CRISPR/Cas9-mediated engineering of Escherichia coli for n-butanol production from xylose in defined medium
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Butanol production from agricultural residues is the most promising alternative for fossil fuels. To reach the economic viability of biobutanol production, both glucose and xylose should be utilized and converted into butanol. Here, we engineered a dual-operon-based synthetic pathway in the genome of E. coli MG1655 to produce n-butanol using CRISPR/Cas9 technology. Further deletion of competing pathway followed by fed-batch cultivation of the engineered strain in a bioreactor with glucose-containing complex medium yielded 5.4 g/L n-butanol along with pyruvate as major co-product, indicating a redox imbalance. To ferment xylose into butanol in redox-balanced manner, we selected SSK42, an ethanologenic E. coli strain engineered and evolved in our laboratory to produce ethanol from xylose, for integrating synthetic butanol cassette in its genome via CRISPR/Cas9 after deleting the gene responsible for endogenous ethanol production. The engineered plasmid- and marker-free strain, ASA02, produced 4.32 g/L butanol in fed-batch fermentation in completely defined AM1–xylose medium.
KeywordsMetabolic engineering Escherichia coli CRISPR/Cas9 Biofuels Butanol Xylose
The authors thank Prof. Ramon Gonzalez for providing E. coli LA07 strain. ASA is a recipient of Arturo Falaschi Postdoctoral Fellowship from ICGEB and KJ is a recipient of Research Fellowship from CSIR.
KJ, ASA and SSY designed the study. ASA and KJ executed all the experiments. ASA, KJ and SSY analyzed all the data. SSY, ASA and KJ wrote the manuscript. All authors read and approved the final manuscript.
This study was funded by Department of Biotechnology, Government of India, via Grant no. BT/PR/Centre/03/2011.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no competing interests.
- 10.Fontaine L, Meynial-Salles I, Girbal L, Yang X, Croux C, Soucaille P (2002) Molecular characterization and transcriptional analysis of adhE2, the gene encoding the NADH-dependent aldehyde/alcohol dehydrogenase responsible for butanol production in alcohologenic cultures of Clostridium acetobutylicum ATCC 824. J Bacteriol 184:821–830CrossRefGoogle Scholar
- 12.Han GH, Seong W, Fu Y, Yoon PK, Kim SK, Yeom SJ, Lee DH, Lee SG (2017) Leucine zipper-mediated targeting of multi-enzyme cascade reactions to inclusion bodies in Escherichia coli for enhanced production of 1-butanol. Metab Eng 40:41–49. https://doi.org/10.1016/j.ymben.2016.12.012 CrossRefGoogle Scholar
- 19.Mundhada H, Seoane JM, Schneider K, Koza A, Christensen HB, Klein T, Phaneuf PV, Herrgard M, Feist AM, Nielsen AT (2017) Increased production of l-serine in Escherichia coli through adaptive laboratory evolution. Metab Eng 39:141–150. https://doi.org/10.1016/j.ymben.2016.11.008 CrossRefGoogle Scholar
- 28.Tao H, Gonzalez R, Martinez A, Rodriguez M, Ingram LO, Preston JF, Shanmugam KT (2001) Engineering a homo-ethanol pathway in Escherichia coli: increased glycolytic flux and levels of expression of glycolytic genes during xylose fermentation. J Bacteriol 183:2979–2988. https://doi.org/10.1128/JB.183.10.2979-2988.2001 CrossRefGoogle Scholar
- 30.Wiesenborn DP, Rudolph FB, Papoutsakis ET (1988) Thiolase from Clostridium acetobutylicum ATCC 824 and its role in the synthesis of acids and solvents. Appl Environ Microbiol 54:2717–2722Google Scholar