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学者姓名：许炼
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Abstract ：
Norepinephrine, a kind of beta-adrenergic receptor agonist, is commonly used for treating shocks and hypotension caused by a variety of symptoms. The development of a straightforward, efficient and environmentally friendly biocatalytic route for manufacturing norepinephrine remains a challenge. Here, we designed and realized an artificial biocatalytic cascade to access norepinephrine starting from 3, 4-dihydroxybenzaldehyde and L-threonine mediated by a tailored-made L-threonine transaldolase PsLTTA-Mu1 and a newly screened tyrosine decarboxylase ErTDC. To overcome the imbalance of multi-enzymes in a single cell, engineering of PsLTTA for improved activity and fine-tuning expression mode of multi-enzymes in single E.coli cells were combined, leading to a robust whole cell biocatalyst ES07 that could produce 100 mM norepinephrine with 99% conversion, delivering a highest time-space yield (3.38 g/L/h) ever reported. To summarized, the current study proposed an effective biocatalytic approach for the synthesis of norepinephrine from low-cost substrates, paving the way for industrial applications of enzymatic norepinephrine production.
Keyword ：
Biocatalytic cascade Biocatalytic cascade L-threonine transaldolase L-threonine transaldolase Tyrosine decarboxylase Tyrosine decarboxylase
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| GB/T 7714 | Xu, Lian , Shen, Jun-Jiang , Wu, Ming et al. An artificial biocatalytic cascade for efficient synthesis of norepinephrine by combination of engineered L-threonine transaldolase with multi-enzyme expression fine-tuning [J]. | INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES , 2024 , 265 . |
| MLA | Xu, Lian et al. "An artificial biocatalytic cascade for efficient synthesis of norepinephrine by combination of engineered L-threonine transaldolase with multi-enzyme expression fine-tuning" . | INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES 265 (2024) . |
| APA | Xu, Lian , Shen, Jun-Jiang , Wu, Ming , Su, Bing-Mei , Xu, Xin-Qi , Lin, Juan . An artificial biocatalytic cascade for efficient synthesis of norepinephrine by combination of engineered L-threonine transaldolase with multi-enzyme expression fine-tuning . | INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES , 2024 , 265 . |
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Hydroxytyrosol, a naturally occurring compound with antioxidant and antiviral activity, is widely applied in the cosmetic, food, and nutraceutical industries. The development of a biocatalytic approach for producing hydroxytyrosol from simple and readily accessible substrates remains a challenge. Here, we designed and implemented an effective biocatalytic cascade to obtain hydroxytyrosol from 3,4-dihydroxybenzaldehyde and l-threonine via a four-step enzymatic cascade composed of seven enzymes. To prevent cross-reactions and protein expression burden caused by multiple enzymes expressed in a single cell, the designed enzymatic cascade was divided into two modules and catalyzed in a stepwise manner. The first module (FM) assisted the assembly of 3,4-dihydroxybenzaldehyde and l-threonine into (2S,3R)-2-amino-3-(3,4-dihydroxyphenyl)-3-hydroxypropanoic acid, and the second module (SM) entailed converting (2S,3R)-2-amino-3-(3,4-dihydroxyphenyl)-3-hydroxypropanoic acid into hydroxytyrosol. Each module was cloned into Escherichia coli BL21 (DE3) and engineered in parallel by fine-tuning enzyme expression, resulting in two engineered whole-cell catalyst modules, BL21(FM01) and BL21(SM13), capable of converting 30 mM 3,4-dihydroxybenzaldehyde to 28.7 mM hydroxytyrosol with a high space-time yield (0.88 g/L/h). To summarize, the current study proposes a simple and effective approach for biosynthesizing hydroxytyrosol from low-cost substrates and thus has great potential for industrial applications.
