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Open AccessFeature PaperArticle

Efficient Production of (R)-3-Aminobutyric Acid by Biotransformation of Recombinant E. coli

by Hongtao Zhang, Qing Xu, Jiajia Lv, Jiaxing Zhang, Tongyi Dou, Shengping You, Rongxin Su and Wei Qi

Catalysts 2025, 15(5), 466; https://doi.org/10.3390/catal15050466 - 9 May 2025

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Abstract

(R)-3-aminobutyric acid is an important raw material for dolutegravir production, which is a key antiretroviral medicine for AIDS treatment. Currently, the industrial production of (R)-3-aminobutyric acid relies on chiral resolution methods, which are plagued by high pollution and low yield efficiency. Here, we report an efficient pathway for (R)-3-aminobutyric acid production via engineered aspartase-driven biotransformation in recombinant E. coli. The engineered aspartase mutants, obtained through rational design based on catalytic mechanisms, were specifically employed to catalyze the production of (R)-3-aminobutyric acid from crotonic acid. The engineered T187L/N142R/N326L aspartase mutant exhibited the highest enzyme activity of 1516 U/mg. Through cell permeabilization, the system achieved (R)-3-aminobutyric acid yield of 287.6 g/L (96% productivity) within 24 h. Subsequent scale-up in a 7 L fermenter achieved a final product yield of 284 g/L (95% productivity) within 24 h. Economic balance showed that the cost of industrial production (¥116.21/kg) is about 1/4 of the laboratory production (¥479.76/kg). In summary, the engineered aspartase-mediated bioconversion pathway using recombinant E. coli offers an industrially viable approach for (R)-3-aminobutyric acid production, featuring mild reaction conditions, environmental sustainability, streamlined processing, high yield, and cost-effective substrates. Full article

(This article belongs to the Special Issue Biocatalysis—Enzymes in Industrial Applications)

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14 pages, 3119 KB

Open AccessArticle

Cloning, Characterization and Functional Analysis of Caspase 8-like Gene in Apoptosis of Crassostrea hongkongensis Response to Hyper-Salinity Stress

by Jinji Lin, Ziqi Yu, Yang Leng, Jiexiong Zhu, Feifei Yu, Yishan Lu, Jiayu Chen, Wenhao He, Yixin Zhang and Yaoshen Wen

Fishes 2024, 9(5), 172; https://doi.org/10.3390/fishes9050172 - 9 May 2024

Cited by 8 | Viewed by 1951

Abstract

Caspase-8, a member of the caspase family, is an initiating caspase and plays a crucial role in apoptosis. In this study, the full-length cDNA of caspase8-like (CASP8-like) was isolated from Crassostrea hongkongensis (C. hongkongensis) by RACE-PCR. ChCASP8-like contained a 1599-bp open reading frame (ORF) encoding 533 amino acids with two conserved death effector domains (DEDs) and a cysteine aspartase cysteine structural domain (CASc). Amino acid sequence comparison showed that ChCASP8-like shared the highest identity (85.4%) with CASP8-like of C. angulata. The tissue expression profile showed that ChCASP8-like was constitutively expressed in gills, hepatopancreas, mantle, adductor muscle, hemocytes and gonads, and was significantly upregulated in hemocytes, hepatopancreas and gills under hyper-salinity stress. The apoptosis-related genes, including ATR, CHK1, BCL-XL, CASP8-like, CASP9 and CASP3, were significantly activated by hyper-salinity stress, but were remarkably inhibited by ChCASP8-like silencing. The caspase 8 activity was increased by 1.7-fold after hyper-salinity stress, and was inhibited by 9.4% by ChCASP8-like silencing. Moreover, ChCASP8-like silencing clearly alleviated the apoptosis resulting from hyper-salinity stress. These results collectively demonstrated that ChCASP8-like played a crucial role in inducing apoptosis against hyper-salinity stress. Full article

(This article belongs to the Section Aquatic Invertebrates)

