Constructing a methanol-dependent Bacillus subtilis by engineering the methanol metabolism.

Area of Science:

• Synthetic biology
• Microbial engineering
• Green chemistry

Background:

• Methanol is a sustainable feedstock for fuels and chemicals.
• Engineering non-native methylotrophic microorganisms is crucial for methanol utilization.
• Bacillus subtilis is a versatile host for industrial applications.

Purpose of the Study:

• To develop a synthetic methylotrophic Bacillus subtilis capable of utilizing methanol as a sole carbon source.
• To enhance methanol utilization efficiency through metabolic engineering strategies.
• To create robust strains for sustainable production of fuels and chemicals.

Main Methods:

• Heterologous expression of methanol dehydrogenase (Mdh).
• Enhancement of key enzyme expressions (Hps, Phi) and regulation at transcriptional/translational levels.
• Stabilization of gene expression using a dual plasmid-genome system.
• NAD+ recycling strategy to improve Mdh catalytic activity.
• Metabolic engineering by deleting phosphoglucose isomerase (Pgi) and adding co-substrates.

Main Results:

• Engineered B. subtilis achieved a methanol consumption of 4.09 g/L.
• Maximum OD600 increased 2.21-fold compared to wild-type.
• Specific methanol consumption rate increased by 27.54% after Pgi deletion and co-substrate addition.
• Successfully constructed strains requiring methanol for growth.

Conclusions:

• Developed a synthetic methylotrophic B. subtilis with enhanced methanol utilization.
• Metabolic engineering strategies are effective for creating efficient methanol-utilizing microorganisms.
• The developed strains show potential for sustainable production of chemicals and fuels.
• The strategies are broadly applicable to synthetic methylotrophy research.