Engineering Yarrowia lipolytica to Produce l-Malic Acid from Glycerol
Journal article, 2024

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.

adaptive evolution

pathway engineering

Yarrowia lipolytica

transporter engineering

l-malic acid

Author

Yaping Wang

Tianjin University of Technology

Yuqing Han

Tianjin University of Technology

Chang Liu

Tianjin University of Technology

Liyan Cao

Tianjin University of Technology

Qingqing Ye

Tianjin University of Technology

Chen Ding

Tianjin University of Technology

Yuyang Wang

Tianjin University of Technology

Qingeng Huang

Ltd

Jiwei Mao

Chalmers, Life Sciences, Systems and Synthetic Biology

Cui Ying Zhang

Tianjin University of Technology

Aiqun Yu

Tianjin University of Technology

ACS Synthetic Biology

2161-5063 (eISSN)

Vol. 13 11 3635-3645

Subject Categories

Biochemistry and Molecular Biology

Microbiology

Other Industrial Biotechnology

DOI

10.1021/acssynbio.4c00445

More information

Latest update

11/30/2024