Metabolomic Alterations Do Not Induce Metabolic Burden in the Industrial Yeast M2n[pBKD2-Pccbgl1]-C1 Engineered by Multiple δ-Integration of a Fungal β-Glucosidase Gene.

Lorenzo Favaro,Sergio Casella,Luca Roscini,Gianluigi Cardinali,Laura Treu,Laura Corte,Marina Basaglia,Willem H Van Zyl,Lorenzo Cagnin,Stefano Campanaro,Fabio De Pascale

doi:10.3389/fbioe.2019.00376

Abstract

In the lignocellulosic yeast development, metabolic burden relates to redirection of resources from regular cellular activities toward the needs created by recombinant protein production. As a result, growth parameters may be greatly affected. Noteworthy, Saccharomyces cerevisiae M2n[pBKD2-Pccbgl1]-C1, previously developed by multiple δ-integration of the β-glucosidase BGL3, did not show any detectable metabolic burden. This work aims to test the hypothesis that the metabolic burden and the metabolomic perturbation induced by the δ-integration of a yeast strain, could differ significantly. The engineered strain was evaluated in terms of metabolic performances and metabolomic alterations in different conditions typical of the bioethanol industry. Results indicate that the multiple δ-integration did not affect the ability of the engineered strain to grow on different carbon sources and to tolerate increasing concentrations of ethanol and inhibitory compounds. Conversely, metabolomic profiles were significantly altered both under growing and stressing conditions, indicating a large extent of metabolic reshuffling involved in the maintenance of the metabolic homeostasis. Considering that four copies of BGL3 gene have been integrated without affecting any parental genes or promoter sequences, deeper studies are needed to unveil the mechanisms implied in these metabolomic changes, thus supporting the optimization of protein production in engineered strains.

Highlights

Today, bioethanol as major biofuel is mostly obtained from corn, wheat, and sugarcane (Mohanty and Swain, 2019)
The evidence that both parental M2n and recombinant C1 strains displayed similar ethanol yield and growth rate from glucose suggested us that the multiple δ-integration and expression of the BGL3 genes did not result in any evident metabolic burden when strains were grown in glucose under oxygen limiting conditions (Cagnin et al, 2019)
Aerobic growth determined in microtiter plates on glycerol (1.8%) and the equivalent amount of glucose (2%) showed similar μmax values for both strains, confirming that the integration of multiple copies of this cellulase does not impose metabolic burdens on yeast metabolism in terms of growth performances

Summary

Introduction

Bioethanol as major biofuel is mostly obtained from corn, wheat, and sugarcane (Mohanty and Swain, 2019). During pre-treatment, lignocellulosic material is partly degraded to inhibitory compounds, such as furans, weak acids and phenolics, which are toxic to the microbial metabolism. These inhibitors can slow down or even stop the fermentation, limiting the process efficiency (Almeida et al, 2007). The development of engineered S. cerevisiae strains able to produce one or more cellulolytic enzymes is required. Such new phenotypic traits can be obtained by engineering robust yeast strains to produce one or more heterologous cellulases (Van Zyl et al, 2007). Noteworthy advancement has been made, a deeper understanding of the mechanisms governing heterologous protein production in yeast will be crucial for developing more efficient protein production systems

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