Delta-Integration of Single Gene Shapes the Whole Metabolomic Short-Term Response to Ethanol of Recombinant Saccharomyces cerevisiae Strains.

Laura Corte,Debora Casagrande Pierantoni,Carla Emiliani,Roberto Maria Pellegrino,Lorenzo Favaro,Marina Basaglia,Luca Roscini,Gianluigi Cardinali,Sergio Casella

doi:10.3390/metabo10040140

Laura Corte, Debora Casagrande Pierantoni + Show 7 more

Open Access

https://doi.org/10.3390/metabo10040140

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Journal: Metabolites	Publication Date: Apr 3, 2020
Citations: 5	License type: CC BY 4.0

Affiliation: University of Perugia, University of Padua

Abstract

In yeast engineering, metabolic burden is often linked to the reprogramming of resources from regular cellular activities to guarantee recombinant protein(s) production. Therefore, growth parameters can be significantly influenced. Two recombinant strains, previously developed by the multiple δ-integration of a glucoamylase in the industrial Saccharomyces cerevisiae 27P, did not display any detectable metabolic burden. In this study, a Fourier Transform InfraRed Spectroscopy (FTIR)-based assay was employed to investigate the effect of δ-integration on yeast strains’ tolerance to the increasing ethanol levels typical of the starch-to-ethanol industry. FTIR fingerprint, indeed, offers a holistic view of the metabolome and is a well-established method to assess the stress response of microorganisms. Cell viability and metabolomic fingerprints have been considered as parameters to detecting any physiological and/or metabolomic perturbations. Quite surprisingly, the three strains did not show any difference in cell viability but metabolomic profiles were significantly altered and different when the strains were incubated both with and without ethanol. A LC/MS untargeted workflow was applied to assess the metabolites and pathways mostly involved in these strain-specific ethanol responses, further confirming the FTIR fingerprinting of the parental and recombinant strains. These results indicated that the multiple δ-integration prompted huge metabolomic changes in response to short-term ethanol exposure, calling for deeper metabolomic and genomic insights to understand how and, to what extent, genetic engineering could affect the yeast metabolome.

Highlights

Bioethanol obtained from biomass is one of the most promising biofuels to reduce dependence on oil and decrease carbon dioxide emissions [1]
The two recombinant strains F2 and F6 were engineered to ferment raw starch into ethanol through the multiple delta-integration of the sGAI gene of A. awamori under the constitutive transcriptional control of PGK1 [21]. The evidence that both the parental S. cerevisiae 27P and recombinant strains displayed similar ethanol yield and growth rate from glucose indicated that the multiple delta-integration and expression of the sGAI gene did not result in any evident metabolic burden when strains were grown in glucose under oxygen limiting conditions [21]
A Fourier Transform InfraRed Spectroscopy (FTIR)-based assay, already employed as a powerful technique for ecotoxicological assessments [28,29,30], was carried out to estimate the type and extent of perturbations induced by ethanol stress on both parental and recombinant yeast strains

Summary

Introduction

Bioethanol obtained from biomass is one of the most promising biofuels to reduce dependence on oil and decrease carbon dioxide emissions [1]. The main cost in bioethanol and other bio-products production is the substrate. Starchy residual biomass is the most available substrate for bioethanol considering its great availability and limited price [11,12,13]. Regardless of these advantages, starch-to-ethanol processing is very expensive because of the need for bulky amounts of commercial enzymes. More cost-effective methods are needed and the consolidated bioprocessing (CBP)

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Conclusion