Biofuel Research Journal

Biofuel Research Journal

Cell factories and transcription factor engineering for bioproducts: the case of Candida glabrata for α-ketoglutarate production

Document Type : Research Paper

Authors
Pan Zhu 1
Yufei Li 1
Zihan Zhao 2
Xinyi Sun 1
1 School of Life Sciences and Health Engineering, Jiangnan University, Wuxi 214122, China.
2 School of Biotechnology and Key Laboratory of Industrial Biotechnology of Ministry of Education, Jiangnan University, Wuxi 214122, China.
10.18331/BRJ2025.12.4.5
Abstract
α-Ketoglutarate, a key intermediate in the TCA cycle, is crucial for amino acid synthesis and nitrogen transport. However, microbial engineering for α-ketoglutarate production is hindered by the intrinsic inefficiency of the metabolic network. In this study, transcription factor engineering was performed for the reconstruction of the metabolic network to boost α-ketoglutarate biosynthesis in Candida glabrata. Transcription factors GCR2 and RTG1 were first reinstalled to kick-start the glycolytic pathway and the TCA cycle, respectively, and then optimized to redistribute carbon flux between the two pathways. In addition, pyruvate carriers, MPC1 and MPC2, were introduced to facilitate the transport of cytoplasmic pyruvate to mitochondria, thereby feeding it into the TCA cycle for α-ketoglutarate biosynthesis. Next, transcription factor HAP4 was utilized to rewire the electron transport chain for improving redox balance and reducing overflow metabolism, thereby channeling more carbon flux to α-ketoglutarate production. Finally, the engineered strain C. glabrata KGA17 was capable of producing 210.4 g/L α-ketoglutarate in a 5-L bioreactor. This approach showed significant promise for developing efficient microbial cell factories for high-value chemical production.

Graphical Abstract

Cell factories and transcription factor engineering for bioproducts: the case of Candida glabrata for α-ketoglutarate production

Highlights

  • Candida glabrata was used as cell factories for efficient production of α-ketoglutarate (AKG).
  • Transcription factor engineering was conducted to reconstruct metabolic network for AKG production.
  • Transcription factors GCR2 and RTG1 were reinstalled and optimized to redistribute carbon flux.
  • Transcription factor HAP4 was utilized to rewire electron transport chain for improving redox balance.
  • The engineered strain produced 210.4 g/L AKG with a yield of 0.54 g/g and productivity of 0.88 g/L/h.

