Molecular Basis of Methanol-Inducible Gene Expression and Its Application in the Methylotrophic YeastCandida boidinii

Abstract

Methanol is a promising feedstock for biotechnological and chemical processes as well as a primary source of energy to replace coal and petroleum. Methylotrophic yeasts, that can utilize methanol as the sole source of carbon and energy, have been studied intensively in terms of both physiological activities and potential applications. During growth on methanol, the enzymes involved in methanol metabolism are massively produced in these yeasts, indicating that the gene promoters of these enzymes are strong methanol-inducible promoters. Using these promoters, high-level heterologous gene expression systems have been developed in several methylotrophic yeast strains, such as Pichia pastoris, Hansenula polymorpha, and Candida boidinii. To achieve efficient industrial use of methanol and efficient protein production by methylotrophic yeasts, it is important to elucidate the molecular basis of methanolinducible gene expression in these yeasts. This review describes recent advances in understanding of the regulation of methanol-inducible gene expression and the molecular mechanism of transcriptional activation in the methylotrophic yeast C. boidinii. Application of this gained knowledge led to successful production of useful enzymes in this yeast, which is also reviewed.

Molecular Characterization of Hap Complex Components Responsible for Methanol-Inducible Gene Expression in the Methylotrophic Yeast Candida boidinii

“…Efficient production has been achieved with their strong and tightly regulated methanolinducible gene promoters in combination with high-cell-density cultures in methanol-containing medium. The genes encoding methanol-metabolic enzymes are highly induced in the presence of methanol, i.e., alcohol oxidase (AOD), dihydroxyacetone synthase (DAS), glutathione-dependent formaldehyde dehydrogenase (FLD), and formate dehydrogenase (FDH), and these promoter regions have been used commercially for heterologous gene expression (5)(6)(7).…”

Unique C-terminal region of Hap3 is required for methanol-regulated gene expression in the methylotrophic yeast Candida boidinii

“…Exhaustion of glucose releases glucose repression, resulting in activation of methanol-regulated genes by CbTrm2p, which does not require methanol for gene activation (derepression). In addition, the presence of methanol induces maximum activation of methanolregulated genes via CbTrm1p (methanol induction; methanol-specific induction) (Hartner & Glieder, 2006;Sasano et al, 2008;Yurimoto, 2009;Sasano et al, 2010;Zhai et al, 2012).…”

Selection of suitably non-repressing carbon sources for expression of alcohol oxidase isozyme promoters in the methylotrophic yeast Pichia methanolica

“…Moreover, the expressions of MOD1 and MOD2 were respectively controlled through a distinct regulatory mechanism against carbon sources (9), methanol concentration (10), and oxygen concentration (12). Therefore, we believe that P. methanolica has an advantageous ability to develop a unique gene-expression system using the MOD2 promoter, P MOD2 , together with the MOD1 promoter, P MOD1 , compared with other major methylotrophic yeasts, such as P. pastoris, H. polymorpha and C. boidinii.For the industrial use of methylotrophic yeast strains, it is important to understand the growth aspects of each strain on several carbon sources, because the methanol metabolic pathway is induced by methanol and is repressed completely by glucose or other carbon sources (7,9,12,14). For example, in P. pastoris, expression of the AOD promoter is completely repressed by both glucose and glycerol (14).…”

“…For the industrial use of methylotrophic yeast strains, it is important to understand the growth aspects of each strain on several carbon sources, because the methanol metabolic pathway is induced by methanol and is repressed completely by glucose or other carbon sources (7,9,12,14). For example, in P. pastoris, expression of the AOD promoter is completely repressed by both glucose and glycerol (14).…”