生物技术通报 ›› 2022, Vol. 38 ›› Issue (2): 173-183.doi: 10.13560/j.cnki.biotech.bull.1985.2021-0345

• 研究报告 • 上一篇    下一篇

基于Methylovorus sp. J1-1基因组尺度代谢网络优化吡咯喹啉醌合成

寇航1,2(), 王艳梅1, 李彤2, 薄明井2, 张惟材2, 熊向华2(), 黎明1()   

  1. 1.工业发酵微生物教育部重点实验室 天津科技大学生物工程学院,天津 300457
    2.军事科学院军事医学研究院生物工程研究所,北京 100071
  • 收稿日期:2021-03-20 出版日期:2022-02-26 发布日期:2022-03-09
  • 作者简介:寇航,男,硕士研究生,研究方向:微生物发酵;E-mail: 727846160@qq.com
  • 基金资助:
    国家科技重大专项(2018X09J18109-003)

Fermentation Optimization for PQQ Synthesis Based on the Genome-scale Metabolic Model of Methylovorus sp. J1-1

KOU Hang1,2(), WANG Yan-mei1, LI Tong2, BO Ming-jing2, ZHANG Wei-cai2, XIONG Xiang-hua2(), LI Ming1()   

  1. 1. Key Laboratory of Industrial Fermentation Microbiology,Ministry of Education,College of Biotechnology,Tianjin University of Science and Technology,Tianjin 300457
    2. Institute of Biotechnology Academy of Military Medical Science,Beijing 100071
  • Received:2021-03-20 Published:2022-02-26 Online:2022-03-09

摘要:

通过构建Methylovorus sp. J1-1基因组尺度代谢网络模型(genome scale of metabolic network model,GSMM),发掘能够提升吡咯喹啉醌(pyrroloquinoline quinone,PQQ)产量的发酵策略和相关靶基因。基于其注释的基因组和生化信息,构建J1-1的GSMM。再通过COBRApy预测可能使PQQ产量提高的氨基酸和潜在的靶基因并进行验证。构建了Methylovorus sp. J1-1的GSMM模型iKH584,共有584个基因,779个生化反应,121个交换反应与765个代谢产物,且可以用于后续模拟。根据iKH584模拟,外源添加谷氨酸、谷氨酰胺和脯氨酸、过表达glyA与hps1以及敲除hps2基因,均具有促进PQQ合成效果。结果表明:添加谷氨酸与脯氨酸时,PQQ的产量分别提高了10.4%与22.9%;过表达基因glyA与hps1,PQQ产量分别提高20.6%与14.6%;敲除基因hps2,PQQ产量最高可达140.84 mg/L,提高了8.0%,这与模拟结果基本一致,表明构建的模型基本正确。最后,在5 L发酵罐进行J1-1△hps2的分批补料发酵,PQQ的产量为812.64 mg/L,比亲本菌株提高了11.1%。建立的模型可以用来指导J1-1的发酵和菌株改造,提高PQQ产量。

关键词: Methylovorus sp. J1-1, 基因组尺度代谢网络模型, 吡咯喹啉醌, 氨基酸, 靶基因预测

Abstract:

The aim of this study is to explore the fermentation strategies and related target genes that can increase PQQ production by constructing genome-scale metabolic model of Methylovorus sp. J1-1. A genome-scale metabolic model(GSMM)of Methylovorus sp. J1-1 was constructed based on its annotated genome and biochemical information. Subsequently,amino acids and several potential genetic targets that may increase PQQ yield were predicted by COBRApy and validated. The GSMM iKH584 of J1-1 was constructed,containing 584 genes,779 biochemical reactions,121 exchange reactions and 765 metabolites,and it may be used in follow-up simulation. According to the iKH584 simulation,addition of glutamic acid,glutamine and proline,overexpression of the glyA and hps1,and knockout of hps2 all promoted PQQ synthesis. The results showed that the addition of glutamic acid and proline increased PQQ production by 10.4% and 22.9% respectively,overexpression of the glyA and hps1 increased the extracellular concentration of PQQ by 20.6% and 14.6% respectively,and hps2 knockout enhanced PQQ concentration up to 140.84 mg/L and increased by 8.0%,which were consistent with the simulated results,indicating that the metabolic model iKH584 of J1-1 was basically correct. Finally,fed-batch fermentations of J1-1△hps2 in 5 L fermentor were carried out,and the PQQ product reached to 812.64 mg/L,improved 11.1% compared with the parent strain J1-1. Conclusively,the established metabolic model iKH584 of J1-1 can be used to guide the fermentation and strain modification of J1-1 for improving the PQQ yield.

Key words: Methylovorus sp. J1-1, genome-scale metabolic model, pyrroloquinoline quinone, amino acids, target prediction