APPLIED BIOCHEMISTRY AND BIOTECHNOLOGY
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2016
·Vol. 182
·期 3
·898-909
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IF 1.606
Xu Jian-Miao,Cheng Feng,Fu Fang-Tian,Hu Hai-Feng,Zheng Yu-Guo
L -2-aminobutyric acid ( L -ABA) as a precursor for the anticonvulsant and the antituberculotic is a key intermediate in the chemical and pharmaceutical industries. Recently, leucine dehydrogenase (LeuDH) with NAD + regeneration was developed for L -ABA production on a large scale. Previously, the L -ABA yield was improved by optimizing conversion conditions, including cofactor regeneration and enzyme immobilization but not protein engineering on LeuDH due to lacking an applicable high-throughput screening (HTS) method. Recently, an HTS assay was developed by us, which enables researchers to engineer LeuDH in a relatively short period of time. Herein, a semirational engineering was performed on LeuDH to increase the catalytic efficiency of BcLeuDH. Firstly, the structure of wild-type (WT) BcLeuDH was modeled and seven potentially beneficial positions were selected for mutation. Five beneficial variants were then identified from the seven site-saturation mutagenesis (SSM) libraries by HTS and confirmed by rescreening via amino acid analyzer. The “best” variant M5 (WT + Q358N) showed 44.5-fold higher catalytic efficiency ( k
cat / K
M ) than BcLeuDH WT, which suggested that BcLeuDH M5 is an attractive candidate for L -ABA production on a large scale. Furthermore, the structure-functional relationship was investigated based on the docking and kinetic results.
研究领域:
生物技术
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生物化工
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酶学
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药学
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DOI
PubMed
高影响因子
CATALYSIS LETTERS
·
2017
·Vol. 147
·期 4
·837-844
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IF 2.799
An Zhengfang,Gu Xiaoxu,Liu Yue,Ge Jingyan,Zhu Qing
Abstract
l -2-Aminobutyric acid ( l -ABA), an unnatural amino acid, is a key intermediate of several important drugs. Although some methods have been developed to prepare pure chiral l -ABA, there are still many drawbacks, including low catalytic efficiency, cumbersome steps and high cost due to the addition of some expensive catalysts or coenzymes. Herein, with chemical and biological approaches together, we discovered a newly isolated Aspergillus tamarii ZJUT ZQ013 strain containing a microbial lipase which could be employed to resolve racemic methyl N -Boc-2-aminobutyrate to produce l -ABA with high enantioselectivity (e.e. s > 99.9%, E = 257). Moreover, the subsequent gram scale experiment confrimed that A. tamarii ZJUT ZQ013 could be an attractive biocatalyst for the efficient preparation of optically pure acid. Graphical Abstract
研究领域:
生物化学
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制药工程
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微生物学
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DOI
高影响因子
Microbial Cell Factories
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2019
·Vol. 18
·期 1
·1-10
·
IF 4.402
Xu Jian-Miao,Li Jian-Qiang,Zhang Bo,Liu Zhi-Qiang,Zheng Yu-Guo
Background l -2-aminobutyric acid ( l -ABA) is an unnatural amino acid that is a key intermediate for the synthesis of several important pharmaceuticals. To make the biosynthesis of l -ABA environmental friendly and more suitable for the industrial-scale production. We expand the nature metabolic network of Escherichia coli using metabolic engineering approach for the production of l -ABA. Results In this study, Escherichia coli THR strain with a modified pathway for threonine-hyperproduction was engineered via deletion of the rhtA gene from the chromosome. To redirect carbon flux from 2-ketobutyrate (2-KB) to l -ABA, the ilvIH gene was deleted to block the l -isoleucine pathway. Furthermore, the ilvA gene from Escherichia coli W3110 and the leuDH gene from Thermoactinomyces intermedius were amplified and co-overexpressed. The promoter was altered to regulate the expression strength of ilvA* and leuDH . The final engineered strain E. coli THR Δ rhtA Δ ilvIH /Gap- ilvA *-Pbs- leuDH was able to produce 9.33 g/L of l -ABA with a yield of 0.19 g/L/h by fed-batch fermentation in a 5 L bioreactor. Conclusions This novel metabolically tailored strain offers a promising approach to fulfill industrial requirements for production of l -ABA.
