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Precise promoter integration improves cellulose bioconversion and thermotolerance in Clostridium cellulolyticum
Xuanyu Tao1, Tao Xu2, Megan L Kempher1
1Institute for Environmental Genomics, Department of Microbiology and Plant Biology, and School of Civil Engineering and Environmental Sciences, University of Oklahoma, Norman, OK, USA.
Metabolic Engineering
|April 16, 2020
Summary
CRISPR-Cas9 editing enhanced cellulose degradation in Clostridium cellulolyticum by modifying gene regulation. Engineered strains showed improved biomass yield and ethanol production, reducing costs for biofuels and chemicals.
Area of Science:
- Biotechnology
- Microbial Engineering
- Synthetic Biology
Background:
- Lignocellulose bioconversion is key for sustainable biofuels and chemicals.
- Low efficiency of lignocellulose degradation leads to high production costs.
Purpose of the Study:
- To improve cellulose degradation efficiency in Clostridium cellulolyticum using CRISPR-Cas9.
- To enhance the production of biofuels and value-added chemicals from lignocellulose.
Main Methods:
- CRISPR-Cas9 gene editing was used to insert synthetic (P4) and endogenous (P2) promoters into the cip-cel gene cluster of Clostridium cellulolyticum.
- Engineered strains (P4-2866, P2-2866) were evaluated for cellulose degradation, growth rate, biomass yield, and thermotolerance.
- Promoter insertion effects were also tested in wildtype and a lactate-defective mutant (LM) strain.
Main Results:
- Engineered strains showed increased transcript abundance of downstream genes and enhanced cellulosome activity.
- P4-2866 and P2-2866 hydrolyzed 29% and 53% of cellulose at high load, respectively, compared to the parental strain.
- Engineered strains exhibited improved growth rates, biomass yields, and thermotolerance; P2 insertion in LM increased ethanol titer by 65%.
Conclusions:
- Editing regulatory elements of catabolic gene clusters is a viable strategy to improve cellulose bioconversion.
- This approach offers new perspectives for enhancing microbial production of biofuels and chemicals from lignocellulose.
- CRISPR-Cas9 mediated promoter engineering can significantly boost lignocellulose utilization efficiency and product yields.
