Biofuel Research Journal

Biofuel Research Journal

Metabolic engineering of the non-conventional yeast Kluyveromyces marxianus for enhancing the biosynthesis of succinic acid

Document Type : Research Paper

Authors
Ziyun Gu
Deyang Ding
Zhanpeng Shan
Yongsheng Tang
Xiulai Chen
School of Biotechnology and Key Laboratory of Industrial Biotechnology of Ministry of Education, Jiangnan University, Wuxi 214122, China.
10.18331/BRJ2025.12.3.5
Abstract
Succinic acid is a crucial four-carbon dicarboxylic acid with widespread applications in the detergent, food, and pharmaceutical industries. However, its microbial production on a large scale is limited by the utilization of neutralizing agents and high cooling costs. In this study, we conducted metabolic engineering of the thermotolerant and acid-tolerant yeast Kluyveromyces marxianus to facilitate the efficient biosynthesis of succinic acid at high temperature and low pH. A robust genetic manipulation platform for K. marxianus was established by developing gene editing tools, characterizing neutral integration sites, and identifying endogenous promoters. Leveraging this efficient platform, we systematically constructed and optimized the biosynthetic pathway of succinic acid through many metabolic engineering strategies, such as the knockout of byproduct pathways, the redistribution of carbon flux, and the enhancement of the succinic acid transport system. Finally, the engineered strain K. marxianus KmSA12 was able to produce 50.6 g/L succinic acid with a yield of 0.31 g/g and productivity of 0.42 g/L/h in a 5-L bioreactor. These results demonstrate the potential of K. marxianus as a promising platform for the large-scale production of various organic acids.

Graphical Abstract

Metabolic engineering of the non-conventional yeast Kluyveromyces marxianus for enhancing the biosynthesis of succinic acid

Highlights

  • The CRISPR-Cas9 system was developed with 92% knockout efficiency in K. marxianus.
  • 20 promoter and 20 neutral integration sites were identified for stable gene expression.
  • The rTCA pathway and transport system were optimized to enhance succinic acid production.
  • Succinic acid production with K. marxianus. KmSA12 was up to 50.6 g/L at 37°C.
  • Succinic acid production reached 41.2 g/L at 45°C with a limited amount of neutralizing agents.

Keywords
Kluyveromyces marxianus
Succinic acid
Metabolic engineering
Genetic manipulation
Thermophilic yeast
Acid-tolerant fermentation
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Biofuel Research Journal
Volume 12, Issue 3 - Serial Number 3
Summer 2025
Pages 2503-2516