Showing papers by "Ya-Ping Xue published in 2022"

Development of an NAD(H)‐Driven Biocatalytic System for Asymmetric Synthesis of Chiral Amino Acids

Abstract: Chiral amino acids are extensively applied in the pharmaceutical, food, cosmetic, and agricultural industries. As a representative example, l-phosphinothricin (l-PPT, a chiral non-natural amino acid) is a broad-spectrum herbicide. An NAD(H)-driven biocatalytic system for the asymmetric synthesis of chiral amino acids (focused on l-PPT) with high efficiency and low cost is highly desired. The key for the development of such biocatalytic system is to obtain an NADH-dependent biocatalyst with high catalytic performance toward l-PPT pro-ketone PPO. Herein, an engineered glutamate dehydrogenase from Lysinibacillus composti (LcGluDH) with desired activity was obtained by gene mining and protein engineering. In silico analyses suggested that the volume of substrate-binding pocket was substantially enlarged from 330.5 Å3 to 409.6 Å3. The stability of LcGluDH was increased (Tm value increased from 47.3 °C to 65.3 °C) by introducing positively charged amino acids or aromatic amino acids at position 375. The desired biocatalytic system was constructed by coupling the engineered LcGluDH and an NAD+-dependent FDH. Through this biocatalytic system, the batch production of l-PPT exhibited high space-time yield (207.3 g ⋅ L−1 ⋅ day−1) with strict stereoselectivity (ee of l-PPT>99%). Furthermore, eight other chiral amino acids were synthesised by the developed NAD(H)-driven biocatalytic system with high ee values.

[Formate dehydrogenase and its application in biomanufacturing of chiral chemicals].

Abstract: The redox biosynthesis system has important applications in green biomanufacturing of chiral compounds. Formate dehydrogenase (FDH) catalyzes the oxidation of formate into carbon dioxide, which is associated with the reduction of NAD(P)+ into NAD(P)H. Due to this property, FDH is used as a crucial enzyme in the redox biosynthesis system for cofactor regeneration. Nevertheless, the application of natural FDH in industrial production is hampered by low catalytic efficiency, poor stability, and inefficient coenzyme utilization. This review summarized the structural characteristics and catalytic mechanism of FDH, as well as the advances in protein engineering of FDHs toward improved enzyme activity, catalytic efficiency, stability and coenzyme preference. The applications of using FDH as a coenzyme regeneration system for green biomanufacturing of chiral compounds were summarized.

Potential of the Signal Peptide Derived from the PAS_chr3_0030 Gene Product for Secretory Expression of Valuable Enzymes in Pichia pastoris

Abstract: Although P. pastoris is widely used for the secretory production of pharmaceutical proteins, its successful applications in the secretory production of industrial enzymes are limited. The α-mating factor pre-pro leader is the most widely used secretion signal in P. pastoris, but numerous industrial enzymes cannot be secreted using it. ABSTRACT Pichia pastoris is widely used for the production of valuable recombinant proteins. An advantage of P. pastoris over other expression systems is that it secretes low levels of endogenous proteins, which facilitates the purification processes if the desired recombinant proteins are efficiently secreted into the culture medium. However, not all recombinant proteins can be successfully secreted by P. pastoris, especially enzymes that are located in intracellular compartments in their native hosts. Few studies have reported strategies for releasing recombinant proteins which cannot be secreted by standard protocols. Here, we investigated whether this challenge can be addressed using novel secretion leaders. Analysis of the secretome and transcriptome of P. pastoris indicated that the four genes with the highest protein-to-transcript ratios were EPX1, PAS_chr3_0030, SCW10, and UTH1, suggesting that their gene products contain efficient secretion leaders. Our data revealed that the signal peptide derived from the PAS_chr3_0030 gene product conferred secretion competence to certain industrial enzymes, e.g., a nitrilase of Alcaligenes faecalis ZJUTB10, a ribosylnicotinamide kinase of P. pastoris, and a glucose dehydrogenase of Exiguobacterium sibiricum. Therefore, the signal peptide derived from the PAS_chr3_0030 gene product represents a novel secretion sequence for the secretory expression of recombinant enzymes in P. pastoris. IMPORTANCE Although P. pastoris is widely used for the secretory production of pharmaceutical proteins, its successful applications in the secretory production of industrial enzymes are limited. The α-mating factor pre-pro leader is the most widely used secretion signal in P. pastoris, but numerous industrial enzymes cannot be secreted using it. The importance of this study is that we identified a signal peptide derived from the PAS_chr3_0030 gene product which conferred secretion competence to three-quarters of the enzymes tested. This signal peptide derived from the PAS_chr3_0030 gene product may facilitate the application of P. pastoris in industrial biocatalysis.