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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.
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.