CRISPR/Cas9-mediated engineering of Escherichia coli for n-butanol production from xylose in defined medium

  • Ali Samy Abdelaal
  • Kamran Jawed
  • Syed Shams YazdaniEmail author
Bioenergy/Biofuels/Biochemicals - Original Paper


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.


Metabolic 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.

Author contributions

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.

Supplementary material

10295_2019_2180_MOESM1_ESM.pdf (59 kb)
Supplementary file1 (PDF 58 kb)


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Copyright information

© Society for Industrial Microbiology and Biotechnology 2019

Authors and Affiliations

  1. 1.Microbial Engineering GroupInternational Centre for Genetic Engineering and BiotechnologyNew DelhiIndia
  2. 2.DBT-ICGEB Centre for Advanced Bioenergy ResearchInternational Centre for Genetic Engineering and BiotechnologyNew DelhiIndia
  3. 3.Department of Genetics, Faculty of AgricultureDamietta UniversityDamiettaEgypt

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