Computational design of noncanonical amino acid-based thioether staples at N/C-terminal domains of multi-modular pullulanase for thermostabilization in enzyme catalysis.

Enzyme thermostabilization is considered a critical and often obligatory step in biosynthesis, because thermostability is a significant property of enzymes that can be used to evaluate their feasibility for industrial applications. However, conventional strategies for thermostabilizing enzymes generally introduce non-covalent interactions and/or natural covalent bonds caused by natural amino acid substitutions, and the trade-off between the activity and stability of enzymes remains a challenge. Here, we developed a computationally guided strategy for constructing thioether staples by incorporating noncanonical amino acid (ncAA) into the more flexible N/C-terminal domains of the multi-modular pullulanase from (BtPul) to enhance its thermostability.

Highly selective synthesis of D-amino acids via stereoinversion of corresponding counterpart by an in vivo cascade cell factory.

Background: D-Amino acids are increasingly used as building blocks to produce pharmaceuticals and fine chemicals. However, establishing a universal biocatalyst for the general synthesis of D-amino acids from cheap and readily available precursors with few by-products is challenging. In this study, we developed an efficient in vivo biocatalysis system for the synthesis of D-amino acids from L-amino acids by the co-expression of membrane-associated L-amino acid deaminase obtained from Proteus mirabilis (LAAD), meso-diaminopimelate dehydrogenases obtained from Symbiobacterium thermophilum (DAPDH), and formate dehydrogenase obtained from Burkholderia stabilis (FDH), in recombinant Escherichia coli.

2-Ketoglutarate-Generated In Vitro Enzymatic Biosystem Facilitates Fe(II)/2-Ketoglutarate-Dependent Dioxygenase-Mediated C-H Bond Oxidation for (2,3,4)-4-Hydroxyisoleucine Synthesis.

Fe(II)/2-ketoglutarate-dependent dioxygenase (Fe(II)/2-KG DO)-mediated hydroxylation is a critical type of C-H bond functionalization for synthesizing hydroxy amino acids used as pharmaceutical raw materials and precursors. However, DO activity requires 2-ketoglutarate (2-KG), lack of which reduces the efficiency of Fe(II)/2-KG DO-mediated hydroxylation. Here, we conducted multi-enzymatic syntheses of hydroxy amino acids.

Reconstitution of TCA cycle involving l-isoleucine dioxygenase for hydroxylation of l-isoleucine in using CRISPR-Cas9.

l-isoleucine dioxygenase (IDO) is an Fe (II)/α-ketoglutarate (α-KG)-dependent dioxygenase that specifically converts l-isoleucine (l-Ile) to (2S, 3R, 4S)-4-hydroxyisoleucine (4-HIL). 4-HIL is an important drug for the treatment and prevention of type 1 and type 2 diabetes but the yields using current methods are low. In this study, the CRISPR-Cas9 gene editing system was used to knockout and gene in the TCA cycle pathway of ().

Evolutionary coupling saturation mutagenesis: Coevolution-guided identification of distant sites influencing Bacillus naganoensis pullulanase activity.

Pullulanases are well-known debranching enzymes hydrolyzing α-1,6-glycosidic linkages. To date, engineering of pullulanase is mainly focused on catalytic pocket or domain tailoring based on structure/sequence information. Saturation mutagenesis-involved directed evolution is, however, limited by the low number of mutational sites compatible with combinatorial libraries of feasible size.

Highly selective synthesis of d-amino acids from readily available l-amino acids by a one-pot biocatalytic stereoinversion cascade.

d-Amino acids are key intermediates required for the synthesis of important pharmaceuticals. However, establishing a universal enzymatic method for the general synthesis of d-amino acids from cheap and readily available precursors with few by-products is challenging. In this study, we constructed and optimized a cascade enzymatic route involving l-amino acid deaminase and d-amino acid dehydrogenase for the biocatalytic stereoinversions of l-amino acids into d-amino acids.

Highly Regioselective and Stereoselective Hydroxylation of Free Amino Acids by a 2-Oxoglutarate-Dependent Dioxygenase from .

Hydroxyl amino acids have tremendous potential applications in food and pharmaceutical industries. However, available dioxygenases are limited for selective and efficient hydroxylation of free amino acids. Here, we identified a 2-oxoglutarate-dependent dioxygenase from by gene mining and characterized the encoded protein (PH1).