Keywords
Transcription factor engineering
Candida glabrata
&alpha
-ketoglutarate
Pyruvate carrier
Pathway optimization
Bioeconomy
[1] Bork, P., Sander, C., Valencia, A., 1992. An ATPase domain common to prokaryotic cell cycle proteins, sugar kinases, actin, and hsp70 heat shock proteins. Proc. Natl. Acad. Sci. U.S.A. 89(16), 7290-7294. 
[2] Bricker, D.K., Taylor, E.B., Schell, J.C., Orsak, T., Boutron, A., Chen, Y.C., Cox, J.E., Cardon, C.M., Van Vranken, J.G., Dephoure, N., Redin, C., Boudina, S., Gygi, S.P., Brivet, M., Thummel, C.S., Rutter, J., 2012. A mitochondrial pyruvate carrier required for pyruvate uptake in yeast, Drosophila, and humans. Science. 337(6090), 96-100. 
[3] Broun, P., 2004. Transcription factors as tools for metabolic engineering in plants. Curr. Opin. Plant Biol. 7(2), 202-209. 
[4] Chen, X., Dong, X., Liu, J., Luo, Q., Liu, L., 2020. Pathway engineering of Escherichia coli for α-ketoglutaric acid production. Biotechnol. Bioeng. 117(9), 2791-2801. 
[5] Chen, X., Xu, G., Xu, N., Zou, W., Zhu, P., Liu, L., Chen, J., 2013. Metabolic engineering of Torulopsis glabrata for malate production. Metab. Eng. 19, 10-16. 
[6] Clifton, D., Fraenkel, D.G., 1981. The gcr (glycolysis regulation) mutation of Saccharomyces cerevisiae. J. Biol. Chem. 256(24), 13074-13078. 
[7] Courchesne, N.M.D., Parisien, A., Wang, B., Lan, C.Q., 2009. Enhancement of lipid production using biochemical, genetic and transcription factor engineering approaches. J. Biotechnol. 141(1-2), 31-41. 
[8] Dabirian, Y., Teixeira, P.G., Nielsen, J., Siewers, V., David, F., 2019. FadR-based biosensor-assisted screening for genes enhancing fatty acyl-CoA pools in Saccharomyces cerevisiae. ACS Synth. Biol. 8(8), 1788-1800. 
[9] Deng, C., Wu, Y.K., Lv, X.Q., Li, J.H., Liu, Y.F., Du, G.C., Chen, J., Liu, L., 2022. Refactoring transcription factors for metabolic engineering. Biotechnol. Adv. 57, 107935. 
[10] Enkler, L., Richer, D., Marchand, A.L., Ferrandon, D., Jossinet, F., 2016. Genome engineering in the yeast pathogen Candida glabrata using the CRISPR-Cas9 system. Sci. Rep. 6, 35766. 
[11] Finogenova, T.V., Morgunov, I.G., Kamzolova, S.V., Chernyavskaya, O.G., 2005. Organic acid production by the yeast Yarrowia lipolytica: a review of prospects. Appl. Biochem. Microbiol. 41(5), 418-425. 
[12] Grotewold, E., 2008. Transcription factors for predictive plant metabolic engineering: are we there yet?. Curr. Opin. Biotechnol. 19(2), 138-144. 
[13] Gu, Z.Y., Ding, D.Y., Shan, Z.P., Tang, Y.S., Chen, X.L., 2025. Metabolic engineering of the non-conventional yeast Kluyveromyces marxianus for enhancing the biosynthesis of succinic acid. Biofuel Res. J. 12(3), 2503-2516. 
[14] Guo, H., Liu, P., Madzak, C., Du, G., Zhou, J., Chen, J., 2015. Identification and application of keto acids transporters in Yarrowia lipolytica. Sci. Rep. 5, 8138. 
[15] Guo, H., Madzak, C., Du, G., Zhou, J., 2016. Mutagenesis of conserved active site residues of dihydrolipoamide succinyltransferase enhances the accumulation of alpha-ketoglutarate in Yarrowia lipolytica. Appl. Microbiol. Biotechnol. 100(2), 649-659. 
[16] Guo, H., Madzak, C., Du, G., Zhou, J., Chen, J., 2014. Effects of pyruvate dehydrogenase subunits overexpression on the alpha-ketoglutarate production in Yarrowia lipolytica WSH-Z06. Appl. Microbiol. Biotechnol. 98(16), 7003-7012. 
[17] Hauf, J., Zimmermann, F.K., Müller, S., 2000. Simultaneous genomic overexpression of seven glycolytic enzymes in the yeast Saccharomyces cerevisiae. Enzyme Microb. Technol. 26(9-10), 688-698. 
[18] He, H.H., Yang, M.F., Li, S.Y., Zhang, G.Y., Ding, Z.Y., Zhang, L., Shi, G.Y., Li, Y.R., 2023. Mechanisms and biotechnological applications of transcription factors. Syn. Syst. Biotechnol. 8(4), 565-577. 
