利用脂肪分解酵母工程技术从甘油中生产 l-苹果酸。

IF 3.7 2区 生物学 Q1 BIOCHEMICAL RESEARCH METHODS ACS Synthetic Biology Pub Date : 2024-10-23 DOI:10.1021/acssynbio.4c00445
Yaping Wang, Yuqing Han, Chang Liu, Liyan Cao, Qingqing Ye, Chen Ding, Yuyang Wang, Qingeng Huang, Jiwei Mao, Cui-Ying Zhang, Aiqun Yu
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引用次数: 0

摘要

苹果酸是一种重要的四碳二羧酸,广泛应用于食品、化工和制药行业。合成生物学和代谢工程已经实现了 l-苹果酸的高效微生物生产,但在重要的工业微生物脂溶性亚罗威氏菌(Yarrowia lipolytica)中尚未实现。本研究旨在探索这种真菌生产 l-苹果酸的潜力。首先,在脂肪分解酵母中过表达内源生物合成基因和异源转运体基因,以确定在甘油上生长的 l-苹果酸生物合成途径的瓶颈。其次,过量表达乙醛酸循环途径中的异柠檬酸裂解酶、苹果酸合成酶和苹果酸脱氢酶,并从Schizosaccharomyces pombe引入苹果酸转运体,显著提高了l-苹果酸产量,使其达到27.0克/升。随后,通过摇瓶培养基优化,苹果酸产量提高到 37.0 克/升。第三,适应性实验室进化使工程菌株 Po1g-CEE2+Sp 能够耐受较低的 pH 值,并积累更多的苹果酸(56.0 克/升)。最后,当扩大到 5 升生物反应器时,滴度达到了 112.5 克/升。总之,本研究首次展示了通过结合代谢工程和实验室进化,在脂溶性酵母中成功生产出了苹果酸,为大规模可持续生物合成这种酸和其他有机酸铺平了道路。
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Engineering Yarrowia lipolytica to Produce l-Malic Acid from Glycerol.

The declining availability of cheap fossil-based resources has sparked growing interest in the sustainable biosynthesis of organic acids. l-Malic acid, a crucial four-carbon dicarboxylic acid, finds extensive applications in the food, chemical, and pharmaceutical industries. Synthetic biology and metabolic engineering have enabled the efficient microbial production of l-malic acid, albeit not in Yarrowia lipolytica, an important industrial microorganism. The present study aimed to explore the potential of this fungal species for the production of l-malic acid. First, endogenous biosynthetic genes and heterologous transporter genes were overexpressed in Y. lipolytica to identify bottlenecks in the l-malic acid biosynthesis pathway grown on glycerol. Second, overexpression of isocitrate lyase, malate synthase, and malate dehydrogenase in the glyoxylate cycle pathway and introduction of a malate transporter from Schizosaccharomyces pombe significantly boosted l-malic acid production, which reached 27.0 g/L. A subsequent increase to 37.0 g/L was attained through shake flask medium optimization. Third, adaptive laboratory evolution allowed the engineered strain Po1g-CEE2+Sp to tolerate a lower pH and to accumulate a higher amount of l-malic acid (56.0 g/L). Finally, when scaling up to a 5 L bioreactor, a titer of 112.5 g/L was attained. In conclusion, this study demonstrates for the first time the successful production of l-malic acid in Y. lipolytica by combining metabolic engineering and laboratory evolution, paving the way for large-scale sustainable biosynthesis of this and other organic acids.

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来源期刊
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.
期刊最新文献
Application of a Cell-Free Synthetic Biology Platform for the Reconstitution of Teleocidin B and UK-2A Precursor Biosynthetic Pathways. Systematic Evaluation and Application of IDR Domain-Mediated Transcriptional Activation of NUP98 in Saccharomyces cerevisiae. Rational and Semirational Approaches for Engineering Salicylate Production in Escherichia coli. Engineering Yarrowia lipolytica to Produce l-Malic Acid from Glycerol. Precision Transcriptome Editing.
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