JOURNAL OF BIOTECHNOLOGY
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2019
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IF 3.163
Fei Peng,Ying Zhao,Fang-Zhou Li,Xiao-Yang Ou,Ying-Jie Zeng,Min-Hua Zong,Wen-Yong Lou
The asymmetric resolution of racemic 1-phenyl-1,2-ethanediol (PED) to ( S )-PED by Kurthia gibsonii SC0312 ( K. gibsonii SC0312) was conducted in a biphasic system comprised of an organic solvent and aqueous phosphate buffer. The impacts of organic solvents on the whole cell catalytic activity, metabolic activity, membrane integrity, and material distribution were first evaluated. The results showed that all organic solvents, except for dibutyl phthalate, showed a detrimental effect on the metabolic activity of the cells, especially for those with low log P values. All organic solvents were capable of changing the membrane permeability and membrane integrity of the cells. Moreover, some organic solvents showed a good extraction of the oxidation product. Finally, a high yield of 47.7 % of ( S )-PED was obtained by the asymmetric resolution of racemic PED using K. gibsonii SC0312 in a biphasic system under the optimal conditions: racemic PED 120 mM, temperature 35 °C, reaction time 6 h, 180 rpm, and a volume ratio of dibutyl phthalate to aqueous phosphate buffer of 1:1. The optical purity of ( S )-PED increased from 51.3 % to >99 %. This work described an efficient approach to improve reaction efficiency, and constructed a highly effective biphasic reaction system for the fabrication of ( S )-PED via K. gibsonii SC0312.
研究领域:
生物化学
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微生物学
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有机合成
✓ 已验证
产品关联
DOI
PubMed
高影响因子
Frontiers in Bioengineering and Biotechnology
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2020
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IF 3.644
Peng Fei,Su Hui-Hui,Ou Xiao-Yang,Ni Zi-Fu,Zong Min-Hua,Lou Wen-Yong
(R)-1-phenyl-1,2-ethanediol is an important synthon for the preparation of β-adrenergic blocking agents. This study identified a (2R,3R)-butanediol dehydrogenase (KgBDH) from Kurthia gibsonii SC0312, which showed high enantioselectivity for production of (R)-1-phenyl-1,2-ethanediol by reduction of 2-hydroxyacetophenone. KgBDH was expressed in a recombinant engineered strain, purified, and characterized. It showed good catalytic activity at pH 6–8 and better stability in alkaline (pH 7.5–8) than an acidic environment (pH 6.0–7.0), providing approximately 73 and 88% of residual activity after 96 h at pH 7.5 and 8.0, respectively. The maximum catalytic activity was obtained at 45°C; nevertheless, poor thermal stability was observed at >30°C. Additionally, the examined metal ions did not activate the catalytic activity of KgBDH. A recombinant Escherichia coli strain coexpressing KgBDH and glucose dehydrogenase (GHD) was constructed and immobilized via entrapment with a mixture of activated carbon and calcium alginate via entrapment. The immobilized cells had 1.8-fold higher catalytic activity than that of cells immobilized by calcium alginate alone. The maximum catalytic activity of the immobilized cells was achieved at pH 7.5, and favorable pH stability was observed at pH 6.0–9.0. Moreover, the immobilized cells showed favorable thermal stability at 25–30°C and better operational stability than free cells, retaining approximately 55% of the initial catalytic activity after four cycles. Finally, 81% yields (195 mM product) and >99% enantiomeric excess (ee) of (R)-1-phenyl-1,2-ethanediol were produced within 12 h through a fed-batch strategy with the immobilized cells (25 mg/ml wet cells) at 35°C and 180 rpm, with a productivity of approximately 54 g/L per day.
研究领域:
生物化学
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酶工程
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合成生物学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
Frontiers in Bioengineering and Biotechnology
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2021
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IF 5.89
Yang Chen,Liu Yushi,Liu Wan-Qiu,Wu Changzhu,Li Jian
Cell-free systems have been used to synthesize chemicals by reconstitution of in vitro expressed enzymes. However, coexpression of multiple enzymes to reconstitute long enzymatic pathways is often problematic due to resource limitation/competition (e.g., energy) in the one-pot cell-free reactions. To address this limitation, here we aim to design a modular, cell-free platform to construct long biosynthetic pathways for tunable synthesis of value-added aromatic compounds, using (S)-1-phenyl-1,2-ethanediol ((S)-PED) and 2-phenylethanol (2-PE) as models. Initially, all enzymes involved in the biosynthetic pathways were individually expressed by an E. coli-based cell-free protein synthesis (CFPS) system and their catalytic activities were confirmed. Then, three sets of enzymes were coexpressed in three cell-free modules and each with the ability to complete a partial pathway. Finally, the full biosynthetic pathways were reconstituted by mixing two related modules to synthesize (S)-PED and 2-PE, respectively. After optimization, the final conversion rates for (S)-PED and 2-PE reached 100 and 82.5%, respectively, based on the starting substrate of l-phenylalanine. We anticipate that the modular cell-free approach will make a possible efficient and high-yielding biosynthesis of value-added chemicals.
研究领域:
合成生物学
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代谢工程
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生物技术
✓ 已验证
产品关联
DOI
PubMed
高影响因子
INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES
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2020
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IF 5.162
Fei Peng,Xiao-Yang Ou,Ze-Wang Guo,Ying-Jie Zeng,Min-Hua Zong,Wen-Yong Lou
Artificial multienzyme biocatalysts have played a crucial role in biosynthesis because they allow for conducting complex reactions. Here, we reorted a facile approach to fabricate multienzyme nanodevices with high catalytic activity and stability based on protein assembly and chemical crosslinking. The self-assembled partner SpyCatcher and SpyTag were genetically fused with 2,3-butanediol hydrogenase and formate hydrogenase to produce Kg BDH-SC (SpyCatcher-fused 2,3-butanediol hydrogenase) and FDH-ST (SpyTag-fused formate hydrogenase), respectively. After assembling the two fusion proteins, the complexes were then immobilized on the functionalized silicon dioxide nanoparticles by glutaraldehyde, forming Kg BDH-SC-ST-FDH-SiO 2 with the capability of reducing 2-hydroxyacetophenone to ( R )-1-phenyl-1,2-ethanediol with cofactor regeneration. Under the optimal conditions, the created co-immobilized enzymes performed 49% activity recovery compared with the mixture of free enzymes as well as showed 2.9-fold higher catalytic activity than the traditional random co-immobilized enzymes. Moreover, Kg BDH-SC-ST-FDH-SiO 2 showed better pH stability and organic solvents stability than the free enzymes, and remained 52.5% overall catalytic activity after 8 cycles. Finally, the co-immobilized enzymes can reduce 60 mM HAP for fabrication of ( R )-PED with cofactor regeneration in the phosphate buffer reaction system, affording 83.9% yield and above 99% optical purity.
研究领域:
生物技术
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酶学
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纳米技术
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化学工程
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催化
✓ 已验证
产品关联
DOI
PubMed
高影响因子
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