Identification of key DNA elements involved in promoter recognition by Mxr1p, a master regulator of methanol utilization pathway in Pichia pastoris

Abstract Background Methanol expression regulator 1 (Mxr1p) is a key transcription factor that plays a vital role in the methanol utilization pathway in Pichia pastoris ( P. pastoris ). Most genes referred to the methanol utilization pathway were regulated by Mxr1p. However, some genes did not show a significant difference between methanol and glycerol even though they play an important role in the methanol utilization pathway. So far, the regulation mechanism about these genes and the relationship with Mxr1p are still unknown. Results Methanol metabolic pathway analysis revealed that most of the methanol-induced genes were upregulated in transcriptional level when cultured in methanol. Whereas some genes like tkl1 (transketolase 1) did not show significant up-regulation in methanol even though it plays a very important role in Xu5P recycle, the reason is still not clear. To clarify this point, firstly, pull-down and MS experiments were performed. The result shows that Tkl1p protein combined with Mxr1p in vitro . Subsequently, this result was further confirmed by yeast two-hybrid in vivo , and the binding region mainly located from 150AA to 400AA. Moreover, Ser215 phosphorylation did not affect this interaction. In addition, Mxr1p-400AA integration into Δmxr1 could rescue cell growth in methanol. All the above results proved that Mxr1p played a post-translational role in the methanol utilization pathway and Mxr1p-400AA may achieved most of Mxr1p functions. Secondly, the function of Mxr1p-Tkl1p complex was expounded by detecting formaldehyde consumption and xylulose production in cell-free systems. Results showed that Mxr1p-Tkl1p mixture could promote formaldehyde consumption and xylulose production in vitro . Conclusion Mxr1p promotes methanol utilization via combining with Tkl1p to accelerate Xu5P recycle and this interaction was not affected by Ser215 phosphorylation.

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Effect of nitrogen source on gene expression of first steps of methanol utilization pathway in Pichia pastoris

Russian Journal of Genetics ◽

10.1134/s102279541304011x ◽

2013 ◽

Vol 49 (4) ◽

pp. 394-400 ◽

Cited By ~ 2

Author(s):

A. M. Rumjantsev ◽

M. V. Padkina ◽

E. V. Sambuk

Keyword(s):

Gene Expression ◽

Pichia Pastoris ◽

Nitrogen Source ◽

Methanol Utilization ◽

Utilization Pathway

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Characterization of methanol utilization negative Pichia pastoris for secreted protein production: New cultivation strategies for current and future applications

Biotechnology and Bioengineering ◽

10.1002/bit.27303 ◽

2020 ◽

Vol 117 (5) ◽

pp. 1394-1405 ◽

Cited By ~ 1

Author(s):

Domen Zavec ◽

Brigitte Gasser ◽

Diethard Mattanovich

Keyword(s):

Pichia Pastoris ◽

Protein Production ◽

Secreted Protein ◽

Methanol Utilization

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Mixing and matching methylotrophic enzymes to design a novel methanol utilization pathway in E. coli

Metabolic Engineering ◽

10.1016/j.ymben.2020.07.005 ◽

2020 ◽

Vol 61 ◽

pp. 315-325

Author(s):

A. De Simone ◽

C.M. Vicente ◽

C. Peiro ◽

L. Gales ◽

F. Bellvert ◽

...

Keyword(s):

E Coli ◽

Methanol Utilization ◽

Utilization Pathway

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Peroxisomal Targeting, Import, and Assembly of Alcohol Oxidase in Pichia pastoris

The Journal of Cell Biology ◽

10.1083/jcb.139.6.1419 ◽

1997 ◽

Vol 139 (6) ◽

pp. 1419-1431 ◽

Cited By ~ 48

Author(s):

Hans R. Waterham ◽

Kimberly A. Russell ◽

Yne de Vries ◽

James M. Cregg

Keyword(s):