Keyword ：
Biocatalytic cascade Biocatalytic cascade hydroxytyrosol hydroxytyrosol stepwisecatalysis stepwisecatalysis
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| GB/T 7714 | Liu, Wen-Kai , Su, Bing-Mei , Xu, Xin-Qi et al. Multienzymatic Cascade for Synthesis of Hydroxytyrosol via Two-Stage Biocatalysis [J]. | JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY , 2024 , 72 (27) : 15293-15300 . |
| MLA | Liu, Wen-Kai et al. "Multienzymatic Cascade for Synthesis of Hydroxytyrosol via Two-Stage Biocatalysis" . | JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY 72 . 27 (2024) : 15293-15300 . |
| APA | Liu, Wen-Kai , Su, Bing-Mei , Xu, Xin-Qi , Xu, Lian , Lin, Juan . Multienzymatic Cascade for Synthesis of Hydroxytyrosol via Two-Stage Biocatalysis . | JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY , 2024 , 72 (27) , 15293-15300 . |
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The synthesis of steroids is challenging through multistep steroidal core modifications with high site-selectivity and productivity. In this work, a novel enzymatic cascade system was constructed for synthesis of testolactone by specific C17 lactonization/Δ1-dehydrogenation from inexpensive androstenedione using an engineered polycyclic ketone monooxygenase (PockeMO) and an appropriate 3-ketosteroid-Δ1-dehydrogenase (ReKstD). The focused saturation mutagenesis in the substrate binding pocket was implemented for evolution of PockeMO to eliminate the bottleneck effect. A best mutant MU3 (I225L/L226V/L532Y) was obtained with 20-fold higher specific activity compared to PockeMO. The catalytic efficiency (kcat/Km) of MU3 was 171-fold higher and the substrate scope shifted to polycyclic ketones. Molecular dynamic simulations suggested that the activity was improved by stabilization of the pre-lactonization state and generation of productive orientation of 4-AD mediated by distal L532Y mutation. Based on that, the three genes, MU3, ReKstD and a ketoreductase for NADPH regeneration, were rationally integrated in one cell via expression fine-tuning to form the efficient single cell catalyst E. coli S9. The single whole-cell biocatalytic process was scaled up and could generate 9.0 g/L testolactone with the high space time yield of 1 g/L/h without steroidal by-product, indicating the potential for site-specific and one-pot synthesis of steroid. © 2024
Keyword ：
Biosynthesis Biosynthesis Cell engineering Cell engineering Cytology Cytology Escherichia coli Escherichia coli Gene expression Gene expression Ketones Ketones Molecular dynamics Molecular dynamics Molecular orientation Molecular orientation
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| GB/T 7714 | Xu, Xinqi , Zhong, Jinchang , Su, Bingmei et al. Single-cell enzymatic cascade synthesis of testolactone enabled by engineering of polycyclic ketone monooxygenase and multi-gene expression fine-tuning [J]. | International Journal of Biological Macromolecules , 2024 , 275 . |
| MLA | Xu, Xinqi et al. "Single-cell enzymatic cascade synthesis of testolactone enabled by engineering of polycyclic ketone monooxygenase and multi-gene expression fine-tuning" . | International Journal of Biological Macromolecules 275 (2024) . |
| APA | Xu, Xinqi , Zhong, Jinchang , Su, Bingmei , Xu, Lian , Hong, Xiaokun , Lin, Juan . Single-cell enzymatic cascade synthesis of testolactone enabled by engineering of polycyclic ketone monooxygenase and multi-gene expression fine-tuning . | International Journal of Biological Macromolecules , 2024 , 275 . |
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Dehydroepiandrosterone (DHEA) is an important neurosteroid hormone to keep human hormonal balance and reproductive health. However, DHEA was always produced with impurities either by chemical or biological method and required high-cost purification before the medical use. To address this issue, a novel chemo-enzymatic process was proposed and implemented to produce DHEA. An acetoxylated derivate of 4-androstene-3,17-dione (4-AD) was generated by chemical reaction and converted into DHEA by an enzyme cascade reaction combining a hydrolysis reaction with a reduction reaction. The hydrolysis reaction was catalyzed by a com-mercial esterase Z03 while the reduction reaction was catalyzed by E. coli cells co-expressing a 3 beta-hydroxysteroid dehydrogenase SfSDR and a glucose dehydrogenase BtGDH. After the condition optimization, DHEA was syn-thesized at a 100 mL scale under 100 mM of substrate loading and purified as white powder with the highest space-time yield (4.80 g/L/h) and purity (99 %) in the biosynthesis of DHEA. The successful attempt in this study provides a new approach for green synthesis of highly purified DHEA in the pharmaceutical industry.
