Engineering riboflavin-overproducing Bacillus subtilis via pathway gene overexpression

Area of Science:

• Metabolic Engineering
• Synthetic Biology
• Microbial Biotechnology

Background:

• Overexpressing genes at metabolic nodes enhances product yield but often causes growth defects and plasmid instability.
• Bacillus subtilis U3, a riboflavin over-producer, was used as a model system to study these effects.
• Understanding and mitigating these issues is crucial for efficient industrial microbial production.

Purpose of the Study:

• To investigate the impact of overexpressing riboflavin operon genes on Bacillus subtilis strain growth and plasmid stability.
• To identify strategies for resolving growth defects and plasmid instability in metabolically engineered strains.
• To evaluate the utility of the Respiration Activity Monitoring System (RAMOS) in metabolic engineering.

Main Methods:

• Systematic overexpression of riboflavin operon genes in Bacillus subtilis U3.
• Analysis of strain growth, plasmid structural integrity, and product yield.
• Introduction of frameshift mutations in the ribD gene.
• Application of Respiration Activity Monitoring System (RAMOS) for growth evaluation and medium optimization.
• Supplementation studies with specific nutrients (guanine, histidine, uracil, tryptophan).

Main Results:

• Overexpression of rib operon genes enhanced riboflavin yield by 13.2% but led to growth defects and plasmid instability.
• Frameshift mutations in ribD significantly reduced the loss of operon gene fragments by 16.7%.
• RAMOS proved effective for evaluating growth and optimizing media, with specific nutrient supplements improving biomass by up to 71.1%.

Conclusions:

• Metabolic engineering strategies for enhanced product yield must address associated growth defects and plasmid instability.
• Targeted genetic modifications (e.g., ribD frameshift mutations) and nutrient supplementation are viable solutions.
• RAMOS is a valuable tool for optimizing conditions and screening strains in metabolic engineering, providing a framework for resolving growth issues.