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11 pages, 1425 KB

Open AccessArticle

One-Pot Synthesis of β-Alanine from Maleic Acid via Three-Enzyme Cascade Biotransformation

by Jia Wu, Bao-Di Ma and Yi Xu

Catalysts 2023, 13(2), 267; https://doi.org/10.3390/catal13020267 - 24 Jan 2023

Cited by 8 | Viewed by 2763

Abstract

A novel and efficient one-pot three-enzyme cascade method for the synthesis of β-alanine from maleic acid was developed. Two recombinant E. coli strains were constructed. The E. coli (MaiA-AspA) co-expressing maleic cis-trans isomerase (MaiA) and L-aspartase (AspA) catalyzed the biotransformation of maleic acid to L-aspartate via fumaric acid, and E. coli (ADC) expressing L-aspartate-α-decarboxylase (ADC) catalyzed the bioconversion of L-aspartate to β-alanine. After systematic optimization of reaction conditions for each strain, the whole cells of two strains were combined for one-pot synthesis of β-alanine. It was found that the ratio of the two kinds of cells as well as the cell amount play critical roles in the reaction rate and yield of β-alanine. Adding two kinds of cells in one-pot at the beginning of the reaction was better than adding step by step. Under optimal conditions, the concentration of β-alanine reached 751 mM after a 9 h reaction, corresponding to a 93.9% yield and 178 g/L/d space-time yield. The developed new route showed application potential for green and efficient biosynthesis of β-alanine from a cheap substrate by tandem biocatalysts. Full article

(This article belongs to the Special Issue Current State-of-the-Art of Biocatalysts)

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12 pages, 2224 KB

Open AccessArticle

Identification and Molecular Characterization of the Operon Required for L-Asparagine Utilization in Corynebacterium glutamicum

by Koichi Toyoda, Riki Sugaya, Akihiro Domon, Masako Suda, Kazumi Hiraga and Masayuki Inui

Microorganisms 2022, 10(5), 1002; https://doi.org/10.3390/microorganisms10051002 - 10 May 2022

Cited by 1 | Viewed by 3923

Abstract

Understanding the metabolic pathways of amino acids and their regulation is important for the rational metabolic engineering of amino acid production. The catabolic pathways of L-asparagine and L-aspartate are composed of transporters for amino acid uptake and asparaginase and aspartase, which are involved in the sequential deamination to fumarate. However, knowledge of the catabolic genes for asparagine in bacteria of the Actinobacteria class has been limited. In this study, we identified and characterized the ans operon required for L-Asn catabolism in Corynebacterium glutamicum R. The operon consisted of genes encoding a transcriptional regulator (AnsR), asparaginase (AnsA2), aspartase (AspA2), and permease (AnsP). The enzymes and permease encoded in the operon were shown to be essential for L-Asn utilization, but another asparaginase, AnsA1, and aspartase, AspA1, were not essential. Expression analysis revealed that the operon was induced in response to extracellular L-Asn and was transcribed as a leaderless mRNA. The DNA-binding assay demonstrated that AnsR acted as a transcriptional repressor of the operon by binding to the inverted repeat at its 5′-end region. The AnsR binding was inhibited by L-Asn. This study provides insights into the functions and regulatory mechanisms of similar operon-like clusters in related bacteria. Full article

(This article belongs to the Special Issue Microbial Cell Factories: Production of Amino Acids Using Microorganisms)

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23 pages, 6711 KB

Open AccessArticle

Rice Husk as an Inexpensive Renewable Immobilization Carrier for Biocatalysts Employed in the Food, Cosmetic and Polymer Sectors

by Marco Cespugli, Simone Lotteria, Luciano Navarini, Valentina Lonzarich, Lorenzo Del Terra, Francesca Vita, Marina Zweyer, Giovanna Baldini, Valerio Ferrario, Cynthia Ebert and Lucia Gardossi

Catalysts 2018, 8(10), 471; https://doi.org/10.3390/catal8100471 - 19 Oct 2018

Cited by 42 | Viewed by 6935

Abstract

The high cost and environmental impact of fossil-based organic carriers represent a critical bottleneck to their use in large-scale industrial processes. The present study demonstrates the applicability of rice husk as inexpensive renewable carrier for the immobilization of enzymes applicable sectors where the covalent anchorage of the protein is a pre-requisite for preventing protein contamination while assuring the recyclability. Rice husk was oxidized and then functionalized with a di-amino spacer. The morphological characterization shed light on the properties that affect the functionalization processes. Lipase B from Candida antarctica (CaLB) and two commercial asparaginases were immobilized covalently achieving higher immobilization yield than previously reported. All enzymes were immobilized also on commercial epoxy methacrylic resins and the CaLB immobilized on rice husk demonstrated a higher efficiency in the solvent-free polycondensation of dimethylitaconate. CaLB on rice husk appears particularly suitable for applications in highly viscous processes because of the unusual combination of its low density and remarkable mechanical robustness. In the case of the two asparaginases, the biocatalyst immobilized on rice husk performed in aqueous solution at least as efficiently as the enzyme immobilized on methacrylic resins, although the rice husk loaded a lower amount of protein. Full article

(This article belongs to the Special Issue Immobilization of Enzymes)

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