Biocatalytic cascades with spatial proximity can orchestrate multistep pathways to form metabolic highways, which enhance the overall catalytic efficiency. However, the effect of spatial organization on catalytic activity is poorly understood, and multienzyme architectural engineering with predictable performance remains unrealized. Here, we developed a standardized framework, called iMARS, to rapidly design the optimal multienzyme architecture by integrating high-throughput activity tests and structural analysis. The approach showed potential for industrial-scale applications, with artificial fusion enzymes designed by iMARS significantly improving the production of resveratrol by 45.1-fold and raspberry ketone by 11.3-fold in vivo, as well as enhancing ergothioneine synthesis in fed-batch fermentation. In addition, iMARS greatly enhanced the in vitro catalytic efficiency of the multienzyme complexes for PET plastic depolymerization and vanillin biosynthesis. As a generalizable and flexible strategy at molecular level, iMARS could greatly facilitate green chemistry, synthetic biology, and biomanufacturing.

Industrial wastewater discharge, agricultural production, marine shipping, oil extraction, and other activities have caused serious marine pollution, including microplastics, petroleum and its products, heavy metals, pesticides, and other organics. Efficiency of bioremediation of marine pollutions may be limited by high salt concentrations (>1%, w/v), which can cause an apparent loss of microbial activities. In this study, functional promoters P1, P2-1, and P2-2 censoring salt stress were isolated and identified from a Vibrio natriegens strain Vmax. Three salt-induced degradation models were constructed to degrade polyethylene terephthalate (PET), chlorpyrifos (CP), and hexabromocyclododecanes (HBCDs) using the marine strain Vmax. The engineered strains are efficient for degradation of the corresponding substrates, with the degradation rates at 15 mg/L PET in 8 d, 50 mg/L CP in 24 h, and 1 mg/L HBCDs in 4 h, respectively. In addition, an immobilization strategy for recycling and reusing of engineered strains was realized by expressing the chitin-binding protein GbpA. This study may help answer the usage of rapidly growing marine bacteria such as V. natriegens Vmax to degrade marine pollution efficiently.