Abstract:Saccharomyces cerevisiae has recently been engineered to use acetate, a primary inhibitor in lignocellulosic hydrolysates, as a cosubstrate during anaerobic ethanolic fermentation. However, the original metabolic pathway devised to convert acetate to ethanol uses NADH-specific acetylating acetaldehyde dehydrogenase and alcohol dehydrogenase and quickly becomes constrained by limited NADH availability, even when glycerol formation is abolished. We present alcohol dehydrogenase as a novel target for anaerobic re… Show more
“…On the other hand, the ethanol yield was maintained at 0.42 ± 0.04 g ethanol g glucose −1 throughout the evolution process in spite of the increase in dilution rate and decrease in pH. Moreover, the concentration of acetate remained constant at 6 g L −1 , as it could not be consumed as a source of carbon by S. cerevisiae under anaerobic conditions (Henningsen et al 2015) (Fig. 6b).Fig.…”
Lignocellulosic bioethanol from renewable feedstocks using Saccharomyces cerevisiae is a promising alternative to fossil fuels owing to environmental challenges. S. cerevisiae is frequently challenged by bacterial contamination and a combination of lignocellulosic inhibitors formed during the pre-treatment, in terms of growth, ethanol yield and productivity. We investigated the phenotypic robustness of a brewing yeast strain TMB3500 and its ability to adapt to low pH thereby preventing bacterial contamination along with lignocellulosic inhibitors by short-term adaptation and adaptive lab evolution (ALE). The short-term adaptation strategy was used to investigate the inherent ability of strain TMB3500 to activate a robust phenotype involving pre-culturing yeast cells in defined medium with lignocellulosic inhibitors at pH 5.0 until late exponential phase prior to inoculating them in defined media with the same inhibitor cocktail at pH 3.7. Adapted cells were able to grow aerobically, ferment anaerobically (glucose exhaustion by 19 ± 5 h to yield 0.45 ± 0.01 g ethanol g glucose−1) and portray significant detoxification of inhibitors at pH 3.7, when compared to non-adapted cells. ALE was performed to investigate whether a stable strain could be developed to grow and ferment at low pH with lignocellulosic inhibitors in a continuous suspension culture. Though a robust population was obtained after 3600 h with an ability to grow and ferment at pH 3.7 with inhibitors, inhibitor robustness was not stable as indicated by the characterisation of the evolved culture possibly due to phenotypic plasticity. With further research, this short-term adaptation and low pH strategy could be successfully applied in lignocellulosic ethanol plants to prevent bacterial contamination.Electronic supplementary materialThe online version of this article (doi:10.1186/s13568-016-0234-8) contains supplementary material, which is available to authorized users.
“…On the other hand, the ethanol yield was maintained at 0.42 ± 0.04 g ethanol g glucose −1 throughout the evolution process in spite of the increase in dilution rate and decrease in pH. Moreover, the concentration of acetate remained constant at 6 g L −1 , as it could not be consumed as a source of carbon by S. cerevisiae under anaerobic conditions (Henningsen et al 2015) (Fig. 6b).Fig.…”
Lignocellulosic bioethanol from renewable feedstocks using Saccharomyces cerevisiae is a promising alternative to fossil fuels owing to environmental challenges. S. cerevisiae is frequently challenged by bacterial contamination and a combination of lignocellulosic inhibitors formed during the pre-treatment, in terms of growth, ethanol yield and productivity. We investigated the phenotypic robustness of a brewing yeast strain TMB3500 and its ability to adapt to low pH thereby preventing bacterial contamination along with lignocellulosic inhibitors by short-term adaptation and adaptive lab evolution (ALE). The short-term adaptation strategy was used to investigate the inherent ability of strain TMB3500 to activate a robust phenotype involving pre-culturing yeast cells in defined medium with lignocellulosic inhibitors at pH 5.0 until late exponential phase prior to inoculating them in defined media with the same inhibitor cocktail at pH 3.7. Adapted cells were able to grow aerobically, ferment anaerobically (glucose exhaustion by 19 ± 5 h to yield 0.45 ± 0.01 g ethanol g glucose−1) and portray significant detoxification of inhibitors at pH 3.7, when compared to non-adapted cells. ALE was performed to investigate whether a stable strain could be developed to grow and ferment at low pH with lignocellulosic inhibitors in a continuous suspension culture. Though a robust population was obtained after 3600 h with an ability to grow and ferment at pH 3.7 with inhibitors, inhibitor robustness was not stable as indicated by the characterisation of the evolved culture possibly due to phenotypic plasticity. With further research, this short-term adaptation and low pH strategy could be successfully applied in lignocellulosic ethanol plants to prevent bacterial contamination.Electronic supplementary materialThe online version of this article (doi:10.1186/s13568-016-0234-8) contains supplementary material, which is available to authorized users.
