Enhanced poly-γ-glutamic acid synthesis in Corynebacterium glutamicum by reconstituting PgsBCA complex and...

Area of Science:

• Microbiology
• Biotechnology
• Synthetic Biology

Background:

• Poly-γ-glutamic acid (γ-PGA) is a biodegradable polymer with diverse applications.
• The γ-PGA synthetase complex (PgsBCA) from Bacillus species is crucial for γ-PGA biosynthesis.
• Previous work established a Corynebacterium glutamicum strain for de novo γ-PGA production.

Purpose of the Study:

• To enhance γ-PGA synthesis by reconstituting and overexpressing the PgsBCA complex in C. glutamicum.
• To investigate the effects of individual subunit expression levels on γ-PGA production.
• To explore the impact of orthologous genes from different Bacillus species on γ-PGA synthesis.

Main Methods:

• Reconstitution and overexpression of the PgsBCA complex in C. glutamicum.
• Tuning the transcription levels of pgsB, pgsC, and pgsA genes.
• Testing orthologous PgsB and PgsC genes from various Bacillus species.
• Optimization of fermentation conditions, including dissolved oxygen and glucose supplementation.

Main Results:

• All three components (PgsB, PgsC, PgsA) are essential for γ-PGA synthesis in C. glutamicum.
• Optimizing pgsC and pgsB transcription significantly increased γ-PGA yield, with pgsC having the most substantial impact.
• Replacing B. licheniformis PgsB with B. methylotrophicus PgsB increased γ-PGA titer to 17.14 g/L.
• Achieved a record de novo γ-PGA titer of 50.2 g/L from glucose in a 5L fermentor.

Conclusions:

• The expression levels of PgsBCA subunits critically influence γ-PGA production.
• Orthologous PgsC genes generally inhibited synthesis, while B. methylotrophicus PgsB showed promise.
• This study presents a novel and highly efficient strategy for high-titer de novo γ-PGA production.