基于工程化毕赤酵母一锅法合成硫酸软骨素A
作者:
基金项目:

国家重点研发计划(2021YFC2103100);江苏省杰出青年基金项目(BK20200025);江苏省科技支撑项目(BE2019630)


One-pot synthesis of chondroitin sulfate A by engineered Pichia pastoris
Author:
  • SHENG Jingyu

    SHENG Jingyu

    Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, Jiangsu, China;Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, Jiangsu, China
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  • JIN Xuerong

    JIN Xuerong

    Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, Jiangsu, China;Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, Jiangsu, China
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  • XU Ruirui

    XU Ruirui

    Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, Jiangsu, China;Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, Jiangsu, China
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  • WANG Yang

    WANG Yang

    Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, Jiangsu, China;Science Center for Future Foods, Jiangnan University, Wuxi 214122, Jiangsu, China;Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, Jiangsu, China
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  • KANG Zhen

    KANG Zhen

    Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, Jiangsu, China;Science Center for Future Foods, Jiangnan University, Wuxi 214122, Jiangsu, China;Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, Jiangsu, China
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  • 摘要
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    摘要:

    硫酸软骨素(chondroitin sulfate,CS)是一种线性多糖,广泛应用于医疗和保健等领域。相比于传统动物组织提取法,微生物合成硫酸软骨素具有可控、易规模化放大等优势。为实现硫酸软骨素A (CSA)的高效合成,本研究首先通过整合软骨素合酶编码基因kfoC、kfoA以及UDP-葡萄糖脱氢酶编码基因tuaD至毕赤酵母GS115基因组中,构建了以甘油为唯一碳源发酵生产软骨素的毕赤酵母工程菌株。通过进一步优化软骨素合成途径,软骨素分批补料发酵水平达到2.6 g/L。在进一步整合表达软骨素-4-O-磺基转移酶的基础上,本研究通过向生产软骨素毕赤酵母工程菌株破碎液中添加3'-磷酸腺苷-5'-磷酰硫酸和软骨素-4-O-磺基转移酶,成功建立了CSA的一锅法生物合成体系。通过优化,最终实现0-40%不同磺酸化水平CSA的可控合成。本研究中CSA的一锅法生物合成体系操作简便、易放大,更适用于工业化大规模生产。本研究结果也为肝素等其他糖胺聚糖的合成提供了思路。

    Abstract:

    Chondroitin sulfate(CS) is a linear polysaccharide, which is widely used in medical, health care and other fields. Compared with the traditional animal tissue extraction method, microbial synthesis of CS has the advantages of controllability and easiness of scaling-up. In order to achieve an efficient synthesis of chondroitin sulfate A(CSA), we constructed a recombinant Pichia pastoris GS115 strain capable of synthesizing chondroitin(Ch) from glycerol by introducing the Ch synthase coding genes kfoC, kfoA and UDP-glucose dehydrogenase coding gene tuaD into the P. pastoris chromosome. The titer of Ch reached 2.6 g/L in fed-batch cultures upon optimizing the synthesis pathway of Ch. After further expressing the chondroitin-4-O-sulfotransferase(C4ST), we developed a one-pot biosynthesis system for CSA production by directly adding 3'-adenosine-5'-phosphoryl sulfate and C4ST into the high-pressure homogenized recombinant P. pastoris cells. Eventually, controllable synthesis of 0-40%CSA with different sulfation degrees were achieved by optimizing the catalytic conditions. The one-pot biosynthesis system constructed here is easy to operate and easy to scale up for industrial production of CSA. The idea of the present study may also facilitate the biosynthesis of other glycosaminoglycan, for instance, heparin.

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盛靖雨,金学荣,胥睿睿,王阳,康振. 基于工程化毕赤酵母一锅法合成硫酸软骨素A[J]. 生物工程学报, 2022, 38(7): 2594-2605

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  • 收稿日期:2022-02-28
  • 在线发布日期: 2022-07-25
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