Dataset Information

Mit1 Transcription Factor Mediates Methanol Signaling and Regulates the Alcohol Oxidase 1 (AOX1) Promoter in Pichia pastoris.

ABSTRACT: The alcohol oxidase 1 (AOX1) promoter (P AOX1) of Pichia pastoris is the most powerful and commonly used promoter for driving protein expression. However, mechanisms regulating its transcriptional activity are unclear. Here, we identified a Zn(II)2Cys6-type methanol-induced transcription factor 1 (Mit1) and elucidated its roles in regulating PAOX1 activity in response to glycerol and methanol. Mit1 regulated the expression of many genes involved in methanol utilization pathway, including AOX1, but did not participate in peroxisome proliferation and transportation of peroxisomal proteins during methanol metabolism. Structural analysis of Mit1 by performing domain deletions confirmed its specific and critical role in the strict repression of P AOX1 in glycerol medium. Importantly, Mit1, Mxr1, and Prm1, which positively regulated P AOX1 in response to methanol, were bound to P AOX1 at different sites and did not interact with each other. However, these factors cooperatively activated P AOX1 through a cascade. Mxr1 mainly functioned during carbon derepression, whereas Mit1 and Prm1 functioned during methanol induction, with Prm1 transmitting methanol signal to Mit1 by binding to the MIT1 promoter (P MIT1), thus increasingly expressing Mit1 and subsequently activating P AOX1.

SUBMITTER: Wang X

PROVIDER: S-EPMC4813576 | biostudies-literature | 2016 Mar

REPOSITORIES: biostudies-literature

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Mit1 Transcription Factor Mediates Methanol Signaling and Regulates the Alcohol Oxidase 1 (AOX1) Promoter in Pichia pastoris.

Wang Xiaolong X Wang Qi Q Wang Jinjia J Bai Peng P Shi Lei L Shen Wei W Zhou Mian M Zhou Xiangshan X Zhang Yuanxing Y Cai Menghao M

The Journal of biological chemistry 20160131 12

The alcohol oxidase 1 (AOX1) promoter (P AOX1) of Pichia pastoris is the most powerful and commonly used promoter for driving protein expression. However, mechanisms regulating its transcriptional activity are unclear. Here, we identified a Zn(II)2Cys6-type methanol-induced transcription factor 1 (Mit1) and elucidated its roles in regulating PAOX1 activity in response to glycerol and methanol. Mit1 regulated the expression of many genes involved in methanol utilization pathway, including AOX1, b ...[more]

PMID: 26828066

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Project description:BackgroundThe methanol-regulated AOX1 promoter (PAOX1) is the most widely used promoter in the production of recombinant proteins in the methylotrophic yeast Pichia pastoris. However, as the tight regulation and methanol dependence of PAOX1 restricts its application, it is necessary to develop a flexible induction system to avoid the problems of methanol without losing the advantages of PAOX1. The availability of synthetic biology tools enables researchers to reprogram the cellular behaviour of P. pastoris to achieve this goal.ResultsThe characteristics of PAOX1 are highly related to the expression profile of methanol expression regulator 1 (Mxr1). In this study, we applied a biologically inspired strategy to reprogram regulatory networks in P. pastoris. A reprogrammed P. pastoris was constructed by inserting a synthetic positive feedback circuit of Mxr1 driven by a weak AOX2 promoter (PAOX2). This novel approach enhanced PAOX1 efficiency by providing extra Mxr1 and generated switchable Mxr1 expression to allow PAOX1 to be induced under glycerol starvation or carbon-free conditions. Additionally, the inhibitory effect of glycerol on PAOX1 was retained because the synthetic circuit was not activated in response to glycerol. Using green fluorescent protein as a demonstration, this reprogrammed P. pastoris strain displayed stronger fluorescence intensity than non-reprogrammed cells under both methanol induction and glycerol starvation. Moreover, with single-chain variable fragment (scFv) as the model protein, increases in extracellular scFv productivity of 98 and 269% were observed in Mxr1-reprogrammed cells under methanol induction and glycerol starvation, respectively, compared to productivity in non-reprogrammed cells under methanol induction.ConclusionsWe successfully demonstrate that the synthetic positive feedback circuit of Mxr1 enhances recombinant protein production efficiency in P. pastoris and create a methanol-free induction system to eliminate the potential risks of methanol.

Project description:Methanol, which produced in large quantities from low-quality coal and the hydrogenation of CO2, is a potentially renewable one-carbon (C1) feedstock for biomanufacturing. The methylotrophic yeast Pichia pastoris is an ideal host for methanol biotransformation given its natural capacity as a methanol assimilation system. However, the utilization efficiency of methanol for biochemical production is limited by the toxicity of formaldehyde. Therefore, reducing the toxicity of formaldehyde to cells remains a challenge to the engineering design of a methanol metabolism. Based on genome-scale metabolic models (GSMM) calculations, we speculated that reducing alcohol oxidase (AOX) activity would re-construct the carbon metabolic flow and promote balance between the assimilation and dissimilation of formaldehyde metabolism processes, thereby increasing the biomass formation of P. pastoris. According to experimental verification, we proved that the accumulation of intracellular formaldehyde can be decreased by reducing AOX activity. The reduced formaldehyde formation upregulated methanol dissimilation and assimilation and the central carbon metabolism, which provided more energy for the cells to grow, ultimately leading to an increased conversion of methanol to biomass, as evidenced by phenotypic and transcriptome analysis. Significantly, the methanol conversion rate of AOX-attenuated strain PC110-AOX1-464 reached 0.364 g DCW/g, representing a 14% increase compared to the control strain PC110. In addition, we also proved that adding a co-substrate of sodium citrate could further improve the conversion of methanol to biomass in the AOX-attenuated strain. It was found that the methanol conversion rate of the PC110-AOX1-464 strain with the addition of 6 g/L sodium citrate reached 0.442 g DCW/g, representing 20% and 39% increases compared to AOX-attenuated strain PC110-AOX1-464 and control strain PC110 without sodium citrate addition, respectively. The study described here provides insight into the molecular mechanism of efficient methanol utilization by regulating AOX. Reducing AOX activity and adding sodium citrate as a co-substrate are potential engineering strategies to regulate the production of chemicals from methanol in P. pastoris.

Dataset Information

Mit1 Transcription Factor Mediates Methanol Signaling and Regulates the Alcohol Oxidase 1 (AOX1) Promoter in Pichia pastoris.

Publications

Mit1 Transcription Factor Mediates Methanol Signaling and Regulates the Alcohol Oxidase 1 (AOX1) Promoter in Pichia pastoris.

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