研究领域:
生物技术
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代谢工程
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微生物生物技术
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DOI
PubMed
高影响因子
BMC BIOTECHNOLOGY
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2021
·Vol. 21
·期 1
·1-9
·
IF 2.563
Zhang Zhiwei,Liu Yang,Zhao Jing,Li Wenqiang,Hu Ruiwen,Li Xia,Li Aitao,Wang Yaping,Ma Lixin
Background The unnatural amino acid, L-2-aminobutyric acid (L-ABA) is an essential chiral building block for various pharmaceutical drugs, such as the antiepileptic drug levetiracetam and the antituberculosis drug ethambutol. The present study aims at obtaining variants of ω-transaminase from Ochrobactrum anthropi (OATA) with high catalytic activity to α-ketobutyric acid through protein engineering.Results Based on the docking model using α-ketobutyric acid as the ligand, 6 amino acid residues, consisting of Y20, L57, W58, G229, A230 and M419, were chosen for saturation mutagenesis. The results indicated that L57C, M419I, and A230S substitutions demonstrated the highest elevation of enzymatic activity among 114 variants. Subsequently, double substitutions combining L57C and M419I caused a further increase of the catalytic efficiency to 3.2-fold. This variant was applied for threonine deaminase/OATA coupled reaction in a 50-mL reaction system with 300 mM L-threonine as the substrate. The reaction was finished in 12 h and the conversion efficiency of L-threonine into L-ABA was 94%. The purity of L-ABA is 75%, > 99% ee. The yield of L-ABA was 1.15 g.Conclusion This study provides a basis for further engineering of ω-transaminase for producing chiral amines from keto acids substrates.
研究领域:
生物化学
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蛋白质工程
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酶学
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PubMed
高影响因子
Drug Metabolism and Pharmacokinetics
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2019
·
IF 1.874
Yang Zhang,Chen Su,Jinxiu Lei,Lu Chen,Haihong Hu,Su Zeng,Lushan Yu
Mandelic acid (MA) is generally used as a biomarker of the exposure of styrene , which is classified as a class of hazardous environmental pollutants, and also used as an important chiral intermediate in pharmaceutical industry. The previous studies have found the excretion of phenylglyoxylic acid (PGA) in human and rat, a metabolite of MA, was mainly from S- MA rather than R -MA. The metabolic mechanism, however, is not clear. In order to explore the possible metabolic mechanism, the enzyme types involved in the stereoselectivity metabolism of MA were firstly studied, and then human and rat long-chain 2-hydroxy-acid oxidase 2 (HAO2) were recombinantly expressed to study the metabolic profiles of S- MA and its analogues. The results indicated that HAO2 might catalyze the stereoselectivity metabolism of S- MA in rats. Human HAO2 ( h HAO2) and rat HAO2 ( r HAO2) isozymes β 1 and β 2 were successfully cloned and expressed with high purity and good enzyme activities. The enzyme kinetic profiles of these enzymes were different for S- MA and analogues. The order of catalytic efficiency for h HAO2 and r HAO2, however, was reverse. It might be relevance to the difference in active amino acid residues and loop 4 in human and rat L-2-hydroxy acid oxidase isozyme B crystal structures.
研究领域:
环境科学
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生物化学
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毒理学
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药物化学
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PubMed
高影响因子
BIORESOURCE TECHNOLOGY
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2021
·
IF 9.642
Jiajie Chen,Rongshuai Zhu,Junping Zhou,Taowei Yang,Xian Zhang,Meijuan Xu,Zhiming Rao
Leucine dehydrogenase (LDH) is widely used in the preparation of L-2-aminobutyric acid (L-2-ABA), however its wide application is limited by 2-ketobutyric acid (2-OBA) inhibition. Firstly, a novel high-throughput screening method of LDH was established, specific enzyme activity and 2-OBA tolerance of Lys72Ala mutant were 33.3% higher than those of the wild type. Subsequently, we constructed a single cell comprised of ivlA , Esldh K72A , fdh and optimized expression through fine-tuning RBS intensity, so that the yield of E. coli BL21/pET28a-R3 ivlA-Esldh K72A -fdh was 2.6 times higher than that of the original strain. As a result, 150 g L-threonine was transformed to 121 g L-2-ABA in 5 L fermenter with 95% molar conversion rate, and a productivity of 5.04 g·L -1 ·h −1 , which is the highest productivity of L-2-ABA currently reported by single-cell biotransformation. In summary, our research provided a green synthesis for L-2-ABA, which has potential for industrial production of drug precursors.