Keyword ：
3?-hydroxysteroid dehydrogenase 3?-hydroxysteroid dehydrogenase Chemoenzymatic process Chemoenzymatic process Dehydroepiandrosterone Dehydroepiandrosterone Enzyme cascade Enzyme cascade Hydrolysis Hydrolysis
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| GB/T 7714 | Su, Bing-Mei , Shi, Yi-Bing , Lin, Wei et al. A chemoenzymatic process for preparation of highly purified dehydroepiandrosterone in high space-time yield [J]. | BIOORGANIC CHEMISTRY , 2023 , 133 . |
| MLA | Su, Bing-Mei et al. "A chemoenzymatic process for preparation of highly purified dehydroepiandrosterone in high space-time yield" . | BIOORGANIC CHEMISTRY 133 (2023) . |
| APA | Su, Bing-Mei , Shi, Yi-Bing , Lin, Wei , Xu, Lian , Xu, Xin-Qi , Lin, Juan . A chemoenzymatic process for preparation of highly purified dehydroepiandrosterone in high space-time yield . | BIOORGANIC CHEMISTRY , 2023 , 133 . |
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2'-deoxyguanosine is a key medicinal intermediate that could be used to synthesize anti-cancer drug and biomarker in type 2 diabetes. In this study, an enzymatic cascade using thymidine phosphorylase from Escherichia coli (EcTP) and purine nucleoside phosphorylase from Brevibacterium acetylicum (BaPNP) in a one-pot whole cell catalysis was proposed for the efficient synthesis of 2'-deoxyguanosine. BaPNP was semi-rationally designed to improve its activity, yielding the best triple variant BaPNP-Mu3 (E57A/T189S/L243I), with a 5.6-fold higher production of 2'-deoxyguanosine than that of wild-type BaPNP (BaPNP-Mu0). Molecular dynamics simulation revealed that the engineering of BaPNP-Mu3 resulted in a larger and more flexible substrate entrance channel, which might contribute to its catalytic efficiency. Furthermore, by coordinating the expression of BaPNP-Mu3 and EcTP, a robust whole cell catalyst W05 was created, capable of producing 14.8 mM 2'-deoxyguanosine (74.0% conversion rate) with a high time-space yield (1.32 g/L/h) and therefore being very competitive for industrial applications.
