Reverse-Engineered Gas-Fermenting Acetogen Strains Recover Enhanced Phenotypes From Autotrophic Adaptive Laboratory...

Area of Science:

• Microbial biotechnology
• Synthetic biology
• Circular economy

Background:

• Gas-fermenting acetogens are key for converting CO and CO2 into fuels and chemicals.
• Understanding gene function in acetogens is crucial for developing efficient cell factories.
• Adaptive laboratory evolution (ALE) identified potential gene targets for improved autotrophic growth.

Purpose of the Study:

• To genetically engineer and characterize Clostridium autoethanogenum strains with enhanced autotrophic phenotypes.
• To validate the role of specific genes identified through ALE in improving industrial traits.
• To gain insights into genotype-phenotype relationships in acetogens for metabolic engineering.

Main Methods:

• Reverse engineering of three specific gene deletion/mutation targets (CLAU_0471, CLAU_3129, CLAU_1957) in C. autoethanogenum.
• Extensive characterization of autotrophic growth in batch and continuous bioreactor cultures.
• Proteome expression analysis and bioinformatic analysis of engineered strains.

Main Results:

• Reverse-engineered strains RE1, RE2, and RE3 recovered superior phenotypes from ALE isolates, including faster growth and robustness.
• Strain RE3 showed increased 2,3-butanediol production, while RE1 matched the performance of a leading ALE isolate.
• Targeted genes appear to be involved in overlapping regulatory networks, influencing key metabolic traits.

Conclusions:

• Targeted genetic modification of acetogens can effectively restore and enhance industrially desirable phenotypes.
• Understanding gene function and regulatory networks is vital for optimizing acetogen cell factories.
• This study provides valuable genotype-phenotype insights for advancing gas fermentation technology.