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Updated: Mar 13, 2026

Methanol Independent Expression by Pichia Pastoris Employing De-repression Technologies

Methanol Independent Expression by Pichia Pastoris Employing De-repression Technologies

Published on: January 23, 2019

Scaffoldless engineered enzyme assembly for enhanced methanol utilization.

J Vincent Price¹, Long Chen¹, W Brian Whitaker^1,2

¹Department of Chemical and Biomolecular Engineering, University of Delaware, Newark, DE 19716.

Proceedings of the National Academy of Sciences of the United States of America

|October 30, 2016

Summary

This summary is machine-generated.

Researchers engineered a supramolecular enzyme complex for efficient methanol conversion. This strategy significantly boosted methanol consumption and fructose-6-phosphate production in vitro and in vivo.

Keywords:

methane methylotophs scaffold substrate channeling supramolcular

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Published on: October 24, 2016

Area of Science:

Biotechnology
Metabolic Engineering
Synthetic Biology

Background:

Methanol is a key feedstock, but its biological conversion into metabolites is limited.
Natural methanol dehydrogenase favors the reverse reaction, hindering formaldehyde sequestration.
Spatial organization of enzymes can enhance metabolic pathway efficiency.

Purpose of the Study:

To develop engineered microorganisms for efficient methanol utilization.
To create a supramolecular enzyme complex for enhanced methanol conversion.
To improve the production of fructose-6-phosphate (F6P) from methanol.

Main Methods:

Utilized a scaffoldless, self-assembly strategy with SH3-ligand interaction to organize Mdh, Hps, and Phi enzymes.
Implemented an "NADH Sink" using E. coli lactate dehydrogenase to prevent reversible formaldehyde reduction.
Combined supramolecular assembly with NADH sink for enhanced in vitro and in vivo methanol conversion.

Main Results:

Engineered supramolecular enzyme complex enhanced methanol conversion to F6P by 97-fold in vitro compared to unassembled enzymes.
The in vivo whole-cell methanol consumption rate increased ninefold with the engineered enzyme assembly.
Demonstrated efficient substrate channeling and improved carbon flux in the desired direction.

Conclusions:

Scaffoldless self-assembly of enzymes into a supramolecular complex is an effective strategy for enhancing methanol bioconversion.
The combined approach of enzyme assembly and NADH sink significantly boosts methanol consumption and F6P production.
This platform enables direct coupling of F6P synthesis with other metabolic engineering strategies for metabolite production from methanol.