Genomic Science Program
U.S. Department of Energy | Office of Science | Biological and Environmental Research Program

Metabolic Engineering of Issatchenkia orientalis for Cost-Effective Production of Citramalate

Authors:

Daniel Bun1,2* (dtbun@iastate.edu), Zong-Yen Wu1,3, Wan Sun1,2, Nurzhan Kuanyshev1,4, Yihui Shen1,5, Patrick Suthers1,6, Degaulle Dai1,4, Linrui Tan1,4, Jimmy Pratas1,5, Jaewan Jang1,7, Jose Avalos1,7, Costas D. Maranas1,6, Joshua D. Rabinowitz1,5, Yong-Su Jin1,4, Yasuo Yoshikuni1,3, Zengyi Shao1,2, Andrew Leakey1

Institutions:

1DOE Center for Advanced Bioenergy and Bioproducts Innovation; 2Department of Chemical and Biological Engineering, Iowa State University; 3Lawrence Berkeley National Laboratory; 4Department of Food Science and Nutrition, University of Illinois Urbana-Champaign; 5Department of Chemistry, Princeton University; 6Department of Chemical Engineering, The Pennsylvania State University; 7Department of Chemical and Biological Engineering, Princeton University

URLs:

Goals

This project aims to engineer a pH-tolerant strain, Issatchenkia orientalis, capable of using sustainable feedstocks to produce citramalate while achieving successful scale-up. Specifically, the team plans to:

  • Increase citramalate titer and yield from glucose, feedstock hydrolysate, and oil through substrate optimization.
  • Expand the knowledge base and tool repertoire for I. orientalis by implementing advanced toolkits, such as optogenetics and piggyBac transposon technology, and conducting genetic studies by constructing a comprehensive knockout library.
  • Conduct thorough investigations on scale-up conditions for the production process.

Abstract

Methyl methacrylate (MMA) is a building block of poly MMA (PMMA), a material commonly recognized as acrylic glass or plexiglass. The prevailing method to manufacture PMMA utilizes petroleum and the acetone cyanohydrin process, which is considered unsustainable and raises concerns regarding the use of toxic chemicals. An alternative route using semisynthesis, combining converting microbially produced citramalate to methacrylic acid (MA) using a catalyst and final esterification of MA to MMA, could present a viable solution.

Previous studies have shown large titers of citramalate in engineered Escherichia coli. However, the increased production cost and carbon footprint from having a neutralization and reacidification step makes this route less economically attractive. A pH-tolerant strain such as I. orientalis would be an attractive alternative. Through extensive collaborative efforts, the Center for Advanced Bioenergy and Bioproducts Innovation (CABBI) team has previously demonstrated that I. orientalis can withstand citramalate of up to 80 g per liter at pH 3. Additionally, the team engineered a strain that produces 2 g/L citramalate by integrating the citramalate synthase gene (cimA) from Methanocaldococcus jannaschii into the I. orientalis genome using piggyBac.

Examining bottlenecks and increasing flux towards citramalate through metabolomic study and genome-scale modeling was essential in increasing citramalate production. The metabolomics studies found that there is pyruvate overflow, accumulation of intracellular citramalate, and excess ethanol byproduct. Using the genome-scale model, pathways of interest were identified to address these problems. The team simultaneously decreased pyruvate overflow and increased lacking acetyl-CoA by utilizing an aldehyde dehydrogenase gene (ALD6) from Saccharomyces cerevisiae and a mutated acetyl-CoA synthase gene (ACSSEL641P) from Salmonella enterica. Researchers also incorporated a multidrug transporter (QDR3) from S. cerevisiae to increase excretion of citramalate into the growing broth. Using piggyBac, researchers integrated QDR3ALD6ACSSEL641P and a more active cimA (cimA3.7) to create a library of variants. The team selected this library’s top producer (Q42) for further engineering. To address excess ethanol production, the project employed CRISPR to delete the pyruvate decarboxylase gene (PDC). The resulting strain, Q42 Δpdc, produced 18 g/L of citramalate in shake flasks and was scaled up to a 3 L bioreactor to produce 30 g/L using synthetic complete medium with 6% ammonium sulfate, 5% glucose, and a trace metal supplement without needing pH or dissolved oxygen control.

In addition, the CABBI team successfully generated a xylose-utilizing strain, Q42X Δpdc, for future work involving potential feedstocks such as sorghum hydrolysate and sugarcane juice. Currently, the team is actively characterizing the metabolism and genomics of I. orientalis by developing a comprehensive knockout library and updating the genome-scale model. The team is also exploring new metabolic engineering strategies such as eliminating glycerol production, exploring alternative routes for acetyl-CoA production, identifying additional target genes for up- or down-regulation, and developing a light-controlled circuit.

References

Wu, Y., et al. 2023. “Metabolic Engineering of Low-pH-Tolerant Non-Model Yeast, Issatchenkia orientalis, for Production of Citramalate,” Metabolic Engineering Communications. DOI:10.1016/j.mec.2023.e00220.

Funding Information

This research was supported by the DOE CABBI, U.S. DOE, Office of Science, BER program, under award number DE-SC0018420.