Enabling malic acid production from corn-stover hydrolysate in Lipomyces starkeyi via metabolic engineering and bioprocess optimization
Abstract Background Lipomyces starkeyi is an oleaginous yeast with a native metabolism well-suited for production of lipids and biofuels from complex lignocellulosic and waste feedstocks. Recent advances in genetic engineering tools have facilitated the development of L. starkeyi into a microbial ch...
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BMC
2025-05-01
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| Series: | Microbial Cell Factories |
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| Online Access: | https://doi.org/10.1186/s12934-025-02705-0 |
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| author | Jeffrey J. Czajka Ziyu Dai Tijana Radivojević Joonhoon Kim Shuang Deng Teresa Lemmon Marie Swita Meagan C Burnet Nathalie Munoz Yuqian Gao Young-Mo Kim Beth Hofstad Jon K. Magnuson Hector Garcia Martin Kristin E. Burnum-Johnson Kyle R. Pomraning |
| author_facet | Jeffrey J. Czajka Ziyu Dai Tijana Radivojević Joonhoon Kim Shuang Deng Teresa Lemmon Marie Swita Meagan C Burnet Nathalie Munoz Yuqian Gao Young-Mo Kim Beth Hofstad Jon K. Magnuson Hector Garcia Martin Kristin E. Burnum-Johnson Kyle R. Pomraning |
| author_sort | Jeffrey J. Czajka |
| collection | DOAJ |
| description | Abstract Background Lipomyces starkeyi is an oleaginous yeast with a native metabolism well-suited for production of lipids and biofuels from complex lignocellulosic and waste feedstocks. Recent advances in genetic engineering tools have facilitated the development of L. starkeyi into a microbial chassis for biofuel and chemical production. However, the feasibility of redirecting L. starkeyi lipid flux away from lipids and towards other products remains relatively unexplored. Here, we engineer the native metabolism to produce malic acid by introducing the reductive TCA pathway and a C4-dicarboxylic acid transporter to the yeast. Results Heterogeneous expression of two genes, the Aspergillus oryzae malate transporter and malate dehydrogenase, enabled L. starkeyi malic acid production. Overexpression of a third gene, the native pyruvate carboxylase, allowed titers to reach approximately 10 g/L during shaking flasks cultivations, with production of malic acid inhibited at pH values less than 4. Corn-stover hydrolysates were found to be well-tolerated, and controlled bioreactor fermentations on the real hydrolysate produced 26.5 g/L of malic acid. Proteomic, transcriptomic and metabolomic data from real and mock hydrolysate fermentations indicated increased levels of a S. cerevisiae hsp9/hsp12 homolog (proteinID: 101453), glutathione dependent formaldehyde dehydrogenases (proteinIDs: 2047, 278215), oxidoreductases, and expression of efflux pumps and permeases during growth on the real hydrolysate. Simultaneously, machine learning based medium optimization improved production dynamics by 18% on mock hydrolysate and revealed lower tolerance to boron (a trace element included in the standard cultivation medium) than other yeasts. Conclusions Together, this work demonstrated the ability to produce organic acids in L. starkeyi with minimal byproducts. The fermentation characterization and omic analyses provide a rich dataset for understanding L. starkeyi physiology and metabolic response to growth in hydrolysates. Identified upregulated genes and proteins provide potential targets for overexpression for improving growth and tolerance to concentrated hydrolysates, as well as valuable information for future L. starkeyi engineering work. |
| format | Article |
| id | doaj-art-a75b069347b04c55b68d8e0dc87b74ed |
| institution | Kabale University |
| issn | 1475-2859 |
| language | English |
| publishDate | 2025-05-01 |
| publisher | BMC |
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| series | Microbial Cell Factories |
| spelling | doaj-art-a75b069347b04c55b68d8e0dc87b74ed2025-08-20T03:47:45ZengBMCMicrobial Cell Factories1475-28592025-05-0124111410.1186/s12934-025-02705-0Enabling malic acid production from corn-stover hydrolysate in Lipomyces starkeyi via metabolic engineering and bioprocess optimizationJeffrey J. Czajka0Ziyu Dai1Tijana Radivojević2Joonhoon Kim3Shuang Deng4Teresa Lemmon5Marie Swita6Meagan C Burnet7Nathalie Munoz8Yuqian Gao9Young-Mo Kim10Beth Hofstad11Jon K. Magnuson12Hector Garcia Martin13Kristin E. Burnum-Johnson14Kyle R. Pomraning15Energy and Environment Directorate, Pacific Northwest National LaboratoryEnergy and Environment Directorate, Pacific Northwest National LaboratoryDOE Agile BioFoundryEnergy and Environment