Keyword ：
2'-deoxyguanosine 2'-deoxyguanosine Enzymatic cascade Enzymatic cascade Semi-rational design Semi-rational design Whole cell catalysis Whole cell catalysis
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| GB/T 7714 | Xu, Lian , Li, Hui-Min , Lin, Juan . Efficient synthesis of 2'-deoxyguanosine in one-pot cascade by employing an engineered purine nucleoside phosphorylase from Brevibacterium acetylicum [J]. | WORLD JOURNAL OF MICROBIOLOGY & BIOTECHNOLOGY , 2023 , 39 (10) . |
| MLA | Xu, Lian et al. "Efficient synthesis of 2'-deoxyguanosine in one-pot cascade by employing an engineered purine nucleoside phosphorylase from Brevibacterium acetylicum" . | WORLD JOURNAL OF MICROBIOLOGY & BIOTECHNOLOGY 39 . 10 (2023) . |
| APA | Xu, Lian , Li, Hui-Min , Lin, Juan . Efficient synthesis of 2'-deoxyguanosine in one-pot cascade by employing an engineered purine nucleoside phosphorylase from Brevibacterium acetylicum . | WORLD JOURNAL OF MICROBIOLOGY & BIOTECHNOLOGY , 2023 , 39 (10) . |
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Laccase is an efficient biocatalyst for oxidative polymerization of organic substrates. However, cost of enzyme preparation, low stability and residual protein diminish the efficiency of laccase mediated polymerization. In this work, a series of silicon dioxide coated ferroferric oxide magnetic nanoparticles were modified by different functional groups including gamma-methacryloxypropyltrimethoxy, succinic anhydride, glutaraldehyde and poly-ethylene imine. Infrared spectra indicated the magnetic carriers have been successfully modified. Vibrating sample magnetometer (VSM) analysis revealed that all of these carriers showed high magnetic responsiveness after the surface functionalization. Laccase from Cerrena sp. HYB07 was then respectively immobilized covalently on these functionalized magnetic carriers. All the immobilized laccases displayed higher thermostability than free laccase and glutaraldehyde functionalized support (named FSNG) immobilized laccase showed better performance. These immobilized laccases all showed higher efficiency than free laccase for oxidative polymerization of catechol and hydroquinone. The immobilized laccases could be separated from the water insoluble polymerization products. The polymerization product of hydroquinone by FSNG immobilized laccase showed the average polymerization degree of the poly(hydroquinone) was six (DP=6). This work provided a comprehensive exploration of laccase immobilization on magnetic carrier for catalyzing polymerization of phenols.
Keyword ：
Laccase immobilization Laccase immobilization Magnetic nanoparticle Magnetic nanoparticle Phenol polymerization Phenol polymerization
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| GB/T 7714 | Xu, Xinqi , Chen, Tianheng , Xu, Lian et al. Immobilization of laccase on magnetic nanoparticles for enhanced polymerization of phenols [J]. | ENZYME AND MICROBIAL TECHNOLOGY , 2023 , 172 . |
| MLA | Xu, Xinqi et al. "Immobilization of laccase on magnetic nanoparticles for enhanced polymerization of phenols" . | ENZYME AND MICROBIAL TECHNOLOGY 172 (2023) . |
| APA | Xu, Xinqi , Chen, Tianheng , Xu, Lian , Lin, Juan . Immobilization of laccase on magnetic nanoparticles for enhanced polymerization of phenols . | ENZYME AND MICROBIAL TECHNOLOGY , 2023 , 172 . |
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本发明属于生物工程技术领域，具体涉及一种3β‑羟基甾体脱氢酶SfSDR及其在制备去氢表雄酮中的应用。本发明将3β‑羟基甾体脱氢酶SfSDR与葡萄糖脱氢酶BtGDH在大肠埃希氏菌中共表达，并以共表达工程菌的静息细胞作为生物催化剂协同酯酶Z03共同催化3‑乙酰氧基‑△3, 5‑雄甾二烯‑17‑酮合成去氢表雄酮。该生物催化剂具有较高的催化活性、区域选择性和立体选择性，可以在6 h以内完全转化32.8 g/L的3‑乙酰氧基‑△3, 5‑雄甾二烯‑17‑酮生成目标产物去氢表雄酮，无需添加有机溶剂且无副产物产生，经过分离纯化，产物回收率高达95%以上，说明该生物催化剂是去氢表雄酮绿色合成的高效催化剂。
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| GB/T 7714 | 林娟 , 苏冰梅 , 师艺冰 et al. 一种3β-羟基甾体脱氢酶及其在制备去氢表雄酮中的应用 : CN202211028698.9[P]. | 2022-08-25 00:00:00 . |
