Methanol-inducible gene expression and heterologous protein production in the methylotrophic yeast Candida boidinii

ABSTRACT We identified genes encoding components of the Hap complex, CbHAP2 , CbHAP3 , and CbHAP5 , as transcription factors regulating methanol-inducible gene expression in the methylotrophic yeast Candida boidinii . We found that the Cbhap2 Δ, Cbhap3 Δ, and Cbhap5 Δ gene-disrupted strains showed severe growth defects on methanol but not on glucose and nonfermentable carbon sources such as ethanol and glycerol. In these disruptants, the transcriptional activities of methanol-inducible promoters were significantly decreased compared to those of the wild-type strain, indicating that CbHap2p, CbHap3p, and CbHap5p play indispensable roles in methanol-inducible gene expression. Further molecular and biochemical analyses demonstrated that CbHap2p, CbHap3p, and CbHap5p localized to the nucleus and bound to the promoter regions of methanol-inducible genes regardless of the carbon source, and heterotrimer formation was suggested to be necessary for binding to DNA. Unexpectedly, distinct from Saccharomyces cerevisiae , the Hap complex functioned in methanol-specific induction rather than glucose derepression in C. boidinii . Our results shed light on a novel function of the Hap complex in methanol-inducible gene expression in methylotrophic yeasts.

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Differences in the inducible gene expression and protein production of IL-12p40, IL-12p70 and IL-23: Involvement of p38 and JNK kinase pathways

Cytokine ◽

10.1016/j.cyto.2008.04.003 ◽

2008 ◽

Vol 43 (1) ◽

pp. 76-82 ◽

Cited By ~ 21

Author(s):

Zlatka Georgieva Dobreva ◽

Spaska Angelova Stanilova ◽

Lyuba Dineva Miteva

Keyword(s):

Gene Expression ◽

Protein Production ◽

Inducible Gene Expression ◽

Inducible Gene

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Multiplexed CRISPR-mediated engineering of protein secretory pathway genes in the thermotolerant methylotrophic yeast Ogataea thermomethanolica

PLoS ONE ◽

10.1371/journal.pone.0261754 ◽

2021 ◽

Vol 16 (12) ◽

pp. e0261754

Author(s):

Worarat Kruasuwan ◽

Aekkachai Puseenam ◽

Sutipa Tanapongpipat ◽

Niran Roongsawang

Keyword(s):

Protein Production ◽

Secretory Pathway ◽

Genome Engineering ◽

Heterologous Protein ◽

Methylotrophic Yeast ◽

Strain Engineering ◽

Heterologous Protein Production ◽

Heterologous Protein Secretion ◽

Multiple Genes ◽

Simultaneous Activation

CRISPR multiplex gRNA systems have been employed in genome engineering in various industrially relevant yeast species. The thermotolerant methylotrophic yeast Ogataea thermomethanolica TBRC 656 is an alternative host for heterologous protein production. However, the limited secretory capability of this yeast is a bottleneck for protein production. Here, we refined CRISPR-based genome engineering tools for simultaneous mutagenesis and activation of multiple protein secretory pathway genes to improve heterologous protein secretion. We demonstrated that multiplexed CRISPR-Cas9 mutation of up to four genes (SOD1, VPS1, YPT7 and YPT35) in one single cell is practicable. We also developed a multiplexed CRISPR-dCas9 system which allows simultaneous activation of multiple genes in this yeast. 27 multiplexed gRNA combinations were tested for activation of three genes (SOD1, VPS1 and YPT7), three of which were demonstrated to increase the secretion of fungal xylanase and phytase up to 29% and 41%, respectively. Altogether, our study provided a toolkit for mutagenesis and activation of multiple genes in O. thermomethanolica, which could be useful for future strain engineering to improve heterologous protein production in this yeast.

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