研究领域:
生物技术
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生物化学
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微生物学
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PubMed
高影响因子
Molecular Catalysis
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2021
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IF 5.062
Wei Luo,Jiagui Hu,Jiapeng Lu,Huili Zhang,Xiaoping Wang,Yuantao Liu,Liqing Dong,Xiaobin Yu
ω-transaminase can mediate the asymmetric synthesis of chiral amines from aldehydes and ketones , which has important value in the synthesis of pharmaceutical intermediates. A novel ω-transaminase derived from Paracoccus pantotrophus ( Pp TA) was obtained and cloned in E. coli BL21(DE3) for expression. The enzyme has high activity for 2-ketobutyric acid and benzylamine , as well as for aromatic compounds with side chains longer than ethyl aliphatic hydrocarbons . Molecular simulation showed that the S-pocket in the active center is larger than those of other ω-transaminases. Thereafter, a whole-cell catalytic system was designed to prepare l -2-aminobutyric acid by cascading Pp TA and other enzymes. By using several strategies (regulation of RBS intensity, by-product decomposition and cofactor self-sufficiency), whole-cell cascade biocatalysis showed a high ee value (> 99%) and high yield (71%) in one pot reaction . This study therefore proposes an efficient biocatalyst for the synthesis of unnatural amino acids with the participation of ω-transaminase.
研究领域:
生物催化
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酶工程
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药物化学
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分子生物学
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DOI
高影响因子
ENZYME AND MICROBIAL TECHNOLOGY
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2024
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IF 3.4
Lili Zhang,Yu Hong,Jiapeng Lu,Yi Wang,Wei Luo
Transaminases (EC 2.6.1.X, TAs) are important biocatalysts in the synthesis of chiral amines, and have significant value in the field of medicine. However, TAs suffer from low enzyme activity and poor catalytic efficiency in the synthesis of chiral amines or non-natural amino acids, which hinders their industrial applications. In this study, a novel TA derived from Paracoccus pantotrophus ( pp TA) that was investigated in our previous study was employed with a semi-rational design strategy to improve its enzyme activity to 2-ketobutyrate. By using homology modeling and molecular docking, four surrounding sites in the substrate-binding S pocket were selected as potential mutational sites. Through alanine scanning and saturation mutagenesis, the optimal mutant V153A with significantly improved enzyme activity was finally obtained, which was 578 % higher than that of the wild-type pp TA (WT). Furthermore, the mutant enzyme pp TA-V153A also exhibited slightly improved temperature and pH stability compared to WT. Subsequently, the mutant was used to convert 2-ketobutyrate for the preparation of L-2-aminobutyric acid (L-ABA). The mutant can tolerate 300 mM 2-ketobutyrate with a conversion rate of 74 %, which lays a solid foundation for the preparation of chiral amines.
