag reshaped the catalytic microenvironment by remodeling the hydrogen-bonding network and reducing the distance between SA and key substrate-positioning residues, thereby enhancing substrate binding and catalytic turnover. To further enhance MeSA biosynthesis, we reinforced intracellular SAM regeneration and established an in situ two-phase fermentation system using n-dodecane as the extractant to alleviate product volatilization. Finally, fed-batch fermentation achieved a MeSA titer of 5.12 g/L, representing the highest production level reported to date. Collectively, this study highlights the potential of polyhistidine tags in enzyme engineering and provides an efficient platform for the biosynthesis of volatile methylated natural products.
Yuan Gao, Shi-Bo Guo, Jun Wu et al.· Bioresource Technology· 0 citations
Construction of microbial cell factories often requires extensive reconfiguration of metabolic networks, which frequently compromises cell growth. Here, we engineered E. coli to produce dencichine (β-ODAP), a plant-derived hemostatic agent. Knocking out serB blocks the competing L-serine pathway, causing L-serine auxotrophy and severely impairing cell growth. This growth defect was only partially restored by supplementation with L-serine or L-glycine, suggesting the presence of additional growth-limiting factors. Through adaptive laboratory evolution and reverse engineering, we further uncovered that overexpression of serA to enhance the supply of the precursor L-2,3-diaminopropionate led to accumulation of the toxic byproduct 2-hydroxyglutarate (2-HG) from α-ketoglutarate. To resolve these two growth constraints, we developed a growth-phase-dependent dual-dynamic regulation circuit. This circuit gradually activates serA expression and progressively represses serB expression as cells enter the stationary phase, thereby reducing 2-HG accumulation and alleviating L-serine auxotrophy. Combined with pathway balancing and cofactor optimization, the final engineered strain produced 13.46 g L-1 of β-ODAP with a yield of 0.32 g g-1 in 3-L bioreactors. This study reveals a toxicity mechanism in serine-pathway engineering and provides a dynamic regulation strategy applicable to the biosynthesis of serine-pathway-derived metabolites.
Yu Zhang, Jun Wu, Jinghua Yu et al.· The Innovation Life· 0 citations
Daphnetin is a clinically established coumarin, but its native biosynthetic pathway remains elusive. In this study, a 2-oxoglutarate-dependent dioxygenase (2OGD), AtS8H, was identified that hydroxylates umbelliferone (UMB) to daphnetin , enabling the design of an artificial biosynthetic pathway. However, the pathway efficiency was constrained by the poor solubility of AtS8H and the low catalytic activity of the upstream 2OGD enzyme IbC2'H. To enhance AtS8H solubility, we developed an integrated strategy combining ProteinMPNN-guided sequence redesign with surface charge engineering. The obtained quintuple mutant S8H 2-6 exhibited significantly improved solubility and a 7.2-fold increase in catalytic efficiency. For IbC2'H, we developed a fluorescence-based high-throughput screening method, and a quadruple mutant C6 was obtained by directed evolution, which displayed a 2.7-fold higher kcat and a 3.2 °C improvement in thermal stability. Implementing both engineered enzymes into an optimized Escherichia coli strain enabled the de novo production of daphnetin at a titer of 46 mg/L. This work reports, to the best of our knowledge, the first microbial de novo production of daphnetin from a simple carbon source and demonstrates an integrated enzyme engineering approach that synergistically refines biosynthetic pathways for efficient microbial production.
Shunmin Ji, Chong Xie, Yanyan Wang et al.· ACS Synthetic Biology· 0 citations
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