| MLA | 林娟 et al. "一种3β-羟基甾体脱氢酶及其在制备去氢表雄酮中的应用" : CN202211028698.9. | 2022-08-25 00:00:00 . |
| APA | 林娟 , 苏冰梅 , 师艺冰 , 许炼 , 许鑫琦 . 一种3β-羟基甾体脱氢酶及其在制备去氢表雄酮中的应用 : CN202211028698.9. | 2022-08-25 00:00:00 . |
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L-threo-4-methylsulfonylphenylserine ethyl ester [(2S, 3R)-1d], a key building block of florfenicol which synthesized by esterification using ethanol and L-threo-4-methylsulfonylphenylserine [(2S, 3R)-1b] as substrates. L-threonine aldolase (LTA) is a promising biocatalyst for producing (2S, 3R)-1b through a one-step process taking 4-Methylsulphonyl benzaldehyde (1a) and glycine as substrates under the mild condition. However, the moderate C-beta-stereoselective blocked the industrial application of LTA. To address this issue, rational design by combination with prereaction state molecular dynamics (MD) simulation and per-residue energy decomposition algorithm are employed to engineer LTA for enhancing C-beta-stereoselective. As a result, a triple mutant N16A/E98S/Y314R (Mu3) is screened out to produce (2S, 3R)-1b with 93.7% de and 90.2% conversion under the 300 mM 1a substrate loading. Furthermore, esterification is applied to synthesize (2S, 3R)-1d with over 99% chiral purity and 99% product purity. The success of this study provides new insights for the rational design of LTA with improved C-beta-stereoselectivity and proves a chemo-enzymatic route for green synthesis of (2S, 3R)-1d.
Keyword ：
C-beta-stereoselectivity C-beta-stereoselectivity Esterification Esterification L-Threonine Aldolase L-Threonine Aldolase Per-residue Energy Decomposition Per-residue Energy Decomposition Prereaction State Prereaction State
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| GB/T 7714 | Wang, Li-Chao , Xu, Lian , Su, Bing-Mei et al. An Effective Chemo-Enzymatic method with An Evolved L-Threonine Aldolase for Preparing L-threo-4-Methylsulfonylphenylserine Ethyl Ester of High Optical Purity [J]. | MOLECULAR CATALYSIS , 2022 , 525 . |
| MLA | Wang, Li-Chao et al. "An Effective Chemo-Enzymatic method with An Evolved L-Threonine Aldolase for Preparing L-threo-4-Methylsulfonylphenylserine Ethyl Ester of High Optical Purity" . | MOLECULAR CATALYSIS 525 (2022) . |
| APA | Wang, Li-Chao , Xu, Lian , Su, Bing-Mei , Xu, Xin-Qi , Lin, Juan . An Effective Chemo-Enzymatic method with An Evolved L-Threonine Aldolase for Preparing L-threo-4-Methylsulfonylphenylserine Ethyl Ester of High Optical Purity . | MOLECULAR CATALYSIS , 2022 , 525 . |
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Baeyer-Villiger monooxygenase (BVMO) mediated sulfoxidation is a sustainable approach for the synthesis of esomeprazole. In this work, a novel phenylacetone monooxygenase from Limnobacter sp. (LnPAMO) was found to have trace activity for synthesis of enantiopure esomeprazole. Through engineering in the substrate tunnel using a mutagenesis strategy called "nonpolarity paving" and some modifications in cofactor binding domains, a mutant harboring 15 mutations (LnPAMO Mu15) was obtained with 6.6 x 103-fold higher activity to convert omeprazole sulfide into esomeprazole. The activities of the mutant for synthesis of (S)-methyl phenyl sulfoxide and (S)-pantoprazole also increased much, indicating the versatility of the mutant for sulfoxide synthesis. Importantly, no over-oxidation byproduct omeprazole sulfone was detected in the sulfoxidation products by both mass spectrometry and HPLC analysis. Then NADP-dependent Burkholderia stabili formate dehydrogenase was ligated behind Mu15 along with a ribosome binding site sequence in pET-28a for co-expression. By single wholecell of recombinant Escherichia coli BL21 coexpressing Mu15 and formate dehydrogenase, omeprazole sulfide was efficiently converted into esomeprazole without production of sulfone (16 g/L substrate, enantiomeric excess > 99.9% (S) and > 99% conversion) and the space-time-yield reached 1.67 g product/L/h.