研究领域:
生物化学
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酶工程
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药物合成
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DOI
PubMed
高影响因子
CHEMBIOCHEM
·
2023
·
IF 3.2
Xin Li,Changzheng Gao,Wanqing Wei,Wei Song,Weiwei Meng,Jia Liu,Xiulai Chen,Cong Gao,Liang Guo,Liming Liu,Jing Wu
Graphical We developed a pathway for the synthesis of L-ABA in a tri-enzymatic cascade in Escherichia coli BL21 (DE3) using L-Thr, and the expression of three enzymes was optimized by vector design. We solved the crystal structure of the key enzyme LDH and proposed protein engineering strategies based on the structure to improve the catalytic activity of the enzyme. L-2-aminobutyrate (L-ABA) is an important chiral drug intermediate with a key role in modern medicinal chemistry. Here, we describe the development of an efficient method for the asymmetric synthesis of L-ABA in a tri-enzymatic cascade in Escherichia coli BL21 (DE3) using a cost-effective L-Thr. Low activity of leucine dehydrogenase from Bacillus thuringiensis ( Bt LDH) and unbalanced expression of enzymes in the cascade were major challenges. Mechanism-based protein engineering generated the optimal triple variant Bt LDH M3 (A262S/V296C/P150M) with 20.7-fold increased specific activity and 9.6-fold increased k cat / K m compared with the wild type. Optimizing plasmids with different copy numbers regulated enzymatic expression, thereby increasing the activity ratio (0.3 : 1:0.6) of these enzymes in vivo close to the optimal ratio (0.4 : 1 : 1) in vitro . Importing the optimal triple mutant Bt LDH M3 into our constructed pathway in vivo and optimization of transformation conditions achieved one-pot conversion of L-Thr to 130.2 g/L L-ABA, with 95 % conversion, 99 % e.e . and 10.9 g L −1 h −1 productivity (the highest to date) in 12 h on a 500 mL scale. These results describe a potential biosynthesis approach for the industrial production of L-ABA.
研究领域:
生物化学
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生物技术
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蛋白质工程
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酶学
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DOI
PubMed
高影响因子
MACROMOLECULES
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2017
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IF 5.835
Dongdong Zhou,Shaoyong Huang,Jingru Sun,Xinchao Bian,Gao Li,Xuesi Chen
The crystallization behaviors and microstructures of poly(l-2-hydroxyl-3-methylbutanoic acid)/poly(l-lactide) blends [P(L-2H3MB)/PLLA] were investigated by OM, DSC, SAXS, in situ temperature-dependent WAXD, and in situ synchrotron WAXS. The blends exhibited a homogeneous state at 250 °C. In the cooling process, P(L-2H3MB), with higher melting temperature, crystallized first at 166.7 °C and drove the formation of the phase separation and microstructure. And the amorphous P(L-2H3MB) and PLLA were excluded into the interlamellar and interfibrillar regions of the former P(L-2H3MB) crystallites. For P(L-2H3MB)/PLLA (5/5), the amorphous P(L-2H3MB) in the interfibrillar regions continued to form crystallites sequentially at 134.6 °C, which clearly confirmed the fractional crystallization of P(L-2H3MB). In our knowledge, this unique fractional crystallization has not been reported yet, of the component in miscible blend, which crystallized first under little confinement. This work provided a new viewpoint and understanding of the relationship between fractional crystallization and microstructures in crystalline/crystalline blends.
研究领域:
材料科学
·
高分子化学
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产品关联
DOI
高影响因子
INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES
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2024
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IF 7.7
Bingmei Su,Wen Yang,Yi Zhou,Juan Lin
Ectoine, so-called tetrahydropyrimidine, is an important osmotic adjustment solute and widely applied in cosmetics and protein protectant. Some attempts have been made to improve the ectoine productivity. However, the strains with both high ectoine production capacity and high glucose conversion were still absent so far. Aim to construct a strain for efficiently producing ectoine, ectoine synthetic gene cluster ectABC from Pseudomonas stutzeri was overexpressed in E. coli BL21 (DE3). The ection production was improved by 382 % (ectoine titer increased from 1.73 g/L to 8.33 g/L) after the rational design of rate-limiting enzyme L-2,4-diaminobutyrate transaminase EctB ps (protein engineering) combined with the metabolic engineering that focused on the enrichment and conversion of precursors. The final strain YW20 was applied to overproduce ectoine in fed-batch fermentation and yield 68.9 g/L of ectoine with 0.88 g/L/h of space-time yield and the highest glucose conversion reported [34 % (g/g)]. From the fermentation broth, ectoine was purified with 99.7 % purity and 79.8 % yield. This study successfully provided an engineered strain as well as an efficient method for the industrial bio-synthesis and preparation of ectoine.
研究领域:
生物技术
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微生物学
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生物化工
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产品关联
DOI
PubMed
高影响因子
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