Regulating ehrlich and demethiolation pathways for alcohols production by the expression of ubiquitin-protein ligase gene HUWE1.

Quan Zhang,Shi-Tao Xia,Rui-Sang Liu,Kai-Zhi Jia,Ya-Jie Tang,Hong-Mei Li,Yang-Hua Xu

doi:10.1038/srep20828

Quan Zhang, Shi-Tao Xia + Show 5 more

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https://doi.org/10.1038/srep20828

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Journal: Scientific Reports	Publication Date: Feb 10, 2016
Citations: 11	License type: CC BY 4.0

Affiliation: Hubei University of Technology

Abstract

Ehrlich and demethiolation pathways as two competing branches converted amino acid into alcohols. Controlling both pathways offers considerable potential for industrial applications including alcohols overproduction, flavor-quality control and developing new flavors. While how to regulate ehrlich and demethiolation pathways is still not applicable. Taking the conversion of methionine into methionol and methanethiol for example, we constructed two suppression subtractive cDNA libraries of Clonostachys rosea by using suppression subtractive hybridization (SSH) technology for screening regulators controlling the conversion. E3 ubiquitin-protein ligase gene HUWE1 screened from forward SSH library was validated to be related with the biosynthesis of end products. Overexpressing HUWE1 in C. rosea and S. cerevisiae significantly increased the biosynthesis of methanethiol and its derivatives in demethiolation pathway, while suppressed the biosynthesis of methional and methionol in ehrlich pathway. These results attained the directional regulation of both pathways by overexpressing HUWE1. Thus, HUWE1 has potential to be a key target for controlling and enhancing alcohols production by metabolic engineering.

Highlights

Ehrlich pathway genes including aminotransferase genes ARO8, BAT and α -ketoacid decarboxylase gene pyruvate decarboxylase (PDC) and demethiolation pathway gene STR3 are highly conserved in eukaryotes
To enrich the differentially expressed genes associated with alcohols production, suppression subtractive hybridization PCR was adopted in this study
Met addition in fermentation medium made HUWE1 overexpression strain U1 produce trace amount of methional and methionol of ehrlich pathway, but much more KMBA of intermediate, MTL and dimethyl sulfide (DMS) of demethiolation pathway (Figs 3A–E and S4A–E). These results suggested that HUWE1 with HECT domain mediate the regulation of ehrlich and demethiolation pathways

Summary

Introduction

Ehrlich pathway genes including aminotransferase genes ARO8, BAT and α -ketoacid decarboxylase gene PDC and demethiolation pathway gene STR3 are highly conserved in eukaryotes Their coordinated expression at transcriptional level controlled the degradation of amino acids[14,15]. To achieve rational regulation of the two pathways, we constructed a suppression subtractive cDNA library and screened differentially expressed genes especially regulator genes associated with the biosynthesis of end products. The effects of these regulators on the production of ehrlich and demethiolation pathway metabolites were analyzed in Clonostachys rosea and Saccharomyces cerevisiae. Our findings support the rational regulation of ehrlich and demethiolation pathways for enhancing alcohols production, the quality optimization and fermentation control

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