# Methanol-induced transcription factor Mpp1 regulates the coordinated expression of multiple genes to achieve a balanced C1 metabolism in the methylotrophic yeast Candida boidinii

## TL;DR

This study shows how a methanol-induced transcription factor helps balance metabolism in a yeast used for protein production.

## Contribution

The study identifies CbMpp1 as a key regulator of methanol-induced gene expression in Candida boidinii, revealing its role in balanced C1 metabolism.

## Key findings

- CbMpp1 expression is regulated by CRMI and is essential for optimal methanol-based growth.
- Excessive CbMpp1 represses methanol-inducible genes and causes metabolic imbalances.
- CRMI-dependent regulation of CbMpp1 and KpMit1 is crucial for coordinated gene expression.

## Abstract

Methylotrophic yeasts can grow on methanol as the sole carbon and energy source. They have been used as hosts for heterologous protein production, which is achieved by their strongly and tightly regulated methanol-induced gene promoters. The expression levels of methanol-induced genes, such as AOD1 and DAS1, are regulated by concentration-regulated methanol induction (CRMI). In this study, we identified CbMpp1 as a methanol-induced transcription factor necessary for the expression of multiple genes in the methylotrophic yeast Candida boidinii and identified the upstream activating sequences in the CbMPP1 promoter. The expression of CbMPP1 was induced by methanol under the control of CRMI and was dependent on other transcription factors, CbTrm1, CbTrm2, and CbHap complex but not on CbMpp1 itself. To investigate the physiological significance of the CRMI-dependent expression of CbMPP1, we constructed strains overexpressing or constitutively expressing CbMPP1 with varying copy numbers. Analysis of growth with methanol and transcript levels of methanol-induced genes of these strains revealed that an appropriate amount of CbMpp1 was required for optimal growth with methanol and that excessive amounts of CbMpp1 repressed the expression of methanol-inducible genes. Such abnormal expression of CbMPP1 in these strains caused accumulation of formaldehyde and growth defects on methanol. In addition, the Komagataella phaffii strain constitutively expressing KpMIT1 (a homolog of CbMPP1) at low levels exhibited retarded growth with methanol. Our results demonstrate that CbMpp1 and KpMit1, whose expression is regulated by CRMI, regulate the coordinated expression of multiple methanol-induced genes to achieve a balanced one-carbon metabolism.

Elucidating the molecular mechanism of how methanol-induced gene expression is regulated to attain a balanced one-carbon (C1) metabolism is important for determining the efficiency of heterologous protein production by methylotrophic yeasts. Although transcription factors involved in methanol-induced gene expression in these yeasts have been studied extensively, how these transcription factors regulate the expression of multiple methanol-induced genes in a concentration-regulated methanol induction (CRMI)-dependent manner has been unclear. In this study, we focused on the physiological significance of methanol inducibility of CbMpp1 and KpMit1 and found that CRMI-dependent CbMPP1 and KpMIT1 expressions are important for the coordinated regulation of multiple methanol-induced genes to facilitate a balanced C1 metabolism in the methylotrophic yeast.