Triggering Respirofermentative Metabolism in the Crabtree-negative Yeast Pichia Guilliermondii by Disrupting the CAT8 Gene

Overview

Journal Appl Environ Microbiol

Specialties Environmental Health
Microbiology

Date 2014 Apr 22

PMID 24747899

Citations 5

Authors

Kai Qi

Jian-Jiang Zhong

Xiao-Xia Xia

Affiliations

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Abstract

Pichia guilliermondii is a Crabtree-negative yeast that does not normally exhibit respirofermentative metabolism under aerobic conditions, and methods to trigger this metabolism may have applications for physiological study and industrial applications. In the present study, CAT8, which encodes a putative global transcriptional activator, was disrupted in P. guilliermondii. This yeast's ethanol titer increased by >20-fold compared to the wild type (WT) during aerobic fermentation using glucose. A comparative transcriptional analysis indicated that the expression of genes in the tricarboxylic acid cycle and respiratory chain was repressed in the CAT8-disrupted (ΔCAT8) strain, while the fermentative pathway genes were significantly upregulated. The respiratory activities in the ΔCAT8 strain, indicated by the specific oxygen uptake rate and respiratory state value, decreased to one-half and one-third of the WT values, respectively. In addition, the expression of HAP4, a transcriptional respiratory activator, was significantly repressed in the ΔCAT8 strain. Through disruption of HAP4, the ethanol production of P. guilliermondii was also increased, but the yield and titer were lower than that in the ΔCAT8 strain. A further transcriptional comparison between ΔCAT8 and ΔHAP4 strains suggested a more comprehensive reprogramming function of Cat8 in the central metabolic pathways. These results indicated the important role of CAT8 in regulating the glucose metabolism of P. guilliermondii and that the regulation was partially mediated by repressing HAP4. The strategy proposed here might be applicable to improve the aerobic fermentation capacity of other Crabtree-negative yeasts.

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References

Kocharin K, Chen Y, Siewers V, Nielsen J . Engineering of acetyl-CoA metabolism for the improved production of polyhydroxybutyrate in Saccharomyces cerevisiae. AMB Express. 2012; 2(1):52. PMC: 3519744. DOI: 10.1186/2191-0855-2-52. View

Shi N, Davis B, Sherman F, Cruz J, Jeffries T . Disruption of the cytochrome c gene in xylose-utilizing yeast Pichia stipitis leads to higher ethanol production. Yeast. 1999; 15(11):1021-30. DOI: 10.1002/(SICI)1097-0061(199908)15:11<1021::AID-YEA429>3.0.CO;2-V. View

Tachibana C, Yoo J, Tagne J, Kacherovsky N, Lee T, Young E . Combined global localization analysis and transcriptome data identify genes that are directly coregulated by Adr1 and Cat8. Mol Cell Biol. 2005; 25(6):2138-46. PMC: 1061606. DOI: 10.1128/MCB.25.6.2138-2146.2005. View

Raab A, Gebhardt G, Bolotina N, Weuster-Botz D, Lang C . Metabolic engineering of Saccharomyces cerevisiae for the biotechnological production of succinic acid. Metab Eng. 2010; 12(6):518-25. DOI: 10.1016/j.ymben.2010.08.005. View

Boretsky Y, Pynyaha Y, Boretsky V, Kutsyaba V, Protchenko O, Philpott C . Development of a transformation system for gene knock-out in the flavinogenic yeast Pichia guilliermondii. J Microbiol Methods. 2007; 70(1):13-9. DOI: 10.1016/j.mimet.2007.03.004. View

Papon N, Savini V, Lanoue A, Simkin A, Creche J, Giglioli-Guivarch N . Candida guilliermondii: biotechnological applications, perspectives for biological control, emerging clinical importance and recent advances in genetics. Curr Genet. 2013; 59(3):73-90. DOI: 10.1007/s00294-013-0391-0. View

Zou Y, Qi K, Chen X, Miao X, Zhong J . Favorable effect of very low initial K(L)a value on xylitol production from xylose by a self-isolated strain of Pichia guilliermondii. J Biosci Bioeng. 2010; 109(2):149-52. DOI: 10.1016/j.jbiosc.2009.07.013. View

Ostergaard S, Olsson L, Johnston M, Nielsen J . Increasing galactose consumption by Saccharomyces cerevisiae through metabolic engineering of the GAL gene regulatory network. Nat Biotechnol. 2000; 18(12):1283-6. DOI: 10.1038/82400. View

Freese S, Passoth V, Klinner U . A mutation in the COX5 gene of the yeast Scheffersomyces stipitis alters utilization of amino acids as carbon source, ethanol formation and activity of cyanide insensitive respiration. Yeast. 2011; 28(4):309-20. DOI: 10.1002/yea.1840. View

10.