[19] Herzig, S., Raemy, E., Montessuit, S., Veuthey, J.L., Zamboni, N., Westermann, B., Kunji, E.R., Martinou, J.C., 2012. Identification and functional expression of the mitochondrial pyruvate carrier. Science. 337(6090), 93-96.
[20] Holz, M., Otto, C., Kretzschmar, A., Yovkova, V., Aurich, A., Potter, M., Marx, A., Barth, G., 2011. Overexpression of alpha-ketoglutarate dehydrogenase in Yarrowia lipolytica and its effect on production of organic acids. Appl. Microbiol. Biotechnol. 89(5), 1519-1526. 
[21] Huang, H.J., Liu, L.M., Li, Y., Du, G.C., Chen, J., 2006. Redirecting carbon flux in Torulopsis glabrata from pyruvate to α-ketoglutaric acid by changing metabolic co-factors. Biotechnol. Lett. 28(2), 95-98. 
[22] Jia, Y., Rothermel, B., Thornton, J., Butow, R.A., 1997. A basic helix-loop-helix-leucine zipper transcription complex in yeast functions in a signaling pathway from mitochondria to the nucleus. Mol. Cell. Biol. 17(3), 1110-1117. 
[23] Kamzolova, S.V., Chiglintseva, M.N., Lunina, J.N., Morgunov, I.G., 2012. α-Ketoglutaric acid production by Yarrowia lipolytica and its regulation. Appl. Microbiol. Biotechnol. 96(3), 783-791. 
[24] Lee, Y.B., Jo, J.H., Kim, M.H., Lee, H.H., Hyun, H.H., 2013. Enhanced production of alpha-ketoglutarate by fed-batch culture in the metabolically engineered strains of Corynebacterium glutamicum. Biotechnol. Bioprocess. Eng. 18(4), 770-777. 
[25] Li, J.W., Zhang, X.Y., Wu, H., Bai, Y.P., 2020. Transcription factor engineering for high-throughput strain evolution and organic acid bioproduction: a review. Front. Bioeng. Biotechnol. 8, 98. 
[26] Li, S., Liu, L., Chen, J., 2015. Compartmentalizing metabolic pathway in Candida glabrata for acetoin production. Metab. Eng. 28, 1-7. 
[27] Liu, L., Li, Y., Zhu, Y., Du, G., Chen, J., 2007. Redistribution of carbon flux in Torulopsis glabrata by altering vitamin and calcium level. Metab. Eng. 9(1), 21-29. 
[28] Liu, L.M., Li, Y., Li, H.Z., Chen, J., 2004. Manipulating the pyruvate dehydrogenase bypass of a multi-vitamin auxotrophic yeast Torulopsis glabrata enhanced pyruvate production. Lett. Appl. Microbiol. 39(2), 199-206. 
[29] Luo, Z.S., Zeng, W.Z., Du, G.C., Chen, J., Zhou, J.W., 2020. Enhancement of pyruvic acid production in Candida glabrata by engineering hypoxia-inducible factor 1. Bioresour. Technol. 295, 122248. 
[30] Nishi, K., Park, C.S., Pepper, A.E., Eichinger, G., Innis, M.A., Holland, M.J., 1995. The GCR1 requirement for yeast glycolytic gene expression is suppressed by dominant mutations in the SGC1 gene, which encodes a novel basic-helix-loop-helix protein. Mol. Cell. Biol. 15(5), 2646-2653. 
[31] Olesen, J.T., Guarente, L., 1990. The HAP2 subunit of yeast CCAAT transcriptional activator contains adjacent domains for subunit association and DNA recognition: model for the HAP2/3/4 complex. Genes Dev. 4(10), 1714-1729.
[32] Otto, C., Yovkova, V., Aurich, A., Mauersberger, S., Barth, G., 2012. Variation of the by-product spectrum during alpha-ketoglutaric acid production from raw glycerol by overexpression of fumarase and pyruvate carboxylase genes in Yarrowia lipolytica. Appl. Microbiol. Biotechnol. 95(4), 905-917. 
[33] Otto, C., Yovkova, V., Barth, G., 2011. Overproduction and secretion of alpha-ketoglutaric acid by microorganisms. Appl. Microbiol. Biotechnol. 92(4), 689-695. 
[34] Qin, Y., Johnson, C.H., Liu, L.M., Chen, J.A., 2011. Introduction of heterogeneous NADH reoxidation pathways into Torulopsis glabrata significantly increases pyruvate production efficiency. Korean J. Chem. Eng. 28(4), 1078-1084. 
[35] Qiu, X.L., Xu, P., Zhao, X.R., Du, G.C., Zhang, J., Li, J.H., 2020. Combining genetically-encoded biosensors with high throughput strain screening to maximize erythritol production in Yarrowia lipolytica. Metab. Eng. 60, 66-76. 