“…Candida and S. cerevisiae , which can convert carbohydrates to endogenous alcohol, were detected in his intestinal secretions and stool. S. cerevisiae is known to use acetate for anaerobic alcohol fermentation 2 5. A large Middle Eastern study involving 1400 subjects who were teetotalers detected very small endogenously produced alcohol levels using gas chromatography and mass spectrometry.…”
Auto-brewery syndrome (ABS), also known as gut fermentation syndrome, is a rarely diagnosed medical condition in which the ingestion of carbohydrates results in endogenous alcohol production. The patient in this case report had fungal yeast forms in the upper small bowel and cecum, which likely fermented carbohydrates to alcohol. Treatment with antifungal agents allowed subsequent ingestion of carbohydrates without symptoms. He had been exposed to a prolonged course of antibiotics before this occurred. We postulate that the antibiotic altered his gut microbiome, allowing fungal growth. This diagnosis should be considered in any patient with positive manifestations of alcohol toxicity who denies alcohol ingestion. The aim of this case report was confirmation and treatment of ABS using a standardised carbohydrate challenge test followed by upper and lower endoscopy to obtain intestinal secretions to detect fungal growth. These fungi were speciated and antifungal sensitivity performed. This allowed the use of appropriate therapy. The patient was kept on a carbohydrate-free diet during the initial 6-week period of therapy. A single-strain probiotic for competitive inhibition of fungal growth was given to the patient. This probiotic was later replaced by a multistrain bacterial probiotic hoping that the multiple bacteria would inhibit fungi better than a single-strain. The beneficial role of probiotics in this condition has not been studied. The patient was rechallenged for endogenous alcohol production prior to reintroducing carbohydrates in his diet.
“…Recently, an alternative metabolic engineering strategy to increase the reduction of acetate to ethanol was described [ 24 ]. This alternative strategy is based on introduction of a heterologous NADPH-dependent alcohol dehydrogenase in combination with overexpression of ZWF1 and/or ACS2.…”
BackgroundAcetic acid, an inhibitor of sugar fermentation by yeast, is invariably present in lignocellulosic hydrolysates which are used or considered as feedstocks for yeast-based bioethanol production. Saccharomyces cerevisiae strains have been constructed, in which anaerobic reduction of acetic acid to ethanol replaces glycerol formation as a mechanism for reoxidizing NADH formed in biosynthesis. An increase in the amount of acetate that can be reduced to ethanol should further decrease acetic acid concentrations and enable higher ethanol yields in industrial processes based on lignocellulosic feedstocks. The stoichiometric requirement of acetate reduction for NADH implies that increased generation of NADH in cytosolic biosynthetic reactions should enhance acetate consumption.ResultsReplacement of the native NADP+-dependent 6-phosphogluconate dehydrogenase in S. cerevisiae by a prokaryotic NAD+-dependent enzyme resulted in increased cytosolic NADH formation, as demonstrated by a ca. 15 % increase in the glycerol yield on glucose in anaerobic cultures. Additional deletion of ALD6, which encodes an NADP+-dependent acetaldehyde dehydrogenase, led to a 39 % increase in the glycerol yield compared to a non-engineered strain. Subsequent replacement of glycerol formation by an acetate reduction pathway resulted in a 44 % increase of acetate consumption per amount of biomass formed, as compared to an engineered, acetate-reducing strain that expressed the native 6-phosphogluconate dehydrogenase and ALD6. Compared to a non-acetate reducing reference strain under the same conditions, this resulted in a ca. 13 % increase in the ethanol yield on glucose.ConclusionsThe combination of NAD+-dependent 6-phosphogluconate dehydrogenase expression and deletion of ALD6 resulted in a marked increase in the amount of acetate that was consumed in these proof-of-principle experiments, and this concept is ready for further testing in industrial strains as well as in hydrolysates. Altering the cofactor specificity of the oxidative branch of the pentose-phosphate pathway in S. cerevisiae can also be used to increase glycerol production in wine fermentation and to improve NADH generation and/or generation of precursors derived from the pentose-phosphate pathway in other industrial applications of this yeast.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0465-z) contains supplementary material, which is available to authorized users.
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