Agrimi G, Brambilla L, Frascotti G, Pisano I, Porro D, Vai M . Deletion or overexpression of mitochondrial NAD+ carriers in Saccharomyces cerevisiae alters cellular NAD and ATP contents and affects mitochondrial metabolism and the rate of glycolysis. Appl Environ Microbiol. 2011; 77(7):2239-46. PMC: 3067453. DOI: 10.1128/AEM.01703-10. View

11.

Pronk J, Steensma H, van Dijken J . Pyruvate metabolism in Saccharomyces cerevisiae. Yeast. 1996; 12(16):1607-33. DOI: 10.1002/(sici)1097-0061(199612)12:16<1607::aid-yea70>3.0.co;2-4. View

12.

Brons J, de Jong M, Valens M, Grivell L, Bolotin-Fukuhara M, Blom J . Dissection of the promoter of the HAP4 gene in S. cerevisiae unveils a complex regulatory framework of transcriptional regulation. Yeast. 2002; 19(11):923-32. DOI: 10.1002/yea.886. View

13.

Sybirna K, Guiard B, Li Y, Bao W, Bolotin-Fukuhara M, Delahodde A . A new Hansenula polymorpha HAP4 homologue which contains only the N-terminal conserved domain of the protein is fully functional in Saccharomyces cerevisiae. Curr Genet. 2004; 47(3):172-81. DOI: 10.1007/s00294-004-0556-y. View

14.

Fan C, Qi K, Xia X, Zhong J . Efficient ethanol production from corncob residues by repeated fermentation of an adapted yeast. Bioresour Technol. 2013; 136:309-15. DOI: 10.1016/j.biortech.2013.03.028. View

15.

van Dam K . Role of glucose signaling in yeast metabolism. Biotechnol Bioeng. 1996; 52(1):161-5. DOI: 10.1002/(SICI)1097-0290(19961005)52:1<161::AID-BIT16>3.0.CO;2-R. View

16.

Charbon G, Breunig K, Wattiez R, Vandenhaute J, Noel-Georis I . Key role of Ser562/661 in Snf1-dependent regulation of Cat8p in Saccharomyces cerevisiae and Kluyveromyces lactis. Mol Cell Biol. 2004; 24(10):4083-91. PMC: 400452. DOI: 10.1128/MCB.24.10.4083-4091.2004. View

17.

Christen S, Sauer U . Intracellular characterization of aerobic glucose metabolism in seven yeast species by 13C flux analysis and metabolomics. FEMS Yeast Res. 2011; 11(3):263-72. DOI: 10.1111/j.1567-1364.2010.00713.x. View

18.

Aguilaniu H, Gustafsson L, Rigoulet M, Nystrom T . Protein oxidation in G0 cells of Saccharomyces cerevisiae depends on the state rather than rate of respiration and is enhanced in pos9 but not yap1 mutants. J Biol Chem. 2001; 276(38):35396-404. DOI: 10.1074/jbc.M101796200. View

19.

Raab A, Hlavacek V, Bolotina N, Lang C . Shifting the fermentative/oxidative balance in Saccharomyces cerevisiae by transcriptional deregulation of Snf1 via overexpression of the upstream activating kinase Sak1p. Appl Environ Microbiol. 2011; 77(6):1981-9. PMC: 3067313. DOI: 10.1128/AEM.02219-10. View

20.

Bonander N, Ferndahl C, Mostad P, Wilks M, Chang C, Showe L . Transcriptome analysis of a respiratory Saccharomyces cerevisiae strain suggests the expression of its phenotype is glucose insensitive and predominantly controlled by Hap4, Cat8 and Mig1. BMC Genomics. 2008; 9:365. PMC: 2536679. DOI: 10.1186/1471-2164-9-365. View