Keyword ：
Esomeprazole Esomeprazole Phenylacetone monooxygenase Phenylacetone monooxygenase Single -cell sulfoxidation Single -cell sulfoxidation Substrate tunnel Substrate tunnel
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| GB/T 7714 | Xu, Xinqi , Zhang, Yajiao , Wang, Shaoyu et al. ?Nonpolarity paving? in substrate tunnel of a Limnobacter sp. Phenylacetone monooxygenase for efficient single whole-cell synthesis of esomeprazole [J]. | BIOORGANIC CHEMISTRY , 2022 , 125 . |
| MLA | Xu, Xinqi et al. "?Nonpolarity paving? in substrate tunnel of a Limnobacter sp. Phenylacetone monooxygenase for efficient single whole-cell synthesis of esomeprazole" . | BIOORGANIC CHEMISTRY 125 (2022) . |
| APA | Xu, Xinqi , Zhang, Yajiao , Wang, Shaoyu , Xu, Lian , Su, Bingmei , Wang, Lichao et al. ?Nonpolarity paving? in substrate tunnel of a Limnobacter sp. Phenylacetone monooxygenase for efficient single whole-cell synthesis of esomeprazole . | BIOORGANIC CHEMISTRY , 2022 , 125 . |
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The synthesis of chloramphenicol, a kind of amphenicol antibiotic with broad-spectrum antibacterial activity, is challenging due to its stereochemistry. Here, we have designed a four-step chemoenzymatic strategy, including a biocatalytic step mediated by L-threonine transaldolase from Pseudomonas sp. (PsLTTA) to convert 4-nitrobenzaldehyde (1) to (25,3R)-2-amino-3-hydroxy-3-(4-nitrophenyl)propanoic acid (2) followed by a three-step chemical reaction to obtain chloramphenicol. A rational design of PsLTTA was devised by reshaping the substrate binding pocket and substrate access channel, resulting in the best variant PsLTTA-N35A/C57I/F59A/H69F (PsLTTA-Mu9), which achieved a 7.1-fold higher yield of 2 than wild-type PsLTTA. After coupling with ScADH/CbFDH to remove the byproduct acetaldehyde and optimizing the reaction conditions, the whole-cell catalyst BL21(PsLTTA-Mu9/ScADH/CbFDH) could synthesize 200 mM of 2 in four hours with 99% conversion and 97.7% de, delivering the highest time-space yield (11.3 g L-1 h(-1)) ever reported. Finally, the chemoenzymatic approach was applied for the gram-scale synthesis of 5 with a high overall yield (54%). The success of this strategy demonstrates the great advantage of the chemoenzymatic approach in the asymmetric synthesis of chloramphenicol and may contribute to its industrial synthesis.
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| GB/T 7714 | Xu, Lian , Nie, Dan , Su, Bing-Mei et al. A chemoenzymatic strategy for the efficient synthesis of amphenicol antibiotic chloramphenicol mediated by an engineered L-threonine transaldolase with high activity and stereoselectivity [J]. | CATALYSIS SCIENCE & TECHNOLOGY , 2022 , 13 (3) : 684-693 . |
| MLA | Xu, Lian et al. "A chemoenzymatic strategy for the efficient synthesis of amphenicol antibiotic chloramphenicol mediated by an engineered L-threonine transaldolase with high activity and stereoselectivity" . | CATALYSIS SCIENCE & TECHNOLOGY 13 . 3 (2022) : 684-693 . |
| APA | Xu, Lian , Nie, Dan , Su, Bing-Mei , Xu, Xin-Qi , Lin, Juan . A chemoenzymatic strategy for the efficient synthesis of amphenicol antibiotic chloramphenicol mediated by an engineered L-threonine transaldolase with high activity and stereoselectivity . | CATALYSIS SCIENCE & TECHNOLOGY , 2022 , 13 (3) , 684-693 . |
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