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.
Author: | Virginia SchadewegGND, Eckhard BolesORCiD |
---|---|
URN: | urn:nbn:de:hebis:30:3-423838 |
DOI: | https://doi.org/10.1186/s13068-016-0673-0 |
ISSN: | 1754-6834 |
Pubmed Id: | https://pubmed.ncbi.nlm.nih.gov/27924150 |
Parent Title (English): | Biotechnology for biofuels |
Publisher: | BioMed Central |
Place of publication: | London |
Document Type: | Article |
Language: | English |
Date of Publication (online): | 2016/12/01 |
Date of first Publication: | 2016/11/25 |
Publishing Institution: | Universitätsbibliothek Johann Christian Senckenberg |
Release Date: | 2016/12/01 |
Tag: | Acetyl-CoA; Acetylating acetaldehyde dehydrogenase; Coenzyme A; Pantothenate; Saccharomyces; Trans-2-enoyl-CoA reductase; n-Butanol |
Volume: | 9 |
Issue: | 257 |
Page Number: | 11 |
First Page: | 1 |
Last Page: | 11 |
Note: | © The Author(s) 2016. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. |
HeBIS-PPN: | 425198170 |
Institutes: | Biowissenschaften / Biowissenschaften |
Dewey Decimal Classification: | 5 Naturwissenschaften und Mathematik / 57 Biowissenschaften; Biologie / 570 Biowissenschaften; Biologie |
Sammlungen: | Universitätspublikationen |
Open-Access-Publikationsfonds: | Biowissenschaften |
Licence (German): | Creative Commons - Namensnennung 4.0 |