通过表达威氏血红蛋白和调节溶解氧张力来提高氧化葡萄糖酸杆菌中吡咯并喹啉醌依赖性 d-山梨醇脱氢酶的活性，以用于 6-（N-羟乙基）-氨基-6-脱氧-α-l-山梨呋喃糖的生物合成。
Improvement of pyrroloquinoline quinone-dependent d-sorbitol dehydrogenase activity from Gluconobacter oxydans via expression of Vitreoscilla hemoglobin and regulation of dissolved oxygen tension for the biosynthesis of 6-(N-hydroxyethyl)-amino-6-deoxy-α-l-sorbofuranose.
机构信息
Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou, Zhejiang 310014, PR China; Engineering Research Center of Bioconversion and Biopurification of the Ministry of Education, Zhejiang University of Technology, Hangzhou, Zhejiang 310014, PR China.
Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou, Zhejiang 310014, PR China; Engineering Research Center of Bioconversion and Biopurification of the Ministry of Education, Zhejiang University of Technology, Hangzhou, Zhejiang 310014, PR China.
出版信息
J Biosci Bioeng. 2021 May;131(5):518-524. doi: 10.1016/j.jbiosc.2020.12.013. Epub 2021 Jan 21.
The miglitol intermediate, 6-(N-hydroxyethyl)-amino-6-deoxy-α-l-sorbofuranose (6NSL), is catalyzed from N-2-hydroxyethyl glucamine (NHEG) by resting cells of Gluconobacter oxydans. One of the key factors limiting 6NSL production was the availability of oxygen during both cell cultivation and biotransformation of NHEG to 6NSL. Based on G. oxydans/pBBR1-sldAB-pqqABCDE-tldD (G. oxydans/AB-PQQ), the Vitreoscilla hemoglobin (VHb) was heterologously expressed in G. oxydans to enhance oxygen transfer efficiency and improve 6NSL production. The recombinant G. oxydans/AB-PQQ-VHb displayed higher biomass and NHEG oxidation activity than the control stain. The transcription levels of respiratory chain-related enzyme genes in G. oxydans/AB-PQQ-VHb exhibited up-regulation, indicating that the presence of VHb promoted the respiration. The dissolved oxygen (DO) concentration for cell cultivation was optimized in a 5-L stirred bioreactor. At a DO concentration of 20%, the maximum volumetric oxidation activity of NHEG of G. oxydans/AB-PQQ-VHb in the stirred bioreactor reached 168.3 ± 3.2 U/L. Furthermore, the biotransformation of NHEG to 6NSL using G. oxydans/AB-PQQ-VHb was carried out under different oxygen tensions to investigate the effect of oxygen on 6NSL production. Finally, up to 87.5 ± 5.9 g/L 6NSL was accumulated in the reaction mixture within 16 h when the DO was controlled at 30%.
米格列醇中间体 6-(N-羟乙基)-氨基-6-去氧-α-l-山梨呋喃糖（6NSL）由氧化葡萄糖酸杆菌的静止细胞从 N-2-羟乙基葡糖胺（NHEG）催化得到。在细胞培养和 NHEG 向 6NSL 的生物转化过程中，氧气的可用性是限制 6NSL 生产的关键因素之一。基于 Gluconobacter oxydans/pBBR1-sldAB-pqqABCDE-tldD（G. oxydans/AB-PQQ），在氧化葡萄糖酸杆菌中异源表达了 Vitreoscilla 血红蛋白（VHb）以提高氧气传递效率并提高 6NSL 的产量。重组 G. oxydans/AB-PQQ-VHb 显示出比对照菌株更高的生物量和 NHEG 氧化活性。G. oxydans/AB-PQQ-VHb 中与呼吸链相关的酶基因的转录水平上调，表明 VHb 的存在促进了呼吸作用。在 5-L 搅拌式生物反应器中优化了细胞培养的溶解氧（DO）浓度。在 DO 浓度为 20%时，搅拌式生物反应器中 G. oxydans/AB-PQQ-VHb 的 NHEG 最大比体积氧化活性达到 168.3±3.2 U/L。此外，使用 G. oxydans/AB-PQQ-VHb 进行了不同氧分压下的 NHEG 向 6NSL 的生物转化，以研究氧对 6NSL 生产的影响。最后，当 DO 控制在 30%时，在 16 h 内反应混合物中积累了 87.5±5.9 g/L 的 6NSL。
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