Two homologs of the Cat8 transcription factor are involved in the regulation of ethanol utilization in Komagataella phaffii

Diane Barbay,Monika Mačáková,Leander Sützl,Diethard Mattanovich,Sonakshi De,Brigitte Gasser

doi:10.1007/s00294-021-01165-4

Abstract

The transcription factors Cat8 and Sip4 were described in Saccharomyces cerevisiae and Kluyveromyces lactis to have very similar DNA binding domains and to be necessary for derepression of a variety of genes under non-fermentative growth conditions via binding to the carbon source responsive elements (CSREs). The methylotrophic yeast Komagataella phaffii (syn Pichia pastoris) has two transcription factors (TFs), which are putative homologs of Cat8 based on sequence similarity, termed Cat8-1 and Cat8-2. It is yet unclear in which cellular processes they are involved and if one of them is actually the homolog of Sip4. To study the roles of the Cat8 homologs in K. phaffii, overexpression or deletion strains were generated for the two TFs. The ability of these mutant strains to grow on different carbon sources was tested, and transcript levels of selected genes from the carbon metabolism were quantified. Our experiments showed that the TFs are required for the growth of K. phaffii on C2 carbon sources, but not on glucose, glycerol or methanol. K. phaffii deleted for Cat8-1 showed impaired growth on acetate, while both Cat8-1 and Cat8-2 are involved in the growth of K. phaffii on ethanol. Correspondingly, both TFs are participating in the activation of ADH2, ALD4 and ACS1, three genes encoding enzymes important for the assimilation of ethanol. Different from S. cerevisiae and K. lactis, Cat8-1 is not regulating the transcription of the putative Sip4-family member Cat8-2 in K. phaffii. Furthermore, Cat8-1 is necessary for the activation of genes from the glyoxylate cycle, whereas Cat8-2 is necessary for the activation of genes from the carnitine shuttle. Neither Cat8-1 nor Cat8-2 are required for the activation of gluconeogenesis genes. Finally, the CAT8-2 gene is repressed by the Mig1-2 transcription factor on glucose and autorepressed by the Cat8-2 protein on all tested carbon sources. Our study identified the involvement of K. phaffii Cat8-1 and Cat8-2 in C2-metabolism, and highlighted similarities and differences to their homologs in other yeast species.

Highlights

The yeast Komagataella phaffii (Pichia pastoris) adapts to different growth conditions through various mechanisms, including reprogramming of gene expression and protein synthesis (Hartner and Glieder 2006; Lin-Cereghino et al 2006; Prielhofer et al 2015)
We studied the functions of two transcription factors named Cat8-1 and Cat8-2 in K. phaffii, which were identified as homologs of the transcription factor Cat8 from S. cerevisiae based on sequence similarity (Valli et al 2016)
The present study elucidated the requirement of the two Cat8 homologs Cat8-1 and Cat8-2 for activation of the ethanol assimilation pathway in K. phaffii, and their differential involvement in regulating the carnitine shuttle and the glyoxylate shunt, respectively

Summary

Introduction

The yeast Komagataella phaffii (Pichia pastoris) adapts to different growth conditions through various mechanisms, including reprogramming of gene expression and protein synthesis (Hartner and Glieder 2006; Lin-Cereghino et al 2006; Prielhofer et al 2015). Cat (CATabolite repression) and Sip (Snf interacting protein) possess a highly similar N-terminal zinc cluster (Zn(II)2Cys6) binding domain (Rahner et al 1996), but they share only little similarity in the rest of their protein sequences (Mehlgarten et al 2015; Turcotte et al 2010) Both Cat and Sip were shown to bind the CSRE (consensus sequence: YCCRTTNRNCGG) (Roth et al 2004; Vincent and Carlson 1998), Sip recognizes and binds to a more specific CSRE motif than Cat, which probably explains why Cat and Sip contribute unequally to gene activation via binding to this motif (Hiesinger et al 2001). In S. cerevisiae and K. lactis, Cat and Sip were described to be activators of transcription, but their mechanism of action in these two yeast are slightly different (Mehlgarten et al 2015)

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Conclusion