Genome editing of Clostridium autoethanogenum using CRISPR/Cas9.

ABSTRACT: BACKGROUND:Impactful greenhouse gas emissions abatement can now be achieved through gas fermentation using acetogenic microbes for the production of low-carbon fuels and chemicals. However, compared to traditional hosts like Escherichia coli or yeast, only basic genetic tools exist for gas-fermenting acetogens. To advance the process, a robust genetic engineering platform for acetogens is essential. RESULTS:In this study, we report scarless genome editing of an industrially used model acetogen, Clostridium autoethanogenum, using the CRISPR/Cas9 system. Initial efforts to retrofit the CRISPR/Cas9 system for C. autoethanogenum resulted in poor efficiency likely due to uncontrolled expression of Cas9. To address this, we constructed and screened a small library of tetracycline-inducible promoters that can also be used to fine-tune gene expression. With a new inducible promoter, the efficiency of CRISPR/Cas9-mediated desired gene deletion in C. autoethanogenum was improved to over 50 %, making it a viable tool for engineering C. autoethanogenum. CONCLUSIONS:Addition of both an inducible promoter library and a scarless genome editing tool is an important expansion to the genetic tool box of industrial C. autoethanogenum strain.

SUBMITTER: Nagaraju S 

PROVIDER: S-EPMC5069954 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature