Metabolic Engineering of Yarrowia lipolytica with Massive Gene Assembly and Genomic Integration.

IF 3.7 2区生物学 Q1 BIOCHEMICAL RESEARCH METHODS

ACS Synthetic Biology Pub Date : 2025-07-17 DOI:10.1021/acssynbio.5c00333

Yuhui Cui, Duo Liu, Huimin Xue, Mingshan Li, Wenhong Guo, Cuiqin Huang, Xintian Zheng, Jichao Yang, Hong Liu, Huifang Yin, Hanjie Wang

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引用次数: 0

Abstract

Synthetic biology has developed varied strategies of one-pot multigene assembly and genomic integration to promote the genetic engineering of the chassis. However, such strategies for engineering oleaginous yeast Yarrowia lipolytica is lacking, given the current stage that at most 5 exogenous genes (around 13 kb) can be assembled and integrated into the genome at once. Here, we developed a strategy of massive gene assembly and integration in the Y. lipolytica genome. As a proof of concept, dozen-gene assembly (more than 30 kb) and integration were achieved stably and reproducibly, and a Y. lipolytica chassis containing a total of 35 exogenous genes (a sum of 93.5 kb) was constructed. The introduction of massive genes modulated the synthesis of lycopene, a heterologous natural product, to quite different extents. Ultimately, an optimized constructed strain containing 15 exogenous genes achieved the highest yield of 144.58 mg/g DCW and produced a lycopene titer of 2144.83 mg/L in a 5 L bioreactor. Our strategy significantly expands the capability of Y. lipolytica genetic manipulation and metabolic engineering.

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利用大量基因组装和基因组整合的多脂耶氏菌代谢工程。

合成生物学发展了一锅多基因组装和基因组整合的多种策略，以促进底盘的基因工程。然而，考虑到目前最多5个外源基因（约13kb）可以同时组装并整合到基因组中，这种工程产油酵母的策略是缺乏的。在这里，我们开发了一种大规模基因组装和整合的策略。为了验证这一概念，我们稳定且可重复地实现了12个基因的组装（大于30 kb）和整合，并构建了一个包含35个外源基因（总计93.5 kb）的脂肪瘤菌底盘。大量基因的引入对番茄红素这一异源天然产物的合成产生了不同程度的调节。最终，优化后的菌株含有15个外源基因，在5l生物反应器中产量最高，为144.58 mg/g DCW，番茄红素滴度为2144.83 mg/L。我们的策略极大地扩展了脂解菌基因操作和代谢工程的能力。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

ACS Synthetic Biology 生物-

CiteScore

8.00

自引率

10.60%

发文量

380

审稿时长

6-12 weeks

期刊介绍： The journal is particularly interested in studies on the design and synthesis of new genetic circuits and gene products; computational methods in the design of systems; and integrative applied approaches to understanding disease and metabolism. Topics may include, but are not limited to: Design and optimization of genetic systems Genetic circuit design and their principles for their organization into programs Computational methods to aid the design of genetic systems Experimental methods to quantify genetic parts, circuits, and metabolic fluxes Genetic parts libraries: their creation, analysis, and ontological representation Protein engineering including computational design Metabolic engineering and cellular manufacturing, including biomass conversion Natural product access, engineering, and production Creative and innovative applications of cellular programming Medical applications, tissue engineering, and the programming of therapeutic cells Minimal cell design and construction Genomics and genome replacement strategies Viral engineering Automated and robotic assembly platforms for synthetic biology DNA synthesis methodologies Metagenomics and synthetic metagenomic analysis Bioinformatics applied to gene discovery, chemoinformatics, and pathway construction Gene optimization Methods for genome-scale measurements of transcription and metabolomics Systems biology and methods to integrate multiple data sources in vitro and cell-free synthetic biology and molecular programming Nucleic acid engineering.