在 Pichia pastoris 中从头开始和提高生产 Monacolin J 的途径重建和代谢工程。

IF 3.5 3区 生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Bioprocess and Biosystems Engineering Pub Date : 2024-11-01 Epub Date: 2024-07-31 DOI:10.1007/s00449-024-03069-2
Yiping Wu, Xuefang Peng, Dexun Fan, Shuangyan Han, Xiaorong Yang
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引用次数: 0

摘要

他汀类药物是主要的降胆固醇药物。莫纳可林 J(MJ)是他汀生物合成途径中的一个关键中间体。在工业中,它是通过碱性水解洛伐他汀获得的。水解过程会产生多种副产品,废水处理成本高昂。在这项工作中,我们使用 Pichia pastoris 作为宿主来生产 MJ。在 P. pastoris 中建立了 MJ 的生物合成途径。通过CRISPR(clustered regularly interspaced short palindromic repeats)-Cas9基因组编辑工具获得了稳定的重组菌株MJ2,并产生了153.6 ± 2.4 mg/L的MJ滴度。利用代谢工程提高了 MJ 的产量,并优化了发酵条件。带有 ATP 依赖性柠檬酸裂解酶(ACL)、葡萄糖-6-磷酸脱氢酶(ZWF1)和 4 个 lovB 基因的重组菌株 MJ5-AZ 的 MJ 滴度为 357.5 ± 5.0 mg/L,比原始菌株 MJ2 高出 132.7%。重组菌株 MJ5-AZ 在 7 升发酵罐中培养,MJ 滴度达到 1493.0 ± 9.2 mg/L。结果表明,增加化学物质生物合成途径中限速步骤的基因剂量可提高生产滴度。这可能适用于其他多酮代谢物的生产优化。
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Pathway reconstruction and metabolic engineering for the de novo and enhancing production of monacolin J in Pichia pastoris.

The statin is the primary cholesterol-lowering drug. Monacolin J (MJ) is a key intermediate in the biosynthetic pathway of statin. It was obtained in industry by the alkaline hydrolysis of lovastatin. The hydrolysis process resulted in multiple by-products and expensive cost of wastewater treatment. In this work, we used Pichia pastoris as the host to produce the MJ. The biosynthesis pathway of MJ was built in P. pastoris. The stable recombinant strain MJ2 was obtained by the CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 genome-editing tool, and produced the MJ titer of 153.6 ± 2.4 mg/L. The metabolic engineering was utilized to enhance the production of MJ, and the fermentation condition was optimized. The MJ titer of 357.5 ± 5.0 mg/L was obtained from the recombinant strain MJ5-AZ with ATP-dependent citrate lyase (ACL), glucose-6-phosphate dehydrogenase (ZWF1) and four lovB genes, 132.7% higher than that from the original strain MJ2. The recombinant strain MJ5-AZ was cultured in a 7-L fermenter, and the MJ titer of 1493.0 ± 9.2 mg/L was achieved. The results suggested that increasing the gene dosage of rate-limiting step in the biosynthesis pathway of chemicals could improve the titer of production. It might be applicable to the production optimization of other polyketide metabolites.

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来源期刊
Bioprocess and Biosystems Engineering
Bioprocess and Biosystems Engineering 工程技术-工程:化工
CiteScore
7.90
自引率
2.60%
发文量
147
审稿时长
2.6 months
期刊介绍: Bioprocess and Biosystems Engineering provides an international peer-reviewed forum to facilitate the discussion between engineering and biological science to find efficient solutions in the development and improvement of bioprocesses. The aim of the journal is to focus more attention on the multidisciplinary approaches for integrative bioprocess design. Of special interest are the rational manipulation of biosystems through metabolic engineering techniques to provide new biocatalysts as well as the model based design of bioprocesses (up-stream processing, bioreactor operation and downstream processing) that will lead to new and sustainable production processes. Contributions are targeted at new approaches for rational and evolutive design of cellular systems by taking into account the environment and constraints of technical production processes, integration of recombinant technology and process design, as well as new hybrid intersections such as bioinformatics and process systems engineering. Manuscripts concerning the design, simulation, experimental validation, control, and economic as well as ecological evaluation of novel processes using biosystems or parts thereof (e.g., enzymes, microorganisms, mammalian cells, plant cells, or tissue), their related products, or technical devices are also encouraged. The Editors will consider papers for publication based on novelty, their impact on biotechnological production and their contribution to the advancement of bioprocess and biosystems engineering science. Submission of papers dealing with routine aspects of bioprocess engineering (e.g., routine application of established methodologies, and description of established equipment) are discouraged.
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