Increasing n‑butanol production with saccharomyces cerevisiae by optimizing acetyl‑CoA synthesis, NADH levels and trans‑2‑enoyl‑CoA reductase expression

Background: n-Butanol can serve as an excellent gasoline substitute. Naturally, it is produced by some Clostridia species which, however, exhibit only limited suitability for industrial n-butanol production. The yeast Saccharomyces cerevisiae would be an ideal host due to its high robustness in fermentation processes. Nevertheless, n-butanol yields and titers obtained so far with genetically engineered yeast strains are only low. Results: In our recent work, we showed that n-butanol production via a clostridial acetoacetyl-CoA-derived pathway in engineered yeast was limited by the availability of coenzyme A (CoA) and cytosolic acetyl-CoA. Increasing their levels resulted in a strain producing up to 130 mg/L n-butanol under anaerobic conditions. Here, we show that under aerobic conditions. this strain can even produce up to 235 mg/L n-butanol probably due to a more efficient NADH re-oxidation. Nevertheless, expression of a bacterial water-forming NADH oxidase (nox) significantly reduced n-butanol production although it showed a positive effect on growth and glucose consumption. Screening for an improved version of an acetyl-CoA forming NAD+-dependent acetylating acetaldehyde dehydrogenase, adhEA267T/E568K/R577S, and its integration into n-butanol-producing strain further improved n-butanol production. Moreover, deletion of the competing NADP+-dependent acetaldehyde dehydrogenase Ald6 had a superior effect on n-butanol formation. To increase the endogenous supply of CoA, amine oxidase Fms1 was overexpressed together with pantothenate kinase coaA from Escherichia coli, and could completely compensate the beneficial effect on n-butanol synthesis of addition of pantothenate to the medium. By overexpression of each of the enzymes of n-butanol pathway in the n-butanol-producing yeast strain, it turned out that trans-2-enoyl-CoA reductase (ter) was limiting n-butanol production. Additional overexpression of ter finally resulted in a yeast strain producing n-butanol up to a titer of 0.86 g/L and a yield of 0.071 g/g glucose. Conclusions: By further optimizing substrate supply and redox power in the form of coenzyme A, acetyl-CoA and NADH, n-butanol production with engineered yeast cells could be improved to levels never reached before with S. cerevisiae via an acetoacetyl-CoA-derived pathway in synthetic medium. Moreover, our results indicate that the NAD+/NADH redox balance and the trans-2-enoyl-CoA reductase reaction seem to be bottlenecks for n-butanol production with yeast

n-Butanol production in Saccharomyces cerevisiae is limited by the availability of coenzyme A and cytosolic acetyl-CoA

Background: Butanol isomers are regarded as more suitable fuel substitutes than bioethanol. n-Butanol is naturally produced by some Clostridia species, but due to inherent problems with clostridial fermentations, industrially more relevant organisms have been genetically engineered for n-butanol production. Although the yeast Saccharomyces cerevisiae holds significant advantages in terms of scalable industrial fermentation, n-butanol yields and titers obtained so far are only low. Results: Here we report a thorough analysis and significant improvements of n-butanol production from glucose with yeast via the acetoacetyl-CoA-derived pathway. First, we established an improved n-butanol pathway by testing various isoenzymes of different pathway reactions. This resulted in n-butanol titers around 15 mg/L in synthetic medium after 74 h. As the initial substrate of the n-butanol pathway is acetyl-coenzyme A (acetyl-CoA) and most intermediates are bound to coenzyme A (CoA), we increased CoA synthesis by overexpression of the pantothenate kinase coaA gene from Escherichia coli. Supplementation with pantothenate increased n-butanol production up to 34 mg/L. Additional reduction of ethanol formation by deletion of alcohol dehydrogenase genes ADH1-5 led to n-butanol titers of 71 mg/L. Further expression of a mutant form of an ATP independent acetylating acetaldehyde dehydrogenase, adhEA267T/E568K, converting acetaldehyde into acetyl-CoA, resulted in 95 mg/L n-butanol. In the final strain, the n-butanol pathway genes, coaA and adhE A267T/E568K, were stably integrated into the yeast genome, thereby deleting another alcohol dehydrogenase gene, ADH6, and GPD2-encoding glycerol-3-phosphate dehydrogenase. This led to a further decrease in ethanol and glycerol by-product formation and elevated redox power in the form of NADH. With the addition of pantothenate, this strain produced n-butanol up to a titer of 130 ± 20 mg/L and a yield of 0.012 g/g glucose. These are the highest values reported so far for S. cerevisiae in synthetic medium via an acetoacetyl-CoA-derived n-butanol pathway. Conclusions: By gradually increasing substrate supply and redox power in the form of CoA, acetyl-CoA, and NADH, and decreasing ethanol and glycerol formation, we could stepwise increase n-butanol production in S. cerevisiae. However, still further bottlenecks in the n-butanol pathway must be deciphered and improved for industrially relevant n-butanol production levels

MOESM2 of n-Butanol production in Saccharomyces cerevisiae is limited by the availability of coenzyme A and cytosolic acetyl-CoA

Additional file 2: Table S1. Yeast promoters and terminators used for the expression of n-butanol pathway genes in this study. Table S2. Genes used in this study and their corresponding identifiers. Table S3. Relevant primers for this study. Table S4. Statistical analysis of n-butanol production

Identification of a glucose-insensitive variant of Gal2 from Saccharomyces cerevisiae exhibiting a high pentose transport capacity

As abundant carbohydrates in renewable feedstocks, such as pectin-rich and lignocellulosic hydrolysates, the pentoses arabinose and xylose are regarded as important substrates for production of biofuels and chemicals by engineered microbial hosts. Their efficient transport across the cellular membrane is a prerequisite for economically viable fermentation processes. Thus, there is a need for transporter variants exhibiting a high transport rate of pentoses, especially in the presence of glucose, another major constituent of biomass-based feedstocks. Here, we describe a variant of the galactose permease Gal2 from Saccharomyces cerevisiae (Gal2N376Y/M435I), which is fully insensitive to competitive inhibition by glucose, but, at the same time, exhibits an improved transport capacity for xylose compared to the wildtype protein. Due to this unique property, it significantly reduces the fermentation time of a diploid industrial yeast strain engineered for efficient xylose consumption in mixed glucose/xylose media. When the N376Y/M435I mutations are introduced into a Gal2 variant resistant to glucose-induced degradation, the time necessary for the complete consumption of xylose is reduced by approximately 40%. Moreover, Gal2N376Y/M435I confers improved growth of engineered yeast on arabinose. Therefore, it is a valuable addition to the toolbox necessary for valorization of complex carbohydrate mixtures

iGEM Frankfurt 2012 Stevia Poster

<p>The project tried to transform the biosynthesis pathway of Rebaudioside A from the stevia plant into yeast cells.</p