Increased fermentative adenosine production by gene-targeted Bacillus subtilis mutation

Area of Science:

• Biotechnology
• Microbial genetics
• Metabolic engineering

Background:

• Adenosine is crucial for cardiovascular disease therapy and widely used as an antiarrhythmic agent.
• Microbial fermentation is a primary source for adenosine production.
• Optimizing adenosine biosynthesis pathways in microorganisms is essential for industrial applications.

Purpose of the Study:

• To enhance adenosine production in Bacillus subtilis (B. subtilis) through metabolic engineering.
• To investigate the genetic mechanisms underlying increased adenosine yields.
• To develop a practical strategy for industrial-scale nucleoside production.

Main Methods:

• Gene targeting to inactivate the guanosine 5 -monophosphate (GMP) synthetase gene (guaA) in B. subtilis A509.
• Metabolic flux analysis to assess the impact of guaA inactivation on adenosine synthesis.
• Central composite design (CCD) for optimizing fermentation conditions.
• Quantitative analysis of adenosine production levels.

Main Results:

• Inactivation of the guaA gene in B. subtilis mutant M3-3 increased adenosine production from 7.40 to 10.45 g/L.
• Further optimization using central composite design enhanced adenosine production to a maximum of 14.39 g/L.
• Up-regulated transcription of the purA gene, encoding adenylosuccinate synthetase, was identified as a potential mechanism for increased adenosine synthesis, as it catalyzes the rate-limiting step.

Conclusions:

• Genetic manipulation of B. subtilis by inactivating the guaA gene is an effective strategy to boost adenosine production.
• The purA gene's transcriptional upregulation plays a key role in enhancing adenosine biosynthesis.
• This study provides a viable approach for the industrial production of nucleosides using engineered B. subtilis strains.