Improving Saccharomyces cerevisiae acid and oxidative stress resistance using a prokaryotic gene identified by functional metagenomics

Abstract

Innovations in obtaining products from lignocellulosic biomass have been largely based on the improvement of microorganisms and enzymes capable of degrading these materials. To complete the whole process, microorganisms must be able to ferment the resulting sugars and tolerate high concentrations of product, osmotic pressure, ion toxicity, temperature, toxic compounds from lignocellulose pretreatment, low pH, and oxidative stress. In this work, we engineered laboratory and industrial Saccharomyces cerevisiae strains by combining a gene (hu) recovered from a metagenomic approach with different native and synthetic promoters to obtain improved acid and oxidative stress resistance. Laboratorial strains harboring hu gene under the control of the synthetic stress responsive PCCW14v5 showed increased survival rates after 2 h exposure to pH 1.5. The hu gene was also able to significantly enhance the tolerance of the industrial strain to high concentrations of H₂O₂ when combined with PTEF1, PYGP1 or PYGP1v7 after 3 h exposure.

Keywords: Low pH; Metagenomics; Oxidative stress; Stress-responsive promoters; Yeast engineering.

Fig. 1

Schematic representation of the pCfB general vector backbone showing the cassettes cloned in the USER site. Functional elements of the plasmid backbone are shown: Amp^R, ampicillin resistance gene; pUC ori, origin of replication for E.coli; ADH1_t and CYC1_t, transcriptional terminators; UP and DOWN, integration targeting sequences; loxP, sites that allow marker rescue mediated by CreA site-specific recombinase; NotI, restriction sites recognized by the endonuclease NotI; Marker cassette, Klleu2 (auxotrophic marker in pCfB2192 – used in CEN.PK113-32D strain) and kanMX (resistance to G418 in pCfB2225 – used in Ethanol red strain). Cloned in the USER site is the hu gene under control of constitutive promoter PTEF1 or the stress-responsive promoters PYGP1, PYGP1v7, or PCCW14v5. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 2

Survival rates of S. cerevisiae CEN.PK113-32D and Ethanol red strains after exposure to extremely low pH. Survival percentages of the engineered S. cerevisiae CEN.PK113-32D harboring hu gene under control of four different promoters after A. 1 h and B. 2 h exposure to pH 1.5. Survival percentages of the engineered S. cerevisiae Ethanol red harboring hu gene under control of four different promoters after C. 1 h and D. 2 h exposure to pH 1.5. The boundaries of the boxes indicate the 25th and 75th percentiles; the lines within the boxes indicate the median, and the whiskers mark the minimum and maximum values obtained. “+” indicate the means. P-values comparing promoters with empty cassette values (t-test) are shown in the graphics when P < 0.05. The experiments were carried out in YNB media pH 1.5 supplemented with 2% glucose and without selective pressure. Independent experiments were carried out at least three times. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 3

Survival rates of S. cerevisiae CEN.PK113-32D and Ethanol red strains after exposure to H₂O₂. A. Survival percentages of the engineered S. cerevisiae CEN.PK113-32D harboring hu gene under control of four different promoters after 3 h exposure to H₂O₂ 2.5 mM. B. Survival percentages of the engineered S. cerevisiae Ethanol red harboring hu gene under control of four different promoters after 3 h exposure to H₂O₂ 11.5 mM. The boundaries of the boxes indicate the 25th and 75th percentiles; the lines within the boxes indicate the median, and the whiskers mark the minimum and maximum values obtained. “+” indicate the means. P-values comparing promoters with empty cassette values (t-test) are shown in the graphics when P < 0.05. The experiments were carried out in YNB media supplemented with 2% glucose and without selective pressure. Independent experiments were carried out at least three times. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 4

Glucose consumption and ethanol production during fermentation/recycling cycles. S. cerevisiae CEN.PK113-32D control (harboring empty cassette – in absence of gene and promoters) and engineered with hu gene under the control of TEF1 and CCW14v5 promoters showing A. glucose consumption and B. ethanol production on fermentation/recycling cycles 1, 6 and 10 after 6 h of fermentation. C. Glucose consumption and D. ethanol production in cycle 11 (after 1 week of acid treatment) after 24 and 48 h fermentation. Independent experiments were carried out in biological triplicates.