Pichia Pastoris ◽

Matrix Protein ◽

Alcohol Oxidase ◽

Mutant Form ◽

Wild Type ◽

Epitope Tag ◽

Peroxisomal Targeting ◽

Targeting Signal ◽

Terminal Amino ◽

Utilization Pathway

Alcohol oxidase (AOX), the first enzyme in the yeast methanol utilization pathway is a homooctameric peroxisomal matrix protein. In peroxisome biogenesis-defective (pex) mutants of the yeast Pichia pastoris, AOX fails to assemble into active octamers and instead forms inactive cytoplasmic aggregates. The apparent inability of AOX to assemble in the cytoplasm contrasts with other peroxisomal proteins that are able to oligomerize before import. To further investigate the import of AOX, we first identified its peroxisomal targeting signal (PTS). We found that sequences essential for targeting AOX are primarily located within the four COOH-terminal amino acids of the protein leucine-alanine-arginine-phenylalanine COOH (LARF). To examine whether AOX can oligomerize before import, we coexpressed AOX without its PTS along with wild-type AOX and determined whether the mutant AOX could be coimported into peroxisomes. To identify the mutant form of AOX, the COOH-terminal LARF sequence of the protein was replaced with a hemagglutinin epitope tag (AOX–HA). Coexpression of AOX–HA with wild-type AOX (AOX-WT) did not result in an increase in the proportion of AOX–HA present in octameric active AOX, suggesting that newly synthesized AOX–HA cannot oligomerize with AOX-WT in the cytoplasm. Thus, AOX cannot initiate oligomerization in the cytoplasm, but must first be targeted to the organelle before assembly begins.

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Enhancement of thermostable β-glucosidase production in a slow methanol utilization strain of Pichia pastoris by optimization of the specific methanol supply rate

Biotechnology and Bioprocess Engineering ◽

10.1007/s12257-014-0686-0 ◽

2015 ◽

Vol 20 (2) ◽

pp. 315-323 ◽

Cited By ~ 1

Author(s):

Theppanya Charoenrat ◽

Kanyalak Sangprapai ◽

Peerada Promdonkoy ◽

Kanokarn Kocharin ◽

Sutipa Tanapongpipat ◽

...

Keyword(s):

Pichia Pastoris ◽

Supply Rate ◽

Methanol Utilization

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Methanol-Dependent Gene Expression Demonstrates that Methyl-Coenzyme M Reductase Is Essential in Methanosarcina acetivorans C2A and Allows Isolation of Mutants with Defects in Regulation of the Methanol Utilization Pathway

Journal of Bacteriology ◽

10.1128/jb.187.16.5552-5559.2005 ◽

2005 ◽

Vol 187 (16) ◽

pp. 5552-5559 ◽

Cited By ~ 28

Author(s):

Michael Rother ◽

Paolo Boccazzi ◽

Arpita Bose ◽

Matthew A. Pritchett ◽

W. W. Metcalf

Keyword(s):

Proteome Analysis ◽

Genetic System ◽

Methanosarcina Acetivorans ◽

Coenzyme M ◽

Prolonged Incubation ◽

Methanol Utilization ◽

Expression Of Genes ◽

Regulatory Mutations ◽

Utilization Pathway ◽

Methyl Coenzyme M Reductase

ABSTRACT Methanosarcina acetivorans C2A is able to convert several substrates to methane via at least four distinct methanogenic pathways. A common step in each of these pathways is the reduction of methyl-coenzyme M (CoM) to methane catalyzed by methyl-CoM reductase (MCR). Because this enzyme is used in each of the known pathways, the mcrBDCGA operon, which encodes MCR, is expected to be essential. To validate this prediction, a system for conditional gene inactivation was developed. A heterologous copy of the mcrBDCGA operon was placed under the control of the highly regulated mtaC1 promoter, which directs the expression of genes involved in methanol utilization, and recombined onto the M. acetivorans chromosome. This allowed for disruption of the endogenous mcr operon in the presence of methanol. Because the PmtaC1 promoter is transcribed only during growth on methanol, mcrBDCGA was rendered methanol dependent and the strain was unable to grow in trimethylamine media, strongly suggesting that mcrBDCGA is essential. Upon prolonged incubation, suppressed mutants which expressed mcrBDCGA constitutively could be selected. Expression analysis of PmtaC1::uidA gene fusions in several isolated suppressed mutants suggests that they carry trans-active mutations leading to deregulation of all genes under control of this promoter. Subsequently, proteome analysis of one such suppressed mutant revealed that all known proteins derived from mtaC1 promoter-dependent expression were constitutively expressed in this mutant. This genetic system can therefore be employed for the testing of essential genes and for the identification of genes under a common regulatory mechanism by making regulatory mutations phenotypically selectable.

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