Directorate, Pacific Northwest National LaboratoryEnergy and Environment Directorate, Pacific Northwest National LaboratoryEnergy and Environment Directorate, Pacific Northwest National LaboratoryEnergy and Environment Directorate, Pacific Northwest National LaboratoryDOE Agile BioFoundryDOE Agile BioFoundryDOE Agile BioFoundryDOE Agile BioFoundryEnergy and Environment Directorate, Pacific Northwest National LaboratoryEnergy and Environment Directorate, Pacific Northwest National LaboratoryDOE Agile BioFoundryDOE Agile BioFoundryEnergy and Environment Directorate, Pacific Northwest National LaboratoryAbstract Background Lipomyces starkeyi is an oleaginous yeast with a native metabolism well-suited for production of lipids and biofuels from complex lignocellulosic and waste feedstocks. Recent advances in genetic engineering tools have facilitated the development of L. starkeyi into a microbial chassis for biofuel and chemical production. However, the feasibility of redirecting L. starkeyi lipid flux away from lipids and towards other products remains relatively unexplored. Here, we engineer the native metabolism to produce malic acid by introducing the reductive TCA pathway and a C4-dicarboxylic acid transporter to the yeast. Results Heterogeneous expression of two genes, the Aspergillus oryzae malate transporter and malate dehydrogenase, enabled L. starkeyi malic acid production. Overexpression of a third gene, the native pyruvate carboxylase, allowed titers to reach approximately 10 g/L during shaking flasks cultivations, with production of malic acid inhibited at pH values less than 4. Corn-stover hydrolysates were found to be well-tolerated, and controlled bioreactor fermentations on the real hydrolysate produced 26.5 g/L of malic acid. Proteomic, transcriptomic and metabolomic data from real and mock hydrolysate fermentations indicated increased levels of a S. cerevisiae hsp9/hsp12 homolog (proteinID: 101453), glutathione dependent formaldehyde dehydrogenases (proteinIDs: 2047, 278215), oxidoreductases, and expression of efflux pumps and permeases during growth on the real hydrolysate. Simultaneously, machine learning based medium optimization improved production dynamics by 18% on mock hydrolysate and revealed lower tolerance to boron (a trace element included in the standard cultivation medium) than other yeasts. Conclusions Together, this work demonstrated the ability to produce organic acids in L. starkeyi with minimal byproducts. The fermentation characterization and omic analyses provide a rich dataset for understanding L. starkeyi physiology and metabolic response to growth in hydrolysates. Identified upregulated genes and proteins provide potential targets for overexpression for improving growth and tolerance to concentrated hydrolysates, as well as valuable information for future L. starkeyi engineering work.https://doi.org/10.1186/s12934-025-02705-0Oleaginous yeastLipomyces starkeyiMalic acid productionMachine learning medium optimization |
| spellingShingle | Jeffrey J. Czajka Ziyu Dai Tijana Radivojević Joonhoon Kim Shuang Deng Teresa Lemmon Marie Swita Meagan C Burnet Nathalie Munoz Yuqian Gao Young-Mo Kim Beth Hofstad Jon K. Magnuson Hector Garcia Martin Kristin E. Burnum-Johnson Kyle R. Pomraning Enabling malic acid production from corn-stover hydrolysate in Lipomyces starkeyi via metabolic engineering and bioprocess optimization Microbial Cell Factories Oleaginous yeast Lipomyces starkeyi Malic acid production Machine learning medium optimization |
| title | Enabling malic acid production from corn-stover hydrolysate in Lipomyces starkeyi via metabolic engineering and bioprocess optimization |
| title_full | Enabling malic acid production from corn-stover hydrolysate in Lipomyces starkeyi via metabolic engineering and bioprocess optimization |
| title_fullStr | Enabling malic acid production from corn-stover hydrolysate in Lipomyces starkeyi via metabolic engineering and bioprocess optimization |
| title_full_unstemmed | Enabling malic acid production from corn-stover hydrolysate in Lipomyces starkeyi via metabolic engineering and bioprocess optimization |
| title_short | Enabling malic acid production from corn-stover hydrolysate in Lipomyces starkeyi via metabolic engineering and bioprocess optimization |
| title_sort | enabling malic acid production from corn stover hydrolysate in lipomyces starkeyi via metabolic engineering and bioprocess optimization |
| topic | Oleaginous yeast Lipomyces starkeyi Malic acid production Machine learning medium optimization |
| url | https://doi.org/10.1186/s12934-025-02705-0 |
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