L-(+)-tartaric acid (L-TA) is a high-value chiral organic acid essential for food and pharmaceuticals. Despite its industrial importance, sustainable green production is constrained by the lack of a fully defined biosynthetic pathway. Here, we report the de novo biosynthesis of L-TA in Saccharomyces cerevisiae through reaction-guided enzyme mining, experimental validation, and Enzyme Commission-specific Catalytic Hybrid Optimizer (ECHO)-assisted enzyme prioritization. We first elucidate the elusive two-step conversion from precursor 5-keto-D-gluconic acid (5-KGA) to L-TA, catalyzed by transketolase (TK) and succinate semialdehyde dehydrogenase (SSDH). To optimize this critical step, we develop the ECHO. This multimodal framework integrates sequence, substrate, and pocket-aware structural information to identify high-performance TK-SSDH pairs. By integrating this pathway with de novo precursor synthesis, cofactor engineering, and semi-rational protein engineering, a final L-TA titer of 6.59 mg L−1 was achieved in a 5-L bioreactor. By connecting computational mining and metabolic assembly through a multi-module engineering strategy, our study establishes a green platform for L-TA production and demonstrates an effective workflow for synthetic pathway design. L-(+)-tartaric acid (L-TA) is a high-value chiral organic acid for food and pharmaceuticals. Here the authors produce L-TA in S. cerevisiae through reaction-guided enzyme mining and Enzyme Commission-specific Catalytic Hybrid Optimizer (ECHO)-assisted enzyme prioritization.
Xuan Zhou, Jiaheng Hou, Zikai Wang et al.· Nature Communications· 0 citations
Difucosyllactose (DFL), a fucosylated human milk oligosaccharide, exhibits significant application potential in the food and pharmaceutical industries. However, the microbial production of DFL faces challenges such as the accumulation of the intermediate 2'-fucosyllactose (2'-FL) and the high costs associated with the use of antibiotics and inducers. In this study, we constructed a plasmid- and inducer-free Escherichia coli MG1655 strain to enhance the biosynthesis of DFL while minimizing 2'-FL accumulation. Initially, the de novo DFL biosynthetic pathway was established by introducing different α-1,3-fucosyltransferases (α-1,3-FucT) into a 2'-FL-producing strain. Combinatorial metabolic engineering strategies were then employed to improve DFL accumulation. Subsequently, guided by the in silico multienzyme assembling by reshaping space (iMARS) framework, a fusion enzyme, FucTaY218K-L60-FutC, was rationally designed to effectively alleviate 2'-FL accumulation. Furthermore, modulation of the hydrophobic microenvironment in the substrate-binding pocket of FucTaY218K yielded the beneficial mutant FucTaY218K/W31R. Finally, modifications to the lactose operon significantly improved strain growth while concurrently enhancing DFL production. The best-performing strain achieved a DFL titer of 74 g/L in a 5-L bioreactor, corresponding to a productivity of 0.96 g/L/h, representing the highest productivity reported to date. This study establishes a robust and environmentally friendly platform for the industrial-scale production of DFL and provides a strategy for the microbial synthesis of other high-value human milk oligosaccharides.
Zhiqiang Liu, Binglin Li, Jieying Deng et al.· ACS Synthetic Biology· 0 citations
Trehalose is a nonreducing disaccharide widely used for its biomolecule-protective properties. However, multienzyme cascade production remains limited by low enzyme expression and suboptimal catalytic performance. To address this, thermostable maltooligosyltrehalose synthase (TreY) and trehalohydrolase (TreZ) from Arthrobacter ramosus were individually expressed intracellularly in Bacillus subtilis, and the crude lysates were combined for trehalose biosynthesis, achieving 281.4 g/L trehalose and a yield of 0.7 g trehalose/g maltodextrin. Integrated computational screening identified MalQ-3 from Cyanobacterium stanieri as a suitable 4-α-glucanotransferase for soluble expression. Subsequent semirational engineering generated MalQ-3-M2 (S54P/V472F), with enhanced activity and stability associated with improved substrate-pocket dynamics, thereby facilitating glucan-chain rearrangement and short-chain reutilization. MalQ-3-M2 was separately expressed in B. subtilis and incorporated into the crude-lysate cascade, increasing the trehalose titer to 338 g/L and the yield to 0.85 g trehalose/g maltodextrin. Overall, this work establishes a scalable B. subtilis platform for efficient trehalose production.
Haidong Huang, Yang-Yang Li, Jin-Song Song et al.· Journal of Agricultural and...· 0 citations
A multilevel engineering strategy to enhance yeast protein production by optimizing precursor supply, translation machinery, and diploid construction provides both improved protein content and key targets for breeding high-protein microbial strains.
Yang Liu, Cai-Ying Jin, Wentao Shen et al.· Bioresource Technology· 1 citation
We use cookies to run the site and, with your consent, for analytics and to show ads.
See our Cookie Policy.
