Sugar aminotransferases (SATs) catalyze the installation of chiral amines onto specific keto sugars, producing bioactive amino sugars. Their activity has been utilized in artificial reactions, such as using the SAT WecE to transform valienone into the valuable α-glucosidase inhibitor valienamine. However, the low thermostability and limited activity on non-natural substrates have hindered their applications. Simultaneously improving stability and enzyme activity is particularly challenging owing to the acknowledged inherent trade-off between stability and activity. A customized combinatorial active-site saturation test-iterative saturation mutagenesis (CAST-ISM) strategy was used to simultaneously enhance the stability and activity of WecE toward valienone. Fourteen hotspots related to improving the stability-\activity trade-off were identified based on evolutionary conservation and the average mutation folding energy assessment of 57 residues in the active site of WecE. Positive mutagenesis and combinatorial mutations of these specific residues were accomplished via site-directed saturation mutagenesis (SSM) and iterative evolution cycles. Compared with those of the wild-type (WT) WecE, the quadruple mutant M4 (Y321F/K209F/V318R/F319V) displayed a 641.49-fold increase in half-life (t_1/2) at 40 °C and a 31.37-fold increase in activity toward the non-natural substrate valienone. The triple mutant M3 (Y321F/K209F/V318R) demonstrated an 83.04-fold increase in (t_1/2) at 40 °C and a 37.77-fold increase in activity toward valienone. The underlying mechanism was dependent on the strengthened interface interactions and shortened transamination reaction catalytic distance, compared with those of the WT, which improved the stability and activity of the obtained mutants. Thus, we accomplished a general target-oriented strategy for obtaining stable and highly active SATs for artificial amino-sugar biosynthesis applications.

The availability of nitrogen (N) is crucial for both the productivity of terrestrial and aquatic ecosystems globally. However, the overuse of artificial fertilizers and the energy required to fix nitrogen have pushed the global nitrogen cycle (N-cycle) past its safe operating limits, leading to severe nitrogen pollution and the production of significant amounts of greenhouse gas nitrous oxide (N₂O). The anaerobic ammonium oxidation (anammox) mechanism can counteract the release of ammonium and N₂O in many oxygen-limited situations, assisting in the restoration of the homeostasis of the Earth’s N biogeochemistry. In this work, we looked into the characteristics of the anammox hotspots’ distribution across various types of ecosystems worldwide. Anammox hotspots are present at diverse oxic-anoxic interfaces in terrestrial systems, and they are most prevalent at the oxic-anoxic transition zone in aquatic ecosystems. Based on the discovery of an anammox hotspot capable of oxidizing ammonium anoxically into N₂ without N₂O by-product, we then designed an innovative concept and technical routes of nature-based anammox hotspot geoengineering for climate change, biodiversity loss, and efficient utilization of water resources. After 15 years of actual use, anammox hotspot geoengineering has proven to be effective in ensuring clean drinking water, regulating the climate, fostering plant and animal diversity, and enhancing long-term environmental quality. The sustainable biogeoengineering of anammox could be a workable natural remedy to resolve the conflicts between environmental pollution and food security connected to N management.

Terpenoids are the largest family of natural products. They are made from the building block isoprene pyrophosphate (IPP), and their bioproduction using engineered cell factories has received a great deal of attention. To date, the insufficient metabolic supply of IPP remains a great challenge for the efficient synthesis of terpenoids. In this work, we discover that the imbalanced metabolic flux distribution between the central metabolism and the IPP supply hinders IPP accumulation in Bacillus subtilis (B. subtilis). Therefore, we remodel the IPP metabolism using a series of genetically encoded two-input-multi-output (TIMO) circuits that are responsive to pyruvate or/and malonyl-CoA, resulting in an IPP pool that is significantly increased by up to four-fold. As a proof-of-concept validation, we design an IPP metabolism remodeling strategy to improve the production of three valuable terpenoids, including menaquinone-7 (MK-7, 4.1-fold), lycopene (9-fold), and β-carotene (0.9-fold). In particular, the titer of MK-7 in a 50-L bioreactor reached 1549.6 mg∙L⁻¹, representing the highest titer reported so far. Thus, we propose a TIMO genetic circuits-assisted IPP metabolism remodeling framework that can be generally used for the synergistic fine-tuning of complicated metabolic modules to achieve the efficient bioproduction of terpenoids.