[36] Qiu, Z.L., Jiang, R.R., 2017. Improving Saccharomyces cerevisiae ethanol production and tolerance via RNA polymerase II subunit Rpb7. Biotechnol. Biofuels 10, 125. 
[37] Reik, A., Zhou, Y., Collingwood, T.N., Warfe, L., Bartsevich, V., Kong, Y., Henning, K.A., Fallentine, B.K., Zhang, L., Zhong, X., Jouvenot, Y., Jamieson, A.C., Rebar, E.J., Case, C.C., Korman, A., Li, X.Y., Black, A., King, D.J., Gregory, P.D., 2007. Enhanced protein production by engineered zinc finger proteins. Biotechnol. Bioeng. 97(5), 1180-1189. 
[38] Stottmeister, U., Aurich, A., Wilde, H., Andersch, J., Schmidt, S., Sicker, D., 2005. White biotechnology for green chemistry: fermentative 2-oxocarboxylic acids as novel building blocks for subsequent chemical syntheses. J. Ind. Microbiol. Biotechnol. 32(11-12), 651-664. 
[39] Sugden, P.H., Newsholme, E.A., 1975. Activities of citrate synthase, NAD+-linked and NADP+-linked isocitrate dehydrogenases, glutamate dehydrogenase, aspartate aminotransferase and alanine aminotransferase in nervous tissues from vertebrates and invertebrates. Biochem. J. 150(1), 105-111. 
[40] Uemura, H., Jigami, Y., 1992. Role of GCR2 in transcriptional activation of yeast glycolytic genes. Mol. Cell. Biol. 12(9), 3834-3842. 
[41] Vom Endt, D., Kijne, J.W., Memelink, J., 2002. Transcription factors controlling plant secondary metabolism: what regulates the regulators?. Phytochemistry. 61(2), 107-114. 
[42] Xu, G., Liu, L., Chen, J., 2012. Reconstruction of cytosolic fumaric acid biosynthetic pathways in Saccharomyces cerevisiae. Microb. Cell. Fact. 11, 24. 
[43] Xu, N., Liu, L.M., 2019. Computational inference of the transcriptional regulatory network of Candida glabrata. FEMS Yeast Res. 19(4), foz036. 
[44] Yin, X., Madzak, C., Du, G., Zhou, J., Chen, J., 2012. Enhanced alpha-ketoglutaric acid production in Yarrowia lipolytica WSH-Z06 by regulation of the pyruvate carboxylation pathway. Appl. Microbiol. Biotechnol. 96(6), 1527-1537. 
[45] Yovkova, V., Otto, C., Aurich, A., Mauersberger, S., Barth, G., 2014. Engineering the alpha-ketoglutarate overproduction from raw glycerol by overexpression of the genes encoding NADP+-dependent isocitrate dehydrogenase and pyruvate carboxylase in Yarrowia lipolytica. Appl. Microbiol. Biotechnol. 98(5), 2003-2013. 
[46] Yu, Z., Du, G., Zhou, J., Chen, J., 2012. Enhanced alpha-ketoglutaric acid production in Yarrowia lipolytica WSH-Z06 by an improved integrated fed-batch strategy. Bioresour. Technol. 114, 597-602. 
[47] Zhang, C.L., Qi, J.S., Li, Y.J., Fan, X.G., Xu, Q.Y., Chen, N., Xie, X.X., 2016. Production of α-ketobutyrate using engineered Escherichia coli via temperature shift. Biotechnol. Bioeng. 113(9), 2054-2059. 
[48] Zhang, D., Liang, N., Shi, Z., Liu, L., Chen, J., Du, G., 2009. Enhancement of α-ketoglutarate production in Torulopsis glabrata: redistribution of carbon flux from pyruvate to α-ketoglutarate. Biotechnol. Bioprocess. Eng. 14(2), 134-139. 
[49] Zhou, J., Dong, Z., Liu, L., Du, G., Chen, J., 2009. A reusable method for construction of non-marker large fragment deletion yeast auxotroph strains: A practice in Torulopsis glabrata. J. Microbiol. Methods. 76(1), 70-74. 
[50] Zhou, J., Yin, X., Madzak, C., Du, G., Chen, J., 2012. Enhanced alpha-ketoglutarate production in Yarrowia lipolytica WSH-Z06 by alteration of the acetyl-CoA metabolism. J. Biotechnol. 161(3), 257-264. 
[51] Zhou, J., Zhou, H., Du, G., Liu, L., Chen, J., 2010. Screening of a thiamine-auxotrophic yeast for alpha-ketoglutaric acid overproduction. Lett. Appl. Microbiol. 51(3), 264-271. 
[52] Zhu, P., Zhang, C., Chen, J.Y., Zeng, X., 2024. Multilevel systemic engineering of Bacillus licheniformis for efficient production of acetoin from lignocellulosic hydrolysates. Int. J. Biol. Macromol. 279(Part 1) ,135142.
Biofuel Research Journal
Volume 12, Issue 4 - Serial Number 4
Autumn 2025
Pages 2569-2581