Pathway Evolution Through a Bottlenecking-Debottlenecking Strategy and Machine Learning-Aided Flux Balancing,Advanced Science

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Pathway Evolution Through a Bottlenecking-Debottlenecking Strategy and Machine Learning-Aided Flux Balancing
Advanced Science ( IF 14.3 ) Pub Date : 2024-02-06 , DOI: 10.1002/advs.202306935
Huaxiang Deng _{1,

2,

3,

4} , Han Yu _{1,

2,

3,

5} , Yanwu Deng _{1,

2,

3} , Yulan Qiu _{1,

2,

3} , Feifei Li _{1,

2,

3} , Xinran Wang _{1,

2,

3} , Jiahui He _{1,

2,

3} , Weiyue Liang _{1,

2,

3,

4} , Yunquan Lan ₆ , Longjiang Qiao ₆ , Zhiyu Zhang ₆ , Yunfeng Zhang _{1,

2,

3} , Jay D Keasling _{3,

7,

8,

9,

10} , Xiaozhou Luo _{1,

2,

3,

5,

6}

Affiliation

Shenzhen Key Laboratory for the Intelligent Microbial Manufacturing of Medicines, Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, Shenzhen, 518055, P. R. China.
CAS Key Laboratory of Quantitative Engineering Biology, Shenzhen Institute of Synthetic Biology, Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, Shenzhen, 518055, P. R. China.
Center for Synthetic Biochemistry, Shenzhen Institute of Synthetic Biology, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen, 518055, P. R. China.
The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, 214122, P. R. China.
University of Chinese Academy of Sciences, Beijing, 100049, P. R. China.
Shenzhen Infrastructure for Synthetic Biology, Shenzhen Institute of Synthetic Biology, Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, Shenzhen, 518055, P. R. China.
Joint BioEnergy Institute, Emeryville, CA, 94608, USA.
Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, CA, 94720, USA.
Department of Chemical and Biomolecular Engineering & Department of Bioengineering, University of California, Berkeley, CA, 94720, USA.
Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kgs. Lyngby, 2800, Denmark.

The evolution of pathway enzymes enhances the biosynthesis of high-value chemicals, crucial for pharmaceutical, and agrochemical applications. However, unpredictable evolutionary landscapes of pathway genes often hinder successful evolution. Here, the presence of complex epistasis is identifued within the representative naringenin biosynthetic pathway enzymes, hampering straightforward directed evolution. Subsequently, a biofoundry-assisted strategy is developed for pathway bottlenecking and debottlenecking, enabling the parallel evolution of all pathway enzymes along a predictable evolutionary trajectory in six weeks. This study then utilizes a machine learning model, ProEnsemble, to further balance the pathway by optimizing the transcription of individual genes. The broad applicability of this strategy is demonstrated by constructing an Escherichia coli chassis with evolved and balanced pathway genes, resulting in 3.65 g L⁻¹ naringenin. The optimized naringenin chassis also demonstrates enhanced production of other flavonoids. This approach can be readily adapted for any given number of enzymes in the specific metabolic pathway, paving the way for automated chassis construction in contemporary biofoundries.

中文翻译：

通过瓶颈-去瓶颈策略和机器学习辅助的通量平衡进行路径演进

途径酶的进化增强了高价值化学品的生物合成，这对于制药和农化应用至关重要。然而，路径基因不可预测的进化景观常常阻碍成功的进化。在这里，在代表性的柚皮素生物合成途径酶中发现了复杂的上位性的存在，阻碍了直接的定向进化。随后，开发了一种用于途径瓶颈和消除瓶颈的生物铸造辅助策略，使所有途径酶能够在六周内沿着可预测的进化轨迹并行进化。然后，这项研究利用机器学习模型 ProEnsemble，通过优化单个基因的转录来进一步平衡该通路。该策略的广泛适用性通过构建具有进化和平衡途径基因的大肠杆菌底盘得到证明，产生 3.65 g L ^-1柚皮素。优化后的柚皮素底盘还显示出其他黄酮类化合物产量的提高。这种方法可以很容易地适应特定代谢途径中任何给定数量的酶，为当代生物铸造厂的自动化底盘构建铺平了道路。

更新